wean 3,90 =< oe sa ie et gota ratens eae ve ii ee Me Hie nt calle Hele vi om ts ce a rf f oe wey Mis “1 (Feu i Mp ih He Prati y vintute i ty catia ia 1 i + mitt a } a Vi ut iy} ati ia - nets hie pa vib au Saye aan i if oe ! a He he ‘a “) ‘ a ve eae sWae i - a fie me ayty aE Why a Wise pit ¥ isu. Hii fi; a oe a ut i eae ins vor that ty Bei ad at re Hef Ane yF rant i tit PG ia th ? ii ae a Seal 7 h fe 7 WIL aes v vo Ay a ie \ Aa 7” Ma a i i Cat TAG AP Da MA uty ] Nay tee Por : ‘ T ‘ ait : : Se THE AMERICAN JOURNAL OF PHYSIOLOGY EDITED FOR Che American Jbpsiological Societp AMERICAN JOURNAL OF PHYSIOLOGY VoLuME XXXII BGSTON, Uss7 TQ13 COPYRIGHT, 1913 BY THE PLIMPTON PRESS > AE oy” THE+PLIMPTON:FPRESS - NORWOOD+MASS+U:S+A CON DE NES Noni. May. 25 16m PAGE THE RELATION OF OSMOTIC PRESSURE TO ABSORPTION PHENOMENA IN THE Magni. Lby'G. G: Scottand W. Denis. 2 bo. 8 ee a ce SE I A DEFINITE Puoysico-CHEMICAL HYPOTHESIS TO EXPLAIN VISUAL RESPONSE PEPE MATI CL el SUlONG! wii ot SO ea Pe ee ee 8 THE UNIFORM RATE OF THE DESTRUCTION OF PEPSIN BY THE PASSAGE OF THE DirEctT ELECTRIC CURRENT. By W. E. Burge ; : 41 THE EFFECT OF ADRENAL SECRETION ON MuSCULAR FatTIGuE. By W. B. AUT PREY NRGE., et) soe ee Cok eon es See ee AE THE RECEPTIVE RELAXATION OF THE Coton. By Henry Lyman. ... 61 REMARKS ON THE ORIGIN OF THE PHRENIC NERVE IN THE RABBIT, CAT, AMO Ce ey Abby Hi: Turner iu. 8 8. gs ens te ew OS ON THE RELATION OF PULSE PRESSURE TO RENAL SECRETION. By Robert PERE ONE ee Sy sg, ER Oe WO Et LES Wad oO No. II, JUNE 2, 1913 DIRECT AND CROSSED RESPIRATION UPON STIMULATION OF THE PHRENIC, THE SCIATIC, AND THE BRACHIAL NERVES. By W.T. Porter and Abby 18! TEOCGTIG? eee OO a ee a ae Cee OS CARBON DIOXIDE PRODUCTION FROM NERVE FIBRES WHEN RESTING AND WHEN STIMULATED; A CONTRIBUTION TO THE CHEMICAL BASIS OF PeEREARLEIN DY SWMOLGSHIUO, foi ede cei eee oS BA 107 A New METHOD AND APPARATUS FOR THE ESTIMATION OF EXCEEDINGLY MINUTE QUANTITIES OF CARBON DioxIDE. By Shiro Tashiro. . . . 137 STUDIES ON THE PHYSICAL PROPERTIES OF PROTOPLASM. I. THE PHYSICAL PROPERTIES OF THE PROTOPLASM OF CERTAIN ANIMAL AND PLANT PREECE UG ECR MME... hae UREN eck e-toc os. aeRO Now iit, JULY ren ON THE RELATION OF THE BLOOD SALTS TO CARDIAC CONTRACTION. By Seen? 27 ieee ee: ), A ee. 2h wee .. oe TOs v1 Contents PAGE THE SUGAR CONSUMPTION IN NORMAL AND DIABETIC (DEPANCREATED) Docs AFTER EVISCERATION. By J. J. R. Macleod and R. G. Pearce 184 ON THE FORMATION OF FAT FROM CARBOHYDRATES. By Sergius Morgulis and Joseph H. Pratt. ..°.. : 5. ee > ee THE ACTION OF THROMBOPLASTIC SUBSTANCE IN THE CLOTTING OF BLOOD. By F. W. MacRae, Jr., ond-A; GuSchnack”. . ee . eee No: IV, Avecusr a, sor STUDIES IN FaTicuE. I. FatiGuE AS AFFECTED BY CHANGES OF ARTERIAL Pressure. By Charles M.Gruber . .. .°. 2-2. ee On CERTAIN DISTINCTIONS BETWEEN TASTE AND SMELL. By George Howard Parker and Eleanor Merritt Stabler’. . . ......:. -. | eee! No. V, SEPTEMBER 2, I913 Is THE PRESSOR EFFECT OF PITUITRIN DUE TO ADRENAL STIMULATION. By R. G. Hoskins and Clayton McPeek. .. =... .. . . seeeeeaE CONTRIBUTIONS TO THE PHYSIOLOGY OF THE STOMACH. V. THE INFLUENCE OF STIMULATION OF THE GASTRIC MUCOSA ON THE CONTRACTIONS OF THE Empty StoMACH (HUNGER CONTRACTIONS) IN MAN. By A. J. Carlson: vu) shes oS em. Ce eee eee Rapm METHOD OF PREPARING THROMBIN. ByW.H. Howell . ... . 264 THE RELATION OF METATHROMBIN TO THROMBIN. By F.W. Weymouth . 266 ON THE ABSORPTION OF WATER BY THE SKIN OF THE Froc. By S. S. Masiell so. ew ee el ee ee No. VI, OcTOBER 1, 1913 PHYSIOLOGICAL OBSERVATIONS FOLLOWING DESCENT FROM PIKE’S PEAK TO CoLoRADO SPRINGS. By Edward C. Schneider... ........ 205 THE EFFECT OF WATER INGESTION ON THE FATTY CHANGES OF THE LIVER IN Fastinc Rapeits. By M.R: Smrnmow. .°. .. . . .1. . . 2) Beg ON THE INFLUENCE OF MuscULAR EXERCISE ON THE ACTIVITY OF BULBAR CENTRES. By E. G. Martin and C. M.Gruber.. ... .. 2... .. gi Contents vil No. VII, NovEMBER 1, 1913 PAGE ELECTROMYOGRAM STUDIES: IT. On Some TECHNICAL PROCEDURES IN THE USE OF THE EINTHOVEN GALVANOMETER. .By Charles D.Snydey .....%:%..... 320 II. ON THE TIME RELATIONS AND FORM OF THE ELECTRIC RESPONSE OF MUSCLE IN THE SINGLE TwitcH. By Charles D. Snyder. . . . 336 THE ENDURANCE OF ANEMIA BY NERVE CELLS IN THE MYENTERIC PLEXUS. mn canrton Gnd 1. KR. BUrRet .5 ke kn Se BAF EVIDENCE OF FAT ABSORPTION BY THE MUCOSA OF THE MAMMALIAN SToMACH. By Charles W. Greene and William F.Skaer ...... . 358 CONTRIBUTIONS TO THE PHYSIOLOGY OF THE STOMACH: VI. A Srupy OF THE MECHANISMS OF THE HUNGER CONTRACTIONS OF THE EmMpTy STOMACH BY EXPERIMENTS ON Docs. By A. J. CASON cP tS io we oa ee ee. 3 2200 VII. Tue INHIBITORY REFLEXES FROM THE GASTRIC Mucosa. By PIES OOTISHIUS ¢ tis x Ores. be) REY. 3) RN B86 THe ToNuS AND HUNGER CONTRACTIONS OF THE Empty STOMACH DURING PARATHYROID TETANY. By A.J. Carison . Ea 398 THE EFFECT OF PITUITARY EXTRACT UPON RENAL Activity. ByC. E. King and O. O. Stoland : - 405 No. VIII, DECEMBER 1, 1913 A Mertuop oF EXCLUDING BILE FROM THE INTESTINE WITHOUT EXTERNAL FistuLa. By Richard M. Pearce and A.B. Eisenbrey. ....... 417 A COMPARISON OF THE AUSCULTATORY BLOOD PRESSURE PHENOMENON IN MAN WITH THE TRACING OF THE ERLANGER SPHYGMOMANOMETER. By mine. Weysse and Brenton Re lade... AT STUDIES IN FaticuE: II. THE THRESHOLD STIMULUS AS AFFECTED BY FATIGUE AND SUBSEQUENT Rest. By Charles M.Gruber ... . . . 438 OE OS a | a ee nn rer eer ERE American Journal of Physiology VOL. XXXII MAY 1, 1913 NO: THE RELATION OF OSMOTIC PRESSURE TO ABSORB-— TION PHENOMENA IN THE DOG FISH By G. G. SCOTT ann W. DENIS [From the Laboratory of the U. S. Bureau of Fisheries, Woods Hole, Mass.] LTHOUGH much work has been done on problems relating to the absorption of inorganic salts, dye stuffs etc. by the skin and mucous membranes of mammals, and the excretion of the same in the urine, but little attention has been directed to this function in marine animals. In connection with the work of one of us (Scott) regarding the behavior of the Elasmobranchs when placed in diluted and concentrated sea water, it occurred to us that it might be of interest to see whether foreign materials such as salts etc. dissolved in the sea water would follow the same laws as apparently apply to the absorption of water. As it has been shown by Botazzi ’06 and others that the blood of Elasmobranchs is about equal in its osmotic pressure to that of the sea water in which they live, and as it is known - that fish of this group will survive, for a time at least, considerable changes in the concentration of this medium, the problem of the absorption is simpler in such a case than in that of the Teleosts. The smooth dog fish “‘Mustelus canis” was used in all our experi- ments, care being taken in every case to use animals in good condition showing no trace of any abrasion of the skin or mucous membranes. Throughout our work the following procedure was employed: First, in all animals the spinal cord was destroyed to about the level of the anterior dorsal fin. The fish was then made to swallow a large 2 G. G. Scott and W. Denis bolus of absorbent cotton saturated with olive oil; to this bolus a long thread was attached so that after the former had passed into the stomach, gentle tractions on the thread would serve to bring the bolus into the viscus. By this device it was possible as was proved by numerous tests to prevent the entrance of any fluid into the stomach. The fish after being subjected to the above treatment was then placed tail up in a tall narrow glass jar of about five litres capacity contain- ing three litres of water and inclined at an angle of about forty degrees. A current of air kept continually bubbling through the water in the jar prevented asphyxiation of the animal. The fish when placed in the jar in the manner described above could be maintained for hours in such a position that only the head was under water. If some nontoxic substance for the qualitative detection of which a delicate method is available be now mixed with the sea water in the jar it is possible to measure the absorption of this body from the water under various experimental conditions. Under the experi- mental conditions‘ outlined above three membranes are exposed to the substance whose absorption is to be observed, viz., the skin of the head, the mucous membrane of the mouth and pharynx, and the gill membranes. As will be shown later on we feel we can surely exclude the skin of the dog fish from any participation in the absorp- tion phenomena taken up in this paper. Regarding the relative importance on the one hand of the buccal mucous membrane and on the other of the gill membranes as paths of absorption we can only say that the evidence presented by Bert ’71, Mosso ’90, Sumner ’o06 indicate the gill membranes to be the seat of osmotic exchange. In a paper to be published soon,! one of us (Scott) will cite experiments which show that Mustelus with its cesophagus ligated, and the head as far back as the fifth gill slit immersed in fresh water, exhibits as great a change in the osmotic pressure of the blood as when: the entire fish is immersed in the experimental solution. In the latter case the skin of the body, the intestinal tract, the gill mem- branes are freely exposed to the experimental medium. In the former case it is the gill membranes which are the chief membranes exposed. Since as great a change occurs in the latter case, the author (Scott) taking into account all the other evidence offered, feels that the only conclusion to be drawn is that the gill membranes constitute by far 1 Annals N. Y. Academy of Sciences. Osmotic Pressure and Absorption 3 the chief passageways through which the substances in the present instance pass into the blood. We however, do not entirely disre- gard the possibility that the mucous membrane of the mouth and the pharynx, which like the gill membrane is continually bathed in the medium, may in part at least be responsible for absorption phenomena, but are satisfied that these membranes at the most are concerned only to a nominal degree. In our first experi- ments we sought to learn whether foreign substances introduced into the sea water but prevented from reaching the stomach could be detected in the body fluids, more especially the coelomic fluid, blood and urine, and the influence if any produced on such absorp- tion by changes in the concentration of the sea water. The urine was collected by means of a canula tied into the urinary papilla. The method of obtaining urine was outlined by Denis ’12. Samples of biood were obtained from the caudal artery. ABSORPTION OF METHYLENE BLUE Experiment I. — Fish 4, length 83.5 cm.; weight 2009 gm. After pithing the animal, tying a canula in the urinary papilla and causing the animal to swallow a bolus of absorbent cotton, the fish was placed in a jar containing three litres of sea water and go mg. of methylene blue. After nine hours the animal while apparently still in good con- dition was killed. The volume of urine collected during this period amounted to 4 c.c. only and contained no trace of methylene blue. Experiment II. — Fish 2, length 71 cm.; weight 1047 gm. This animal was placed for four hours in a mixture of one and one half litres of sea water and one and one half litres of distilled water to which had been added go mg. of methylene blue. At the end of this time the urine was of a distinct green tinge showing that some of the dye stuff had undoubt- edly been absorbed. ABSORPTION OF Boric AcID Experiment IIT. — Fish 42, length 76 cm.; weight 1641 gm. This animal was placed for four hours and fifteen minutes in sea water to which had been added 0.3 gm. boric acid per litre. The volume of urine collected amounted to about 1 c.c. Urine, blood, coelomic fluid and stomach contents all gave negative results when tested for the presence 4 G. G. Scott and W. Dents of boric acid by means of turmeric paper. This test was carried out directly in all the fluids with the exception of the blood; in this case precipitation of the greater part of the coagulable protein was secured by allowing the blood to flow into about three times its volume of 95 per cent alcohol. After standing for about an hour the coagulum was filtered off and the filtrate after evaporation almost to dryness on the water bath was tested for boric acid as described above. Experiment IV. — Fish 43, length 68 cm.; weight, 1188 gm. This animal was placed for four hours and fifteen minutes in a mixture of one volume of distilled water to which had been added 0.3 gm. boric acid _ per litre. The volume of urine collected amounted to 5 c.c. Boric acid was found to be present in the urine and blood, but could not be detected either in the stomach or in the coelomic fluid. ABSORPTION OF POTASSIUM IODIDE Experiment V. — Fish 22, length, 88.5 cm.; weight, 1358 gm. This animal was placed for four and one half hours in sea water to which had been added potassium iodide in a concentration of 1 gm. per litre. At the end of the experiment the presence of potassium iodide could be demonstrated in the blood but not in the urine (volume about 1 c.c.), coelomic fluid or stomach contents. The test used for the detection of potassium iodide was the familiar starch paste reaction, the fluid under examination being first treated with a few drops of a concen- trated solution of potassium nitrate and a little concentrated hydro- chloric acid. Before applying the test to blood the latter was first coagulated with alcohol as described above. Experiment VI. — Fish 15, length, 78 cm.; weight, 1670 gm. This animal was placed for two hours and twenty minutes in a mixture of one volume sea water and one volume distilled water to which had been added 1 gm. of potassium iodide per litre. At the end of the experi- ment the animal was still alive and in good condition; this experi- ment was continued for a shorter time than the preceding one in which undiluted sea water was used, as experience had taught us that if the dog fish be allowed to remain too long in diluted sea water, bleeding at the gills may at times ensue. The urine (volume 5 c.c.), blood and coelomic fluid of this animal all showed the presence of potassium iodide. None was found in the stomach. Experiment VIT.—Fish 7, length, 78 cm.; weight, 2773 gm. ‘This animal was placed for three hours and fifty minutes in sea water which had been concentrated by evaporation until it had obtained a specific Osmotic Pressure and Absorption 5 gravity of 1.035, and to which had been added potassium iodide in a concentration of 1 gm. per litre. At the end of this time no potassium iodide could be detected in the urine (volume about 1 c.c.); coelomic fluid or stomach contents; the blood gave a positive test. All of the experiments described above have been repeated several times but as in every case results practically identical with those reported have been obtained it seems useless to take up further space to report them in detail. As the absorption phenomena described in this paper occur largely by way of the gill membranes, in order to prove definitely that the skin of the dog fish does not in any way act as a path of absorption for salts etc., we have performed the following experiments. Experiment VIIT. — Fish 62, length, 73 cm.; weight 1075 gm. The cord of this animal was destroyed up to the level of the dorsal fin, the opening in the skin made by the pithing was carefully covered with a collodion dressing, and the fish then placed tail down in a tall narrow jar containing a o.1 per cent solution of potassium iodide in sea water. A piece of rubber sheeting with an opening in the centre was then placed around the animal just below the gills, the edges of the rubber being tightly bound to the jar and the whole immersed in a large tank of sea water. By means of this device the epidermis covering two thirds of the body, and the mucous membrane of the cloaca are bathed in a solution of potassium iodide while the gills and mucous membranes of the mouth and pharynx are prevented from coming in contact with the salt. After thirty-five minutes blood drawn from the dorsal aorta gave no test for potassium iodide. In another fish treated in the same way, in which the sea water was diluted with an equal volume of distilled water, a similar nega- tive result was obtained. It has been found by Scott that if the dog fish be placed in diluted sea water, water is absorbed by the animal, the osmotic pressure, the nitrogen and salt content of the blood decreases; by our results it would seem that at the same time salts and-so-forth might be ab- sorbed, and absorbed with greater ease when the animal is surrounded by a medium of less osmotic pressure than its own blood. To further elucidate this point it has seemed worth while to make a few observa- 6 G. G. Scott and W. Denis tions regarding the actual rapidity of absorption of potassium iodide by the dog fish under various osmotic conditions of the external medium. All experiments on this subject were carried out under the conditions described above except that no attempt was made to collect the urine. It was our object in this series of experiments to determine as nearly as possible the actual time required by a fish immersed in a sea water solution of potassium iodide to absorb a sufficient quantity of this salt to enable it to be detected in the systemic blood. After pithing, the tail of the animal was therefore removed and the caudal artery closed by means of a small plug in such a way that the latter could be easily withdrawn when it was desired to collect samples of blood. The amount of blood taken for each test was five cubic centimetres which was allowed to flow into three times its volume of 95 per cent alcohol. The method used for the detection of the iodide has already been described. Taking in each case the average of several determinations it was found that when the head of the dog fish is immersed in sea water containing one gram of potassium iodide per litre this salt can be detected in the blood within four to five minutes. If, as the external medium, we use sea water diluted with its own volume of distilled water, absorption can be demon- strated in two minutes, while if the medium consists of sea water concentrated to a specific gravity of 1.035 (the specific gravity of sea water being 1.025) no potassium iodide can be detected in the blood until the expiration of nine to ten minutes. In all of these experi- ments the stomach was tested for the presence of potassium iodide but invariably with negative results. Certain theoretical considerations seem to be of interest here. It has been said that the gill membranes are by all means the chief structures concerned in these absorption phenomena. In the experi- ments showing the relation of the time of absorption to the concentra- tion of the external medium it would appear that the physical laws of diffusion suffice to explain the results. On the inner side of the gill membranes is the blood which contains no potassium iodide. Outside the membranes the potassium iodide is in solution along with other solutes. In every case the osmotic pressure of potassium iodide in the blood is zero while outside the gill membranes its osmotic pressure is considerably above zero. So that in every solution in Osmotic Pressure and Absorption ‘| which it is dissolved, namely sea water, hypotonic sea water and hypertonic sea water, the potassium iodide will pass into the blood. Its action is independent of the other solutes in the external medium. But in the case of the potassium iodide dissolved in the diluted sea water there is simultaneously a rapid movement of water into the blood through the gill membranes and this will accelerate the move- ment of the salt. In the hypertonic solution there is a rapid movement outward of water and this outward going stream will retard the inward movement of the potassium iodide. Papers CITED Bert, P.: Comptes rendus de l’académie des sciences, 1871, xxiii, p. 382. Bottazzi, F.: Archivio fisiologia, 1906, ili, p. 416. Denis, W.: Journal of biological chemistry, xiii, 1912-1913, p. 225. Mosso, A.: Biologisches Centralblatt, 1890, x, p. 570. SuMNER, F. B.: Bulletin of the United States Bureau of Fisheries, 1905, RORY 253. A DEFINITE PHYSICO-CHEMICAL HYPOTHESIS TO EXPLAIN VISUAL RESPONSE By LEONARD T. TROLAND [From the Biological Laboratories, Massachusetts Institute of Technology.] I. THe PRESENT-DAy SITUATION IN VISUAL PHYSIOLOGY Although Essential, an Adequate Theory of Visual Response Does Not Exist. — The progress of modern physics has conclusively proven that there is but one way in which we can develop a syste- matic account of the phenomena natural to a particular field of scientific investigation: we must advance structural hypotheses of such a character as to provide us with a basis for the deduction of these phenomena in accordance with known dynamical laws. The field of visual physiology is one which embraces a large variety of closely interconnected and very definitely observable processes, and it is one in which — it would seem — the speculative method could be applied with great advantage; but the fact remains that in spite of the repeated efforts which have been made in the direction of an hypothesis to explain the phenomena of visual response, there is extant to-day no system which is capable of doing this. As a result visual physiology is in a state which may not unfairly be character- ized as chaotic; chaotic in the lack of organization among its con- stituent and varied facts, and chaotic in the disagreements of its special authorities. Necessity of a Physical Viewpoint. — It is the belief of the present writer that most of the extant theories of visual response — e.g., those of Hering, Donders, Mrs. Ladd-Franklin, etc. —err in a quanti- tative rather than in a qualitative way; they are on the right track, but have failed in progressiveness. It seems inconceivable that we should be able to develop a satisfactory theory of the nature of visual response, if we neglect to consider the properties of radiant energy and the mechanism of its interaction with matter in general, A Definite Physico-Chemical Hypothesis 9 as these are revealed by modern researches. It is also incredible that we should obtain such a theory without close attention to the fundamental laws of chemistry as these are exemplified or are exempli- fiable in the physiological processes of the retina. And yet, so far as the writer has been able to discover, the extant hypotheses of visual response involve only very vaguely, or not at all, the salient concepts and principles of modern theoretical physics and chemistry. Hence it is the purpose of the present paper to indicate — of course, very schematically — the possible fruitfulness of the application of these concepts and principles to the speculative field in question. It is perhaps impossible to overemphasize the importance of the physical viewpoint in respect to the attainment of any finally satis- factory notion of the mechanism of visual — or, in fact, of any other physiological — processes; if we believe, as we profess, that life is an organic complex of physical and chemical reactions, we must surely be intellectually blind if, in the theoretical study of these reactions —or of life—we overlook the fundamentals of the electrical and molecular conception of matter and of energy. The Extant Hypotheses. The extant hypotheses concerning the mechanism underlying visual sensation may be divided into three classes: the mechanical, the chemical and the electrical. Among the mechanical hypotheses we may count the original proposals of Young,! the views of Charpentier,” G. Stanley Hall,* William Patten,‘ Antoine Pizon,> H. M. Bernard,® Adolph Stohr,’ and others. In each of these hypotheses it is supposed that molar masses of matter —of microscopic size —, fibrillae, granules, etc., are set into vibra- tion by the action of the light waves which impinge upon the retina, 1 YounG, THomas: Lectures on Natural Philosophy, 1807, London. * CHARPENTIER, AUGUSTIN: La lumiére et les couleurs, 1888, chap. xii, pp. 265-204. 3 Hatt, G. STANLEY: Proceedings of the American Academy of Arts and Sciences, 1878, xiii, p. 402. 4 PaTTEN, WILLIAM: American Naturalist, 1898, xxxii, pp. 832-857. > P1zon, ANTOINE: Comptes rendus, Académie des sciences, 1901, Cxxxiii, pp. 835-837. 6 BERNARD, H. M.: Annals and Magazine of Natural History, 1896, xvii, pp. 162-167. 7Sr6HR, ADOLPH: Zur Hypothese der Sehstoffe und Grundfarben, 1808, Leipzig. Cf. LADD-FRANKLIN, C.: Psychological Review, 1900, vii, p. 415. 10 Leonard T. Troland these vibrations forming the essence of the response. Much more closely in line with the probabilities of the case are the chemical theories, the most important of which are the Young-Helmholtz,’ Hering,’ and Ladd-Franklin ® hypotheses. Derivatives of these are to be found in the suppositions made by A. Konig," H. Ebbing- haus,” G. E. Miller,” F. C. Donders, Schenck,” Wundt™ andee von Kries.’ Chemical hypotheses have also been advanced by F. W. Edridge-Green,’® E. R. Oppolzer, and W. Preyer. While differing with respect to details, these theories all agree in supposing that the light rays act upon molecular, rather than upon molar units of matter. Framed more with reference to electro-physiological phe- nomena than with respect to the electro-magnetic character of light are the electrical hypotheses of Edridge-Green,” Preyer,?? William 8 HermuHoLtz, H. von: Handbuch der physiologischen Optik, 3rd edition, IQII, li, pp. 119 ff. ° HERING, EwaLp: Grundziige der Lehre vom Lichtsinn, 1905 and 1907, Leipzig. Also: Lehre vom Lichtsinne, 1878, Vienna. 10 LADD-FRANKLIN, CHRISTINE: Zeitschrift fiir Psychologie und Physiologie der Sinnesorgane, iv, pp. 211-221. 4 Konic, A. and Drererict, C.: Zeitschrift fiir Psychologie und Physiologie der Sinnesorgane, 1893, iv, pp. 241-348. Also in: Sitzungsbericht, Akademie der Wissenschaften, Berlin, 1886, pp. 805-830. ” EBBINGHAUS, H.: Zeitschrift fiir Psychologie und Physiologie der Sinnes- organe, 1893, v, pp. 145-238. 8 MULtER, G. E.: Zeitschrift fiir Psychologie und Physiologie der Sinnes- organe, 1896-1897, x, pp. [and 321; xiv, pp. land 161. 4 DonvERS, F. C.: Graefe’s Archiv fiir Ophthalmologie, 1881, xxvii, 1, p. 55. 19 ScHENCK, F.: Archiv fiir die gesammte Physiologie, 1907, cxviii, pp. 129-181. 6 Wunpt, W.: Physiologische Psychologie, 1893, i, p. 535. Kries, J. vON: Zeitschrift fiir Psychologie und Physiologie der Sinnesorgane, 1899, xix, pp. 175-191. See also: zbid., ix, p. 81. 18 EDRIDGE-GREEN, F. W.: The Hunterian Lectures on Colour-vision and Colour-blindness, delivered before the Royal College of Surgeons of England, Feb. 1 and 3, 1911; 1911, London. ‘9 Oppoizer, E. R. von: Zeitschrift fiir Psychologie und Physiologie der Sinnesorgane, 1902, xxix, pp. 183-203. 20 PREYER, W.: Archiv fiir die gesammte Physiologie, 1881, xxv, p. 31. *1 See reference 18, supra. * Preyer, W.: Zeitschrift fiir Psychologie und Physiologie der Sinnesorgane, 1894, Vil, p. 241. A Definite Physico-Chemical Hypothesis ig Peddie,” G. G. Stokes *4 and William Nicati.? In addition to these there exist two theories based upon purely optical considerations; these have been published by Gdller *° and G. Darzens.”” The most im- portant of the above mentioned hypotheses are adequately presented in current text-books of physiology, and hence even if space per- mitted it would be supererogatory for us to review them here. Weaknesses of Extant Visual Hypotheses. —. The most general criticism which can legitimately be made of the above listed theories is that of inexactness and superficiality. In the present paper we shall pass by as unworthy of notice all attempts to justify hypotheses upon a purely pragmatic basis; the view that scientific hypotheses are merely aids in the systematization of phenomena may to-day be regarded as irrelevant, if not antique. If any hypothetical account of the mechanism of visual response is to be seriously considered it must employ the definite structural and quantitative concepts of theoretical physics and chemistry, and the dynamical interactions which are involved must not only lead deductively to results which explain the psycho-physical phenomena, but they must be consistent with the other logical contents of the “universe of discourse’? which they are forced to enter. Now none of the extant visual theories fulfill these requirements. Most of them are not only vaguely formu- lated, and contain no distinct reference to general physics and chemis- try — to say nothing of the special physical chemistry of light and of nervous response — but they often flatly contradict both physical and physiological principles. The physical conception of resonance lies at the bottom of practically all of the hypothetical accounts which have been given of the process of visual stimulation; but it requires only a very simple calculation to show that if any microscopically *3 PEDDIE, W.: Proceedings of the Royal Society of Edinburgh, 1903, xxiv, pp. 448-440. 24 SToKEs, G. G.: Nature, 1895, lili, pp. 66-68. 25 Nicati, W.: La psychologie naturelle, 1898, Paris. Also: Archives d’oph- thalmologie, 1895, xv, pp. 1-44. _ %6 G6rtLtER: Du Bois-Reymond’s Archiv, 1888. ; 27 DarzeNs, G.: Comptes rendus, Académie des sciences, 1895, cxxi, pp. 133-135. Reference should here also be made to the recent electro-optical hy- pothesis of Mersrinc, A. A.: Zeitschrift fiir Sinnesphysiologie, 1907, xlii, pp. 229-249; especially as interpreted by E. B. Holt, in ‘The New Realism,” 1912, New York, pp. 312 ff. The present writer has not seen Meisling’s article. 12 Leonard T. Troland observable structure is to resonate in tune with even the longest light waves the material substance involved must possess a modulus of elasticity two hundred million times greater than that of hard-drawn steel.*> This is a reductio ad absurdum of all theories of mechanical visual stimulation which depend upon resonance, and as yet there has appeared but one theory of any sort (the theory of Darzens, supra) which does-not employ this latter principle, either explicitly or implicitly. A second and conclusive objection to all purely mechan- ical hypotheses is to be found in the certainty that light can act directly only upon electrical, and not upon neutral mechanical, struc- tures. Accordingly, we are led from molar to molecular systems, from mechanical to chemical hypotheses, and there can be little doubt that here we have entered the appropriate field of investigation. Light is known to originate in processes of chemical change, and even if we had no empirical evidence of photo-chemical resonance, we should be amply justified in the supposition that molecular systems are of the right magnitude to make possible such a selective response. Moreover, at the present time, we have every reason for believing that the forces of chemical affinity are actually electrical in nature, so that the elec- trical forces of the light ray may be expected to bring about the chemical decomposition of the sensitive molecules.. Now nearly any one of the thirteen distinctively chemical hypotheses of visual response which we have mentioned above may be accepted as providing us with a satisfactory general schema of the actual process of the response. But at this point their excellence is apt to end; none of them follows out in detail the obvious implications of the electro-chemical view- point, and only a few of them appear to have been construed in their particulars with careful and comprehensive reference to the actual phenomena of visual sensitivity, whether these phenomena be psy- chological or physiological. In explanatory value the hypothesis of Hering is undoubtably superior to any other extant theory, but in its (pseudo-)chemical, physiological and psychological aspects “8 The formula used in this calculation was: — 4mk2ML SSS the force on the vibrating particle being —Mkx, where x is its displacement. L and a are the dimensions of the elastic structure. For a wave-length of 750 mp, Y (Young’s modulus) =17 x 107. A Definite Physico-Chemical Hypothesis 13 it is guilty of all manner of offense against fact and reason. The scientific value of the more consistent Young-Helmholtz hypoth- esis, on the other hand, is at the present time almost negligible; it explains only the most rudimentary of the phenomena of visual sensation. That the ultimately successful doctrine as to the nature of the visual mechanism must involve the concept of electricity is made obvious by the fact that light is an electro-magnetic process, and consequently can react only with electrical or magnetic systems, as well as by the facts of retinal and general nerve physiology, which all point to electrical factors in stimulation. Never-the-less the extant theories which make use of electrical conceptions are perhaps the most unsatisfactory and vaguest of all, quite failing to take into consideration the established nature of electricity and its relation- ship with matter. Therefore, in view of the tremendous strides which have been made during the past twenty years in our knowledge of the general physics of light, chemical action, and physiological response, it seems advisable that something be attempted in the way of a recti- fication of that chaos of visual theory which has arisen in so unpre- dictable manner from the epoch-making work of Helmholtz. The present paper purports to be modest in its attitude, if not in its intentions. Il. THe PsycHOLOGICAL ANALYSIS AND PSYCHO—PHYSICS OF VISUAL SENSATION The Elementary Visual Sensation and its Attributes. — The problem of the theory of visual response is complicated by the fact that the latter includes within its field that greatest of all philosophical enigmas, the psycho-physical relation. However physiological in its intent, no hypothesis of visual response can neglect to consider the facts of visual psychology, for it is towards the explanation of these facts that the theory ultimately turns. If we assume the sub- jective standpoint of introspective psychology, we find that the concrete subject-matter with which we have to deal is the individual visual field, with its qualitative and quantitative modifications, and we may, for convenience, describe the abstract content of any point in this field as the elementary visual sensation, S. Now the exact character of S may vary both quantitatively and qualitatively, and 14 Leonard T. Troland by studying these variations introspectively we find that they involve six distinct qualities: red, R; green, G; blue, B; yellow, Y; black, B; and white, W; not all of which, however, can be present simul- taneously. Let us denominate these six qualities, the fundamental attributes of S, and as they may exist in varying intensities, their degrees may be symbolized by the letters: r, g, b, y, 6 and w, respectively. These quantitative terms will be spoken of as redness, greenness, etc. It is the opinion of the writer that these are the basic factors of the psychology of visual sensation, and that no others are involved; the ‘“‘brightness”’ of Hering’s theory is merely a whiteness conceptually modified to meet the demands of the hypothesis, or else it is a perceptual element, misplaced.” The Schematic Relations of the Attributes. A study of the modes of occurrence of the fundamental attributes of S reveals the follow- ing correlations: (1) if g>0, r=0; (2) if y>o, b=o; and, conversely: (3) if r>0, g=o, and (4) if b>o, y=o. In other words, the hues: R and G, and Y and B, are mutually exclusive, or ‘‘antagonistic.”’ / BY The hues can be arranged in the cyclic order: R . G in which yee adjacent qualities will fuse, while opposite ones exclude, or cancel each other. The attributes W and B seem to lie outside of the cycle above represented, and in such a way that W may be added to any possible combination of hues, while B is present in strict proportion to the absence of R, G, B, Y, and W, so that we may write: b=k— (r+g+b+y-+w) where k is a constant. These relations are geo- metrically schematized in the well-known ‘‘color pyramid,” and they are derived from a purely subjective, or non-physical, examination of the elementary sensation. General Explanation of the Psycho-physical Situation. — The problem now arises as to the manner in which we shall conceive the relationship which exists between S and the physical processes of response. Corresponding with any specified elementary visual sensa- *8 The exact manner in which the six “attributes” of the visual sensation may be discriminated from each other has been elaborately discussed by the author in his Massachusetts Institute of Technology (Boston) bachelor’s thesis: “Studies in the Theory of Visual Response,” 1912, section LX. This work is not published, but is on file in the Biological Library of the Institute. A Definite Physico-Chemical Hypothesis 15 tion, there exists what we may describe as an elementary visual response, V, which can be analyzed into five parts, succeeding each other in time, as follows: (1) the stimulus, P; (2) the stimulation, or retinal process, KR; (3) the afferent nerve impulse, I; (4) the central brain process, C; and (5) an efferent impulse, M@. Now in the ordi- nary theory of psycho-physical parallelism it is asserted that S, although not a part of V, occurs simultaneously with, and as a con- stant function of C. From the modern panpsychic or ‘“‘psychical- monistic’’ standpoint, however, it appears more likely that in any actual case of observation, C would lag slightly behind S in time, since the entire series, V, is regarded as being merely a perceptual or conceptual representation, in the consciousness of the physiological observer, of an objective, non-physical, process of which S is an inte- gral part.*° This objective series of events, which may be called Q, corresponds, point for point, with V, and in such a manner that S, the elementary visual sensation, finds its physical representation in C, the elementary visual cerebrosis. The postulates of psychical monism, like those of a non-critical parallelism, permit us to write: C={(S), since the essence of the panpsychic doctrine lies in the sup- position that the observable physical world is causally conditioned by the objective or paraphysical world. Specific Psycho-physics of Visual Sensation. — Since it has been found that the elementary visual sensation, S, may exhibit six distinct attributes, it follows from the relation, C=f(S), that C, also, has a six-fold attributive character. Let us designate the factors in ques- tion as follows: C,, C,, C,, C, C,,C,. These terms must evidently be related with R, G, B, Y, B, and W, in the following manner: (r) c,=f{(r), (2) c,=f(g), (3) c»=f(b), (4) cy=fly), (5) c,=£(0) and (6) c,,={(w), the lower-case letters being employed to indicate the quan- titative expression for the qualities, entities or processes which are qualitatively symbolized by the corresponding upper-case letters. For simplicity’s sake, we shall assume in the following discussion that all of the functional relations above represented may be regarded as proportionalities of such a sort that, c,=g, etc. Any error involved 39 An adequate idea of the philosophical position of ‘psychical monism”’ can be obtained from Wm. MacDougall’s recent book: ‘‘Mind and Body,” chapter on “‘automaton theories.”” A more elaborate discussion appears in C. A. Strong’s “‘Why the Mind has a Body” (N. Y., 1903). 16 Leonard T. Troland by this assumption will be such as to not materially affect the general validity of our argument; moreover it is, for several reasons, the most probable hypothesis. III. OUTLINE OF A DEFINITE PHYSICO-CHEMICAL THEORY OF VISUAL RESPONSE Argument from the Physical Nature of Light. — We found that the principal objections which can be raised against a mechanical hypothesis of visual response lie in the fact that mechanical systems are not of the right order of magnitude to resonate in tune with light, and that, as a rule, molar masses of matter do not bear the free elec- trical charges which are essential in order that the forces of the light ray should grip them. In the molecule, however, we find both of the above indicated requirements quite perfectly fulfilled (cf. page 12 supra); we have good reason for declaring that many, if not all, chemical molecules are electrical dyads made up of positively and neg- atively charged atoms or radicles which can be separated from each other by the action of electrical forces. Suppose that we consider a certain molecule, M, which is composed of the positive and negative parts: I+ and I-, respect’vely. Then since the system is not a rigid one, I+ and I- will be capable of vibrating with respect to each other with a certain natural frequency, n. Now if m is also the frequency of some light ray which impinges upon M, the molecule will resonate with respect to this ray, so that under the proper conditions, the constantly increasing amplitude of vibration will result in a final disruption of the molecule. I+ and J-, in the free state, are ions, and the process initiated by the light is, accordingly, one of ionization. Mechanism of Visual Stimulation.— The above argument of course contains nothing of novelty from the general physical point of view, but if we apply it to the physiological case of visual response we arrive at the conclusion that in all probability the immediate effect produced by light upon the retina consists in an increase tn the ionization of certain specific chemical substances there present. Now in harmony with what we know concerning the probable localization of the light sensitivity in the retinal elements, let us suppose that these sub- stances, which may be designated in general by M, are enclosed in A Definite Physico-Chemical Hypothesis 7 the terminal segments of the rod and cone cells.** Now the most re- cent, and thus far the most successful hypothesis to explain the nerve impulse is that of W. Nernst,*” especially as elaborated by A. V. Hill * and R. S. Lillie,** and in accordance with this hypothesis, the stimu- lation of nervous tissue is conditioned by an increase in the ionic concentration of its native dissolved substances, or by some equiva- lent process. It appears, then, that besides corresponding in a general way with the recognized physiological phenomena of electrical varia- tion in the stimulated retina,®® our deductions with reference to the intimate nature of the retinal response are quite in harmony with what now appears to be the correct view concerning the fundamental nature of protoplasmic stimulation and conduction. Certain sub- stances contained in the terminal segments of the rods and cones suffer increased ionization under the influence of light, and this increased ionization, va the mechanism of the Nernst hypothesis (vide infra), initiates the visual impulse in the contiguous nerve fibres. Exact Mechanism of the Visual Impulse. — Previous to becoming acquainted with the Nernst hypothesis, the present writer was led, on purely speculative grounds,” to make the following definite assump- tions concerning the mechanism of the visual impulse. (1) The visual impulse consists in an actual propagation of the positive ion, I,, from the rod and cone cells along the optic nerve and tract, to the cerebrum (especially to the neurons of the cuneus in the cerebral cortex). (2) This propagation takes place with the speed of the visual impulse, and occurs within the neuro-fibrils, which are “‘to be thought of as molecular tubes, specialized protoplasmic structures within which it is possible for even single ions to travel without encountering great 31 The author has discussed the problem of the localization of the sensitive structures in the retina, in the work above referred to, section XVIII. A large number of independent considerations make it impossible to deny that the rods and cones contain the immediately stimulable elements of the retina. 32 See: NeRNsT, W.: Archiv fiir die gesammte Physiologie, 1908, cxxii, pp. 275-315. 33 Hitt, A. V.: Journal of Physiology, 1910, xl, pp. 190-224. 4 Linu, R.S.: This Journal, 1911, xxviii, pp. 197-223. 3° The original memoir in this field is that of James Dewar and J. G. McKEN- DRICK: Transactions of the Royal Society of Edinburgh, 1873, xxvii, p. 141. 36 The writer’s ‘‘Thesis,” already cited, section XXI. 18 Leonard T. Troland resistance. (3) The manner in which an individual ion or a group of ions may be imagined to be propagated . . . will be described as follows. The non-fibrillar portion of the nerve fibre is made up of a mixture of substances certain of which are ionized, and others of which are capable of constituting an osmotic membrane which nor- mally is equally permeable to the positive and to the negative ions. However, when a positive ion comes into contact with one of the neuro-fibrils the surrounding neural substance acquires a slight dif- ferential permeability, so that the negative ions are capable of moving within it more readily than are the positive ions. This being the case, the loss of negative ions into the surrounding tissues — say into the myelin sheath, when this is present — results in the development of a positive charge within the core itself. The original positive ion thus finds itself placed within the influence of a positive electrical field. Since this is a state of disequilibrium, if the ion is free to move — and if, as will be the case, its charge is much smaller than that produced in the nerve — it will travel in one direction or the other along the neuro-fibril. If we suppose the ion to have had an original impetus in the afferent direction, it will move in this sense. The resulting process is obvious. As soon as the ion has moved into a new region of the nerve fibril the permeability of the neural substances about it for negative ions will be altered as before, a new state of disequilibrium will be produced and the process will be repeated, the ion moving continuously in one direction within the fibril.’ The mechanism above described may be aptly characterized as an electrical peristalsis, and is capable of being historically regarded as return to the view of Descartes*’ with reference to the functioning of the motor nerves. At first sight it appears very improbable that any such bodily transfer of the ions as is above imagined should occur; it is well known that the average velocity of ions moving in solutions is about one centi- metre per hour (with a potential gradient of a volt per centimetre), whereas the nerve impulse in man travels 125 metres per second, or about 800,000 times as fast. On the other hand, the negative ions of a Crookes tube or from radium have a velocity often 30,000 times that of the nerve impulse. If we suppose nervous tissue to be specially organized for conduction purposes, no general objection can be raised 7 In his essay entitled, ‘“L’homme.” Cf. HowELt, W. H.: A Text-book of Physiology, 1909, p. 111. ee A Definite Physico-Chemical Hypothesis 19 to the rapid motion of chemical ions within it, for no cause appears why special structures may not reduce the resistance encountered, to practically the zero point. As we shall see, the hypothesis as it stands, possesses great explanatory power, and hence should be retained as long as it proves consistent with known physical and biological principles.** The electro-peristaltic hypothesis makes use of the essential mechanism of the Nernst theory, viz., the presence and action in irritable structures of osmotic membranes endowed with variable differential permeability with respect to the positive and negative constituents of the native electrolytes of the tissues, and the dependence of the selective permeability of such membranes upon their state of polarization, i.e., upon the relative concentration of positive or negative ions in their vicinity. The hypothesis as above stated (quoted from the writer’s Massachusetts Institute of Tech- nology thesis, 1912) does not coincide in every respect with the mechanism described by Lillie ** as that characteristic of general cell irritability and conductivity; it is, however, strictly analogous with the latter; and at the present stage of development of the theory it is gratuitous to suppose that the reactions of all tissues are strictly identical, although the general relationships involved are probably universal. The detailed analysis of the structures and _ processes implied by the electro-peristaltic hypothesis of the visual — and other — impulses shows that the mechanism in question will insure continuous afferent conduction, corresponding at each instant with the intensity of the force acting uniformly upon the sense-organ; the Nernst theory, alone — like the artificial stimulation of nerve fibres under laboratory conditions — gives conduction only when changes 38 The electro-peristaltic hypothesis when applied to other departments of sense and neurology proves equally fruitful in explanations. Consider, for example, its bearing upon the singular fact that the only sensory nerves, the olfactory, whose ends are unprotected, yield a different sense quality for every specific stimulus; our interpretation would obviously be that the stimulating molecules (ions) are transported bodily to the brain. The excitation of mus- cular tissue, on the other hand, can be attributed, on the basis of this hypothesis, to the transmission of small quantities of oxidases or similar enzymes along the motor fibres. The hypothesis also provides a simple explanation for sleep, memory, the delay of the impulse at the synapses, the psycho-physics of affec- tive states, the “‘Ritter-Valli law,” and other psycho-physiological facts. 34 Livuiez, R. S.: This Journal, 1911, xxviii, pp. 197-223. 20 Leonard T. Troland in the nature of the stimulus take place. The laboratory results, however, are also entirely consistent with our hypothesis. Nature and Relations of the Visual Cerebrosis. — The general nature of the visual cerebrosis, C, follows at once from our account of the mechanism of the impulse. The cerebral state corresponding with any condition of retinal stimulation consists simply in the presence in the cerebral cells of the specific tons which are liberated in the retina by the action of the light. ‘The physics of this correlation is made so obvious by our hypothesis concerning the nature of the visual impulse that it requires no expository comment. In the preceding section of this paper we have given reasons for supposing that the visual cerebrosis possesses six more or less independent aspects, which we have symbolized as C,, C,, ce C,, G, and Cy; _ respectivelysaaaie have already stated the hypothetical psycho-physics of these terms, and at present it only remains to indicate their physiological connec- tions. The character of these connections is perhaps obvious. It is clearly implied by the fundamentals of our theory that for each of these components there must be a corresponding — and materially identical — component in the visual impulse, so that the constitution of the latter may be represented as: I= 1I.+1,+4,+77+ Moreover, it is further implied that the retinal process, R, has a similar division. Now with one reservation, this may be taken to mean that there are contained in the external segments of the rods and cones, specific ionizable chemical substances corresponding with each of the components of the visual cerebrosis. The one exception is the case of C;, which in view of our psychological equation: 6= k—(r+g+b+y-+w) (cf. page 14) may be taken to represent the absence of tonic charge in the appropriate cerebral elements. Thus we arrive at the conclusion that there exist in the retina five distinct visual substances: M,,M,, M,, M,, and M,; these the writer has denominated molecular resonators, because they are selectively ionized by lights of specific and differing wave-length, or frequency, and their intrinsic positive ions, L.,, I,,,1,,, L,, and 1,., ere thewexaes psycho-physical correlatives of the fundamental visual qualities, R, G, B, Y, and W, respectively. The Mechanism of Complementation.— In order to take in account the ‘‘antagonistic”’ relations which exist between the hues (cf. page 14), it is necessary to make the following additional hypothe- A Definite Physico-Chemical Hypothesis 21 sis, which is independent of those already stated, but which can be justified upon developmental grounds.*® Within the large “ganglion”’ cells of the inner stratum of the retina there must be supposed to exist a certain substance which we may call the complementation substance. The component molecules of this complementation sub- stance are made up of a nucleus and two side-chains; each of these side-chains is a potential negative ion, the nucleus itself being a doubly charged positive ion. Of these molecules there are two varieties. The first is so constructed chemically that its two negatively charged ionic side-chains are capable of combining simultaneously, but not separately, with the two positive ions of the visual impulse: I,, and I,,. The second reacts in a similar way with the visual ions: I. and I,,. The result is that in each case the positively charged nuclei of the molecules are set free and become a part of the impulse, as it is passing through the ganglion cells. We may speak of the two substances above described as the R-G-complementation substance and the Y-B-complementation substance, respectively. If the R-G- complementation molecule, M,,, has the constitution M,.= I’_(I,,,) I’,-, the complementation reaction for the ions, I,, and I,+ may be written as follows: an * Le ae Mae ras ee a Ct) lee igs.) only I, 4, of course, entering the impulse. As indicated by its sub- rapid. = 21,.. Mathematical and Other Special Properties of the Molecular Resonators. — We shall assume that the five specific molecular resonators, whose presence in the retinal end-cells has been postu- 39 There is reason for believing that color-vision is a secondary sexual char- acter. (Consider, for example, the fact that color-blindness is forty times more common in males than in females.) We must suppose that the complementary colors once represented antagonistic motor tendencies of approach and retreat (love and repulsion), and that the purpose of the complementation reaction is to eliminate the conflict which would thus otherwise arise between two opposed reflexes when, as in white light, both ‘‘colors” are physically present at once. On this basis, of course, we must assign to the mechanism a very ancient line- age. Let us note in passing that the “‘complementation molecule”’ has all of the properties of an Ehrlich amboceptor, and hence may be thought of as a common type of physiological molecule. 22 Leonard T. Troland lated, are so distributed that under normal conditions each of the cones contains M,, M,, M, and M, in equivalent concentrations, while M,, is contained only and alone, in the rods. M,, we shall suppose to be identical with the familiar visual purple. Now since under the action of the light — and even, as we shall see, apart from this — the molecular resonators are constantly being destroyed, there must exist corresponding anabolic reactions which, continually build them up. Like all specific organic reactions, these must be under the control of appropriate enzymes, which we shall designate as aesthesogenases, the substrates “? upon which these enzymes operate being called aesthesogens. Each molecular resonator must be conceived to possess a characteristic set of properties and auxiliaries. Perhaps the most important conception which we must apply is that of specific con- centration, which may be expressed in terms of molecules per retinal element (rod or cone), the concentration of M, being symbolized by m,. Under the action of light, or even in its absence, M, will be decomposing at a definite rate, which may be written as dm,/dt = m,; this has the abstract significance common in chemical kinetics. Another characteristic of each resonator must be its light sensitivity, q,, which is the equiva- lent of dm,/de, where e is the intensity of the light fall- ing upon the molecule. This constant is related with the specific resonance function, which is of the type: m,=f,,(A), and shows how the number of molecules broken down per second varies with the wave-length of the impinging light. The general form of resonance functions (as determined by dynamical reasoning) is illustrated in the separate curves of Fig. 4. The decomposition: M,—I,, + I,_ may be regarded as the analytic aspect of what we shall call a resonance reaction. ‘This reaction, like all of its species, is reversible, and under equilibrium conditions, the reverse change must at any instant be equal to the forward one, so that the position of the equili- brium point will obviously determine the percentage ionization of the resonator at that instant. We have every reason for supposing that this will not be zero even in the absence of light and consequently *° T.e., the specific substances whose reaction velocities are accelerated by the presence of the enzyme. 4 Cf. Encyclopaedia Britannica, 11th ed., vi, p. 27, for a discussion of the theory involved. A Definite Physico-Chemical Hypothesis 23 we are forced to define for each molecular resonator a specific normal tonization ; the effect of the introduction of a light ray into this chemi- cal system is merely to increase the reaction constant of the disintegra- tive or tonizing reaction. We have already spoken of the aesthesogens and these must be supposed to enter into a synthetic reaction which produces the molecular resonators, as follows: (Aesthesogenetic React.) (Resonance Reaction) AB + CS ABC (=M,) SA+BC (=1,, + 1L_) AB and C being the aesthesogens which are specific for M,. The aesthesogenetic reaction, conformably with our symbolism, need not be ionic. Mathematical Properties of the Visual Impulse.—It is only reasonable to suppose that the number of ions leaving a retinal ele- ment, via the neuro-fibrillae, per second is proportional to the number present, or to the concentration, and it is obvious that what may be designated as the intensity of the impulse, or the number of ions passing through any cross-section of the nerve fibre per unit of time, will depend upon the number leaving the retinal element and the number Jost in the process of conduction. If we symbolize a com- ponent of impulse intensity by t,, we may write: t,, = knj,,, where tn, is the impulse intensity at the retina, and tn+ is the concentration of positive ions of the species I,+ in the same region, and k,, is a constant. The generalized impulse intensity, u, will, of course, have five components: 4 =4,,4,,4,,4,, and t,. Another quantitative conception involved in the notion of visual conduction is that of impulse loss; we must suppose that at the synapses, and possibly at other places, some of the traveling ions are permanently lost from the neuro-fibrils. The specific rate of loss at any point in the path of conduction may be symbolized by 7,. The visual cerebrosis, as we have stated, is merely a concentration of the impulse and hence must depend for its intensity, c, — the number of ions per cortical element — upon the intensity of the visual impulse at the cortex. If we make the only probable assumption, that the rate of diffusion of ions from the cortical element is proportional at any time to the number present, we get for equilibrium conditions the following relation: Cc, = 4ic¢/Kyc, Where c, is a component of cerebrosis inten- 24 Leonard T. Troland sity, 4, the corresponding impulse intensity at the cortex and k,. a constant.*” IV. THe EXPLANATION OF CERTAIN VISUAL PHENOMENA The Problem of the Explanation of Visual Phenomena. — The purpose of an hypothesis concerning the intimate mechanism of the visual response is simply to enable us to explain the manifold phenom- ena of that response, that is, to permit us to deduce from the hypothe- sis, as combined with general principles of science and of thought, certain conclusions which coincide with the facts of the particular field which the hypothesis enters. The presumption is that an hypoth- esis which is completely successful as a basis for explanation can only be one which describes or represents the “‘real’”? unseen mechanism. The present section of our paper will be devoted to a very cursory presentation of some of the many theoretical consequences to be drawn from the definite physico-chemical theory of vision outlined on the preceding pages. The Electrical Phenomena in the Stimulated and Unstimulated Eye. — We have supposed that the rods and cones of the retina are the seats of the production of an equal number of positive and nega- tive ions, and that, of these ions, the former are propagated along the optic nerve in the form of the visual impulse. It follows that the negative ions remain unneutralized in the bacillary layer. Since the state of ionization at the retina is not quantitatively zero even in the absence of all light stimulus, it follows that if we examine a fresh and even unstimulated eye we should find the cut surface of the optic nerve to be positive with respect to the layer of rods and cones, the latter being negative. Experiment shows this to be the case.* The fact that the inner layers of the retina are normally positive with respect to the cut surface of the optic nerve may be explained by supposing that there is a large impulse loss (of the positive ions) in © The first proportionality takes the form: ¥" =kycc, where Y, is the rate of diffusion referred to. But at equilibrium the income of the cortical element must equal its outgo, or the impulse intensity at the cortex, ‘4c = Wp, from whence the given relation is obtained. * An excellent brief account of the electrical phenomena of the eye is given by Rivers, W. H. R.: A Text-book of Physiology, edited by E. A. Schaefer, 1900, Ebdinurgh and London, ii, pp. 1050 ff. eee A Definite Physico-Chemical Hypothesis 25 the synapses of these layers (cf. page 23). This corollary also accounts for the negativity of the outer as compared with the inner strata, and of the nerve as compared with the ocular media and cornea. When light falls upon the retina, our postulates demand an immediate increase in the ionization of the molecular resonators in the rod and cone layer, the consequence of which is an increase in the impulse intensity, an increased impulse loss in the synaptic strata, and an increased positivity of the optic nerve endings. If the entire retina is illuminated, the first electrical effect will be an increase in the negativity of the bacillary layer, owing to the departure of a larger number of positive ions per element of time. The second electrical effect will be an augmented positivity of the synaptic layers, owing to the discharge of the above mentioned positive ions into this region. These ions will in part be picked up by the fibrils of the optic nerve fibres, with the result that an increased positivity of the cut surface of this nerve will ensue. Coincident with this, however, there will be a still greater enhancement of the positivity of the ocular media, and hence of the cornea, by virtue of the increased impulse loss. We expect, therefore, that the incidence of light at the retina will result in a positive variation of the current normally established between the cornea of the eye and the cut surface of the nerve, and that with an injured retina this will be immediately followed by a negative variation. Both of these expectations are fulfilled by experi- mental data.** When the stimulus is removed the flow of positive ions along the nerve will immediately decrease, but on account of its relatively great mass the charge of the ocular media wi.l be only slowly lost; consequently the removal of the stimulus wil effect a ‘second “positive variation,” as shown by experiment. Lack of space, alone, forbids a more detailed study of the electrical phenomena o/ the stimulated and unstimulated eye on the basis of our hypotheses. The Physiology of Visual Thresholds. — The explanation of many important factors in visual phys ology involves the conception of threshold differences; these are physical quant'ties which are capable of inducing a specified just perceivable sensory change. We may suppose that the perception or ‘‘discrimination”’ of such change is paralleled psycho-physically by the automatic reaction of a certain cortical element, Z,j;, which takes place whenever there occurs a 44 RIVERS: loc. cit., p. 1051. 26 Leonard T. Troland change ina specific component of the cerebrosis intensity, equal to Ac,. On the basis of our postulates it can be shown that the corresponding change, At,, in the impulse intensity at the retina, will be proportional to Ac,, but the concomitant liminal alteration, Am,, in the rate of ionization of the molecular resonators in the cones will be proportional, as we shall shortly see, to some complex func- tion: fi (c, +Ac,) —f5(c,). It is clear that thresholds for light in'en- sity, Ae, will depend, other factors constant, upon the light sensitivity, dn, (cf. page 22) of the corresponding molecular resonators, M,, so that Ae = Am,/q,. Thresholds for changes in wave-length must be conditioned by the form of the resonance function in such a way that: AA = Am,/f’,,(A), for a specific limited range of wave-lengths, f’,» being the first derivative of the resonance function (cf. page 22). By the use of our conception of impulse loss in the synaptic regions of the retina, and of the brain, and an application of the general princ ples of diffusion it is possible to show that a correlation should exist between the retinal area stimulated and the intensity of light required to just produce sensation. These deductions with reference to area thresholds are in accordance with the empirical determina- tions of Abney * and others; lack of space prevents their exposition here. By similar means we may explain with exactness the relation which exists between the intensity threshold for a given region and the state of stimulation of outlying regions of the retina. It is obvious that the concentration of the molecular resonators, m,, must be involved in the determination of all of the visual thresholds. The psycho-physics of the thresholds is made too obvious by our postu- lates to require discussion (An = Ac,, cf. pages 15-16). The Explanation of Certain Temporal Relationships of Stimulus and Sensation. — It is well known that in order that a given light stimulus should produce visible effects it must act upon the retina for a certain minimum period, the magnitude of which is partly deter- mined by the intensity of the light. For the production of a sensible change our theory demands merely that the per cent ionization of the molecular resonators in the rods or cones should be increased by a certain definite amount, and it is obvious that the increase brought 4° ABNEY, W. DE W.: Proceedings of the Royal Society, London, 1897, lxi, P- 339. A Definite Physico-Chemical Hypothesis 27 about by a given stimulus will be strictly dependent upon the quantity of energy absorbed by the rod or cone, so that it becomes possible to write the following expression for an energy threshold: A(tae), where a is the cross-section of the light ray passing through the rod or cone, ¢ the duration of the stimulus, and e its intensity. This applies, of course, only to stimuli of relatively short duration, and explains the correlations observed by Charpentier.“© The fact that the basis of Visual response is chemical is very 4, clearly indicated by the phe- nomenon of energy threshold. 30 Exner and others’ have made exhaustive studies upon the 20 curve of excitation and de-exci- tation of the retina. According ' to our hypotheses the phe- nomena of inertia and persis- 0 10 = 20N 30 40. 50 60 tence of vision may be both Fic. 1. To illustrate the character of the 5 : eae excitation-de-excitation curve necessitated retinal and cerebral in origin, by our postulates. The abscissae give since the process of excitation units of time elapsed ; the ordinates give a: Set : the impulse intensity. and de-excitation is essentially 4 tight stimulus of constant intensity was applied at Paomeame ia either place. Tf, Sipscwuting acbittay cmccte values m the equitions owing to the establishment of a *’™"°™ constant state of stimulation at the retina, the impulse intensity at the cortex assumes a constant value v,, it follows that if the rate of diffusion of the ions from the cortical element is proportional to their concentration, we may write: dc=(u, — k.c)dt, where k, is the appropriate constant (cf. page 23, bottom). By integration this equation becomes: ~ ,- log (u.—k,.c) =¢, which represents an excitation curve of the same general form as that empirically found by Exner. Similarly, when the stimulus is withdrawn, we have: dc/dt =k,c, or log c=k,f, ¢ in each case being the time after the alteration of the stimulus. The theoretical and experimental curves are compared in Figs. 1 and 2. An extension of this argument 46 CHARPENTIER, A.: Archives d’ophthalmologie, 1890, x, p. 108. Cf. Rivers: loc. cit., pp. 1067-1068. 47 EXNER, SIGMUND: Sitzungsbericht, Kénigliche Akademie der Wissenschaf- ten zu Wien, 1868, lviii, p. 601. Cf. RIVERS: Joc. cit., p. 1066. 28 Leonard T. Troland enables us to account quite perfectly for the phenomena observed “flicker photometry.” * The Explanation of Fechner’s Law. — By an application of the principle of chemical mass action to the double reaction schematized on page 23 (supra), it is possible to deduce an equation which has the form of the Weber-Fechner law connecting the intensity of the stimulating light, e, and the intensity of the corresponding sensation, s. Translated into physiological terms Fechner’s law has the approximate form: c, = k log e, and since, as we have seen (page 23) the cerebrosis in- tensity must be proportional to the impulse intensity at the ret- ina, we may also write: t,, k log e, k being any constant. The mechanism underlying Fech- WIG a2. Exner, showing the state of excitation of An empirical curve obtained by the visual apparatus as a function of the time following the application of a stimulus. The abscissae give the relative time elapsed; the ordinates give the sensory intensity. ner’s law must, accordingly, be retinal, and that this is actually the case 1s evidenced by measure- Norte. — Exner’s curve is an excitation curve only; the curve of de-excitation is interpolated from data by Fick. See TiGERSTEDT, ROBERT: A Text-book of Human Physiology, trans. by J. R. Murlin, 1906, N. Y., pp. 540 and 539. ments upon the action currents of the frog’s eye.“ The chemical equations to which we have just alluded form the basis of the two following equations: (1) m, = (k; +eq)m—kei,i_, for any definite set of ions, where m, is the apparent or average rate of change of the ionization; and (2) m,=k,ab c —ksm; ab and c represent the concentrations of the two aesthesogens. Solving these equations simultaneously, we get an expression of the form: (3) m= Vke/k + ke = kt, =kc, k being 48 The cortical dissipation, ¥, (see note 42 above) = k,c = — dc/dt, so that the time, ff, which can elapse between one pulse of afferent visual ions and the next —dependent upon the frequency of the stimulus — must be t;= Ac/k,c, if flicker is to be exactly upon the verge of disappearance. frequency, Hence the stimulus p= 1/t; = k.c/Ac = constant X c, or the frequency requisite to just abolish flicker is proportional to the cerebrosis intensity, and hence to the inten- sity of the sensation. By Fechner’s law, $= (approx.) constant X log e, which is the result found by: GRUNBAUM, O.: Journal of Physiology, 1898, xxii, p. 433. 49 See RIVERS: Joc. cit., pp. 1050 ff. A Definite Physico-Chemical Hypothesis 29 a different constant in each place. As shown in Fig. 3, equation (3) has the same general form as that of empirically determined curves representing Fechner’s law. The argument, although complicated, is straight-forward.” Explaining the Phenomena of Color Vision. — Most of those who have theorized concerning the visual mechanism have devoted them- selves quite exclusively to the phenomena of color vision. While the above discussion of some of the general correlations exhibited in the visual process is of necessity very schematic, it will, perhaps, suffice to convince the reader of 60 the possibility of making a single physical hypothesis cover with 40 even quantitative precision the general, as well as special, as- pects of the field. It now re- : mains to be seen whether our o 2 #4240 #460 80 100 120 electro-chemical theory will prove Fic.3. Curves illustrative of the explanation : : ; of the Weber-Fechner law deduced from equally fruitful with reference to our hypotheses. The abscissae give the 20 the even more complex phe- light intensity; the ordinates give the im- pulse intensity. nomena of color. It must be en ee Curve A is a plot of the expression ¢, = understood, of course, that our 6-V/ 400 e /(80 + 3e) Curve B isa plot of the expression v, = purpose is more that of the re- viewer than of the expositor; a full, or even an adequate, discussion of the simplest of the problems involved would require in itself a volume of no small size. ihe theoretical situation in visual response is tremendously intricate, — put there is nothing which is intricate which is not, 7pso facto, capable of being analyzed, provided one has sufficient time and patience. In the first place, although the relationships concerned are not simple it is perhaps obvious in what way the character- istics of a light ray which falls upon the retina are able to deter- mine the attributive constitution of the corresponding sensation. In Fig. 4 we have drawn five curves which represent the manner in which the five specific molecular resonators are broken down by light 6+/ gooe/ (50 + 3e) ) 0 The argument is presented in detail in the author’s ‘Thesis,’ already several times referred to. 30 Leonard T. Troland rays of varying wave-frequency. These theoretical curves have the same symmetrical form as curves of resonance in mechanics and they represent what we have called the resonance functions of the specific substances, M,, M,, M,, M,, and M,, but it is important to notice that, while the general distribution of relative sensitivity remains constant, the exact shape of these areas must vary widely with alterations in the concentration of the resonators, in the intensity of the light, etc. For stimuli of high intensity these curves will all- 5 0 750 a700 B 650 a600 D 550 E 500 F 450 G 400H 350 Fic. 4. Graphical representation of the approximate nature of the resonance functions of Mr, Mg, My, Mb, and Mw. The ordinates represent the relative number of molecules decomposed per second per unit light intensity, the concentration of the molecular resonators also being constant and standard. The abscissae give the wave-length of the stimulating light in 10-7 cmss, uniform energy and normal spectrum. be markedly flatted owing to the concomitant influence of the forces expressed in Fechner’s law. Complementation and Some of its Corollaries.—On pages (20-21), we have described a chemical mechanism by the operation of which specific pairs of visual ions would be able to mutually exclude each other from the visual impulse. The law governing the change pro- duced in the constitution of the visual impulse due to the action of the complementation molecules may be exemplified as follows. If the impulse before passing through the ‘‘ganglion cells” has the con- stitution: t+, where t, >t,, the constitution after passing these cells will be: (4, — tg) of I++ 24, of I,4. The corresponding sensa- *! See, e.g., LAMB, Horace: The Dynamical Theory of Sound, 1910, London, Pp. 33: A Definite Physico-Chemical Hypothesis 31 tion, S, then, will be a pink, not a greenish red. An entirely analogous relationship must hold between the impulse components, ty and u,, and it is obvious that when t,=vt, and ty =u the only impulse component reaching the cortex will be v,; these, then, are the condi- tions for complete complementation ; the positions of complementary lights in the spectrum are determined by the retinal and neural conditions underlying and limiting this result, — they find an approx- imate implicit representation in the curves of Fig. 4. All of the familiar effects of “color mixture”’ are similarly represented in this diagram. Suppose, for example, that we stimulate the same cone with lights of 4 =650 and A=550; with appropriate intensities the two elements 4, and wg, will cancel each other leaving only the vu (and tw) which is also introduced by both lights, as necessitated by the form of our curves. The constitution of the resulting sensation will obviously be S = Y + W, although, as we say, “red” and ‘“‘green”’ lights have been mixed. Examination of our diagram will show that it explains very perfectly the fact established by J. J. Miiller and von Kries® that when a heterogeneous light stimulus is made up of two (or more) lights having wave-lengths falling between the limits \ = 760 to 567, or A = 390 to 492 the chroma of the induced sensation does not differ from that of a sensation induced by a homogeneous wave yielding the same hue. It also explains the location of the points of least chroma and greatest luminosity, in the visible spectrum, at X= 575 (approx.) and A= 500; at these points the M, and M,, and M, and M,, curves, respectively, intersect, and hence with these lights the complementation reaction finds its maxima. Fig. 4 is also consonant with the fact that the physical complementary of “green” is necessarily heterogeneous, for a light exciting Mg, also strongly excites My, so that to produce perfect complementation it is requisite that an increment of wt, as well as of 4, be introduced into the impulse at the retina. The relations which the curves bear to each other are such that all other lights can find homogeneous com- plementaries. Another important phenomenon which our general theory permits us to readily account for, is the disappearance of hue with increasing light intensity. Every light stimulus, we suppose, acts upon every molecular resonator, but at low intensities a light 52 See: GREENWOOD, M., Jr.: Studies in Special Sense Physiology, in: Further Advances in Physiology, 1909, New York. 32 Leonard T. Troland of wave-length \ = 655 (say) acts very strongly upon M, and only weakly upon M,, My and My. But as the intensity is increased the increase in the several components of c (at the retina) follows Fechner’s law (qg.v.) —as we have interpreted it — and for this reason each of these components approaches a definite maximum; the nearer any _ component is to the maximum natural to it, the less will any change in the light intensity affect it. The result is that, no matter what the wave-length of a light may be, its effect upon the several resonators at very high intensities is the same. The relative light sensitivities indicated by the maxima of the curves in Fig. 4 are such as to be in harmony with the results of the quantitative study of this phenomenon for different sets of lights. Another interesting point with reference to our diagram is the manner in which it accounts for the repetition of the hue R in the (violet) short-wave end of the spectrum. The relationship figured between the resonance functions of M, and Mg is justified by four or five independent considerations, which, however, cannot be enumerated here.** Our explanation of the peculiar hue relations sustained by the short wave-lengths is in complete accord with the mutations of spectral violet with in- creasing light intensity, fatigue, etc.” Extant visual hypotheses — excepting Hering’s — are to be adversely criticized for their inability to explain the repetition of the hue R in two widely separated parts of the spectrum. Further Notes on the Psycho-physics of the Hues.— The forms of the resonance functions which we have suggested in our figure are such as to make it necessary to suppose that the limits of the visible spectrum are imposed by the selective absorption of the ocular media or of the inner strata of the retina. However, chemical reso- nance curves are seldom symmetrical and consequently it would be quite legitimate to modify our functions to suit the demands which may be made upon them in this respect. Contrary to the assump- 8 See: Roop, O. N.: Modern Chromatics, 1875, New York, p. 18r. *! Among these may be counted the phenomena of selective adaptation, the change of hues with increasing light intensity, and the position of M, in the optical and the evolutionary scales. M,, we must suppose, has the greatest light sensitivity of any of the chromatic resonators; is a peculiarly unstable compound. °° See, e.g.: Hess, Cart: Ueber die Tonanderung der Spectralfarbung durch Ermiidung der Netzhaut, 1890, Leipzig. — OD dagen Alagille mes A Definite Physico-Chemical Hypothesis 33 tions of Kénig and Dieterici®® but in harmony with general chemical analogy we have assumed that any light ray — within the limits of the visible spectrum — can appreciably increase the ionization of each molecular resonator. This factor in our hypothesis not only permits us to satisfactorily account for the phenomena of color mix- ture but it also provides an explanation for the exact nature of the shift occurring in the positions of complementary lights with increas- ing intensities, both in peripheral and foveal vision,®’ as well as for many interesting fatigue phenomena.” In this connection it should be noted that, owing to the excitability of each of the resonators by every visible light, the induction of W by “daylight” depends not only upon the principles of exact complementation, but also upon the action of Fechner’s law, for the effective intensity of “‘daylight”’ is proportional to the energy integral over the entire spectrum. Another psycho-physical relation which is readily taken into account by our hypothesis is the fact that the highest difference sensibility occurs in those parts of the spectrum possessing the highest luminosity and least chroma. A glance at our resonance function diagram (Fig. 4) will show that it is in just these regions—about A = 580 and A = 490 — that the greatest shifting in the composition of the visual impulse should occur, and consequently — in accordance with our interpre- tation of visual thresholds (pages 25-26) — the greatest number of discriminations should be made for a given change in wave- length. The Problem of Sensory Luminosity. — In terms of our theory, sensory luminosity, or ‘‘Helligkeit”’ is identical with the attribute, W, of the visual sensation, and the extent in which this attribute appears is always proportional to u,. This, in turn, is determined by two factors, the complementation reaction, and the excitation of M,. The “specific luminosity”? of the spectral colors obviously » depends upon the manner of superposition of complementary reso- nance functions for the light involved, and the ground of the distribu- tion of these ‘‘specific luminosities”’ we have already indicated. The fact that in the prismatic spectrum the maximum luminosity is in 56 See reference given in note II, supra. 57 Cf. Nicati, W.: La psychologie naturelle, 1898, Paris, pp. 63-65. 58 F.g., those observed by: Burcu, G. J.: Journal of Physiology, 1897, xxi, p. XXvii. 34 Leonard T. Troland the yellow must be assigned to the combination of the curve of dis- persion of the refracting substance with the M, and M, curves of our diagram, rather than to any purely physiological cause. The shift in the location of this maximum which occurs with decreasing illumina- tion is, of course, due to the rise of the M,, or “twilight”’ excitation; this constitutes the familiar and somewhat over-emphasized Purkinje phenomenon, which has been completely explained by the researches of von Kries and others into the properties of the visual purple. The present writer accepts von Kries’ theory and, as already stated, identifies M,, with the substance peculiar to the retinal rods. The measurement of luminosity by the method of “flicker photometry” may be explained upon our hypothesis if it is supposed that the cortical discrimination limen, Ac,,is smaller than that characteristic of the chromatic components; this supposition is, in fact, necessi- tated by the results of Konig. The Explanation of Simultaneous Contrast.— In the original statement of our hypothesis concerning visual stimulation we asserted that, in accordance with general chemical analogies, the ionization of the molecular resonators should not be zero even in the absence of optical stimulus (normal ionization). On this basis it follows that the visual impulse is never null, and so we are at once provided with an explanation of the so-called “‘proper light of the retina.’ Since in the absence of stimulus, one “‘sees gray,” it becomes necessary to account for the fact that one is ever able to “‘see black.”” The mechan- ism by which the elementary visual impulses corresponding to certain retinal cells may suffer a decrease through the stimulation of adjacent cells is that underlying simultaneous contrast, and may be briefly described as follows. We will first take notice of the retinal rods alone, and consider that the only molecular resonator present is M,. Let Sa denote the visual sensation situated at a point in the visual field corresponding with Ry, a stimulated portion of the retina, while S,, an immediately outlying sensation element, corresponds with R,, an outlying retinal activity. Now suppose that the light La, stimulat- ing Ra, has a finite intensity eg, while the outlying light L, has zero intensity, so that we may write: m,,>m,,; hence, t,4 >, - This means that the rate at which the I,,, ions are leaving the stimulated ** Konic, A.: Zeitschrift fiir Psychologie und Physiologie der Sinnesorgane, 1895, Vili, pp. 375-381. A Definite Physico-Chemical Hypothesis 35 region is greater than that at which they are leaving the unstimulated area; but since, in the reaction: M,—I,,+I1,_, I, ions are produced in number equal to that of I,,, when equilibrium is estab- lished, the rate of dissipation of the negative ions through the retinal bacillary layer from the position of Ra must be greater than that taking place in the opposite direction. If this is the case, the con- centration of I,,_ ions in the region of R, must have increased over the concentration of the same ions which existed previously to the stimulation of the adjoining region, Ry. But the state of ionization of the molecular resonator, M,, in any region depends upon the concentration of each of the ions, I,, and I,_, in such a way that: (k, +k;)m, =k,i,.i,_. It is obvious then that an increase in i,- must be accompanied by a decrease in iw;, and consequently 1,, must fall below that normal to the unstimulated retina, so that the corresponding sensation will be “‘darker’’ — contain less W, than the “proper light of the retina.’ This is the mechanism of simple achro- matic contrast. Since the “‘proper light” is normally gray, we must suppose that in the unstimulated retina, the complementary ions, I,, and I,,, and I,, and I,, are exactly balanced in number. Com- plex achromatic contrast must then depend upon a process entirely similar to that above described, in which, however, four different species of ions are involved. The physical mechanism of chromatic contrast may be argued from the above. Suppose that Rg is stimu- lated by a homogeneous light. In this case there will be a spread of specific negative chromatic ions from the stimulated point, the disturbance of the normal ionization of specific chromatic resonators, a failure of the — unstimulated — complementation equilibrium, and the consequent induction of a sensory hue in the region of the visual field which is occupied by S. which is complementary with respect to the — stimulated — sensory hue of Sa. Owing to the fact that the transfer of ions between adjacent retinal elements under the conditions above studied is not a diffusion process but is due primarily to the differences of electrical potential existing between the elements, there must be a motion of positive ions from R, into Ra, as well as of negative ions in the opposite direction. The quantitative measure- ments of the distribution and correlations of contrast are quite in harmony with the above explanation, which is a natural consequence of the form of our original hypotheses. 36 Leonard T. Troland The Explanation of Adaptation. — In the deduction of Fechner’s law (cf. page 28) one develops, as an intermediate step, the equation: k+kabc mn = —_—— k+ke in which ab and ¢ are the concentrations of the aesthesogens (cf. page 22) AB and C, respectively, and m is the concentration of the corres- ponding molecular resonator. Since the rate of dissociation, m,, of the resonator is relatively proportional to m, and since the intensity of the corresponding component of the visual cerebrosis, C, is similarly proportional to m,, it follows that any change occurring in ab and c, will be reflected by a similar change in c. We should expect to find a diminution of ab and c after long continued stimulation, during which the process has been moving constantly in the sense of left to right in the double chemical equation given on page 23. It appears then, that stimulation with “white” light should result in an after-image containing less W than the retinal proper light, whereas stimulation with a homogeneous light should be followed by an after-image complementary in hue to the original, owing to the sub- traction of a specific component of the impulse, the normally balanc- ing complementary of which is then left free. The data obtained in the special qualitative and quantitative study of adaptation phenom- ena prove to be strictly in harmony with the demands of our Fig. 4. Our hypothesis also has the advantage of being able to account for the high luminosity of certain after-images, for as noted in the last paragraph, the effect of local stimulation is to cause a general migra- tion of positive ions into that region, so that those molecular resonators in the stimulated area which are not intensively concerned in the response will increase rather than diminish in concentration during the specialized fatigue process.*' The positive after-image may be attributed to an over-charging of the cortical element with ions of a specific character, and other details of adaptation, such as the func- 69 See notes 54 and 55, above. Several cases are discussed in detail in the author’s “‘ Thesis,” chapter 21. *l The necessity of accounting for the great luminosity of the negative after- image has been emphasized by Hering and by Mrs. Ladd-Franklin. See, e.g., LApD-FRANKLIN, C.: Psychological Review, 1894, 1, pp. 396-399. A Definite Physico-Chemical Hypothesis a7 toning of the M,, or twilight substance, may be accounted for in a way entirely consistent with our postulates. The Problem of Color-blindness. — The majority of theories of visual response find their impetus in the phenomena of color-blind- ness, and yet none of the extant hypotheses is capable of satisfactorily account for the well-known abnormal types of color-vision. These types are the “‘red-blind” or scoferythrous, which is characterized by a low stimulus value for lights of low frequency; the “‘green-blind”’ or photerythrous, for which the limits of the spectrum are the same as for a normal observer, although only two hues can be discriminated; and the so-called yellow-blue-blind, in which red and green can be discriminated, , but not yellow and blue. Both the Young-Helmholtz and the ° Hering hypotheses, which were specially designed to account for 0 40 80 120 160 200 240 Fic. 5. Biometric plot showing the distribu- tion of twenty cases of ‘‘dichromatic” : visual response. these types of color-blindness, 4 are now acknowledged—at least in the most important cases — to be incapable of doing this.” Mrs. Ladd-Franklin’s theory is hardly more successful. The explanation of color-blindness is a complex matter, and one which cannot be accomplished apart from genetic and pathological investigations. It is to be noted, however, that the arrangement of the curves of Fig. 4 is such as to account perfectly for the established facts concerning dichro- matic response if it is supposed that in the photerythrous type there is absent a special cortical mechanism, which we may call the R-G-cortical-chromatic element, the presence of which is essential in order that the I,+ and I,+ ions of the impulse should be received in the visual cerebrosis; while the scoterythrous type may be explained by supposing the concomitant absence of the M, resonator. The positions of ‘‘gray bands,” maxima of luminosity, etc., as observed OrpINATEs : Class frequency. ABSCISSAE: Intensity of light from the lithium line required to match a given intensity of light from the sodium (D) line. Arbitrary units. Data from: Krres, JOHANNES, von: Zeitschrift fiir Psychologie und Physiologie der Sinnesorgane, 1897, xiii, p. 259. ® For a frank confession of the failure of Hering’s hypothesis in this connec- tion, see: TSCHERMAK: Ergebnisse der Physiologie, 1902, i, 2, p. 795. 38 Leonard T. Troland for these two types are in harmony with the resonance functions represented in our diagram. The blue-yellow-blind, moreover, can be classed with equal success as cases of the absence of a Y-B-cortical- chromatic element. Total color-blindness may be due to the absence of both of the cortical elements, or of all of the chromatic molecular resonators, or to other conceivable effective combinations of losses. Obviously the explanatory scheme which is suggested is a very flexi- ble one.® The phases of color-blindness exhibited in the normal peripheral retina may be satisfactorily accounted for in the same terms. Scope of the Above Discussion. — It must not be supposed that the writer regards the above exposition of the consequences of what may be appropriately denominated the photo-ionic theory of visual response, to be sufficient to establish the validity of the hypotheses which he has advanced. On the contrary, the entire paper must be looked upon simply as an abstract of a much more elaborate, but still exceedingly imperfect discussion of the deductions and points of view made possible by the notion that the mechanism of visual response is ionic in character. It is hoped, however, that the paper will at least suffice to dimly suggest, if not to demonstrate, the impor- tance of looking upon these, and other, physiological problems in the light of modern theoretical physics and chemistry. V. SUMMARY At the outset we saw that extant hypotheses to explain visual phenomena not only fail to keep step with the progress of physics and chemistry, but are unduly vague in statement and, in many cases, are inconsistent with the sciences the concepts and laws of which they are bound to employ. Consequently it seemed advisable to formulate a renovated, if not a new, chemical hypothesis to deal with * The explanation here offered of the two distinct types of dichromatic visual response has the appearance of artificiality, largely on account of the fact that, — for purposes of abbreviation, the cortical chromatic elements were not presented as integral parts of our hypothesis. In the explanation of color-blindness, one cannot legitimately expect any primitive simplicity. A simple explanation of these phenomena, even if it were obtainable, would be inconsistent with the general tendency of the field of nature involved. There can be no reasonable A Definite Physico-Chemical Hypothesis 39 these phenomena, in a manner more consonant with the viewpoint and methods of modern physics and chemistry. From the nature of the stimulus we argued that the physical proc- ess of excitation in the retina must be one of tonization, and since we know that the nerve impulse is ionic in its mechanism, it was easy to connect this excitation in a definite way with the visual nerve current and thence with the cerebral and psychical processes. Our assumptions were perhaps over-simple, but they were quantitative in character, wherever possible, and the conclusions drawn from them later appeared to be quite consonant with the facts. We were led to postulate five specific visual substances, or molecular resonators, corresponding with the psychological qualities, red, green, blue, yellow, and white, respectively. By the introduction of a special and clearly defined chemical mechanism which would remove “‘com- plementary components”’ of the visual impulse in pairs and substitute a ‘white component”’ we laid a foundation for the explanation of the phenomena of complementary colors. In every case we sought to make the definition of our hypotheses as sharp as possible. Arriving at the practical problem of applying our assumptions to the facts of visual physiology and psychology, we found, in the first place, that the ionic viewpoint permitted at least a schematic explana- tion of the electrical phenomena exhibited by the stimulated and unstimulated eye. A brief discussion of certain important “visual thresholds” showed again that we were on the right track, and pro- vided a foundation for further argument. Certain calculations based immediately upon our original postulates yielded a theoretical curve for the rise and decay of the visual excitation, coinciding with that experimentally established. Furthermore, we discovered that an application of the law of chemical mass action to the ionization process in the retina provided us with a direct explanation of the well-known Weber-Fechner law. doubt, in view of the ascertained facts, that color-blindness may depend upon either or both retinal or (and) cerebral abnormalities. Neither, in view of such measurements as those represented in the distribution areas of Fig. 5, can it be doubted that there exist distinct visual types, which can only be accounted for upon some presence-absence hypothesis, and not on the basis of a continuous variability of properties such as Hering’s explanation of the inconstancy of “red- green blindness”’ (as due to fluctuations in the color of the lens or ocular media) demands. 40 Leonard T. Troland In the field of “color vision”’ our hypotheses proved equally fruit- ful, their consequences harmonizing even quantitatively not only with the familiar phenomena of “color mixture’? and adaptation, which are more or less adequately accounted for by extant theories, but also giving us an insight into certain of the finer details of the mechanism of color. Among the latter were the mutations and final disappearance of the hues with increasing light intensity, the distribu- tion of brightness and chroma in the spectrum, and in spectrum hues produced by mixture, the fact that “‘green”’ lacks a homogeneous complementary, the repetition of ‘“‘redness”’ in the ‘‘violet end”’ of the spectrum, the distribution of ‘“‘sensibility to differences”? with respect to change in wave-length, the shift in the relative positions of complementary colors with changes in light intensity, etc. The problem of sensory luminosity, or ‘‘ Helligkeit’’ was then discussed in connection with our assumptions, which deal with the phenomena in question more simply than does the theory of Hering. We saw that the ionic hypothesis provides a direct electro-chemical basis for the explanation of simultaneous and successive contrast, and one which avoids the difficulties which have been raised by Hering and others with respect to the relative luminosity of the after-image. In the discussion of these phenomena the physico-chemical view- point was maintained, and proved enlightening. In conclusion, it appeared that an additional assumption with reference to the cerebral connections of the different hues, would permit our hypothesis to account quantitatively for the most familiar types of color-blindness, by means of a doctrine — seemingly necessi- tated by the facts — of the absence of unit characters. THE UNIFORM RATE OF THE DESTRUCTION OF PEPSIN BY THE PASSAGE OF THE DIRECT ELECTRIC CURRENT By W. E. BURGE [From the Physiological Laboratory of the University of Illinois.] T has been shown that the activity of pepsin and of ptyalin is destroyed by the passage of a direct electric current and that the rate of this destruction in the case of ptyalin is uniform per coulomb. The present investigation was begun to determine whether the rate of decrease in peptic activity is also uniform per coulomb. The solution of pepsin used was prepared by dissolving one gram of a commercial preparation of pepsin in too c.c. of distilled water. The digestive activity of equal portions of the solution before and after electrolysis was determined by the amount of digestion in Mett’s tubes in forty-eight hours. The egg-white in these tubes was cooked in boiling water for one minute. The electrolyzing cylinder, a description of which has already been published,! was charged with 5 c.c. of the pepsin solution and placed across the electrodes of a direct electric circuit in series with a poten- tial reducer and a milliammeter. The cylinder was then placed in a shaking machine and shaken at the rate of five hundred single shakes per minute in order to prevent polarization. Portions of the normal solution had already been shaken at this rate for several hours and it was found that this rate of shaking of itself had no effect upon the activity of the enzyme. Twenty-five milliamperes of current were passed through the 5 c.c. of solution for twenty minutes while it was being shaken at the above named rate. At the end of this time the electrolyzed solution was removed from the cylinder, 5 c.c. of fresh solution were introduced, the cylinder was replaced in the shak- ing machine, and twenty-five milliamperes were passed for forty minutes. At the end of this period the solution was removed and the 1 BurGE: This journal, 1913, xxxi, p. 328. 42 W. E. Burge cylinder was recharged. Electrolyses were continued in this manner until solutions were obtained through which the current had passed for twenty, forty, sixty, eighty, one hundred, one hundred twenty and one hundred forty minutes respectively. When the series were complete 3 c.c. of .5 per cent hydrochloric acid were added to 3 c.c. of each of the electrolyzed pepsin solutions and to 3 c.c. of the non- electrolyzed solution which served for comparison. Into each test tube containing the .25 per cent hydrochloric acid pepsin solution a Mett’s tube 3 cm. in length was introduced and these test tubes were placed in a thermostat at 38° C. for forty-eight hours. At the end of this time the Mett’s tubes were removed from the solutions and the amount of digestion was measured. A photograph of a typical experiment is reproduced here showing Mett’s tubes twice the actual size (Fig. 1). The dark portion represents the amount of undigested egg-white and the light portion the empty tube from which the egg had been digested. The results of the experiments are indicated also in the accompanying table. TABLE I Decrease in | Decrease mm. of egg in mm. digested per Q. Coulombs | Mm. of egg Wes passed digested | | | | Non-electrolyzed solutions I Electrolyzed solutions Average As will be seen from the table the amount of egg-white digested was decreased in proportion to the number of coulombs that were allowed to pass. The decrease in digestive power as between Tube I Destruction of Pepsin by Direct Current 43 and Tube II is expressed as a difference of 1.5 mm. in the column of egg-white. As this difference was caused by the passage of 30 coulombs, the quotient of 39 or .o5 mm. expresses the decrease in digestive power per coulomb. The quotients obtained in a similar | | | | Vill Vil vI v IV Il II I Ficure 1.— Photograph of Mett’s tubes magnified twice. The dark portion repre- sents the undigested egg white, the light portion the extent of digestion. way for the other tubes, in comparison with Tube I, are given in the last column of the Table. The average obtained from these figures indicates a diminution in digestive power of 0.04 mm. per coulomb. The agreement among these figures is sufficiently close to justify the conclusion that the digestive activity of a solution of pepsin is decreased by the passage of a direct electric current at a uniform rate per unit of current. THE EFFECT OF ADRENAL SECRETION ON MUSCULAR FATIGUE? By W. B. CANNON anp L. B. NICE [From the Laboratory of Physiology in the Harvard Medical School] N the older literature on the adrenal glands the effect of their ab- sence, or of injected extracts, on skeletal muscle was not unfre- quently noted. As evidence accumulated, however, tending to prove that adrenal secretion has important relations with the sympathetic nervous system, its relations with skeletal muscle began to receive less consideration. The muscular weakness of persons suffering from Addison’s disease was well recognized before experimental work on the adrenals was begun. Experiments on rabbits were reported in 1892 by Alba- nese who showed that muscles stimulated after removal of the adrenal capsules, were much more exhausted than when stimulated the same length of time in the same animal before decapsulation.” Similarly Boinet reported that rats recently decapsulated were much more quickly exhausted in a revolving cage than were normal animals.* More direct evidence of the effect of adrenal extract on skeletal muscle was brought forward by Oliver and Schafer. After injecting the extract subcutaneously into a frog they found that the excised gastrocnemius muscle registered a curve of contraction about 33 per cent higher and about 66 per cent longer than the corresponding muscle not exposed to the action of the extract. Similar prolonga- tion of the muscle curve was observed after injecting the extract intravenously into a dog. A beneficial effect of adrenal extract on 1 A preliminary report of this research was given at the meeting of the Ameri- can Physiological Society, December, 1ro11. See Proceedings, this Journal, 1912, XXIX, Pp. XXIV. * ALBANESE: Archives italiennes de biologie, 1892, xvii, p. 243. ’ BorneT: Comptes rendus, Société de Biologie, 1895, xlvii, pp. 273, 408. 4 OrrverR and ScHAFER: Journal of physiology, 1895, xviii, p. 263. See also RapwWANsKA, Anzeiger der Akademie, Krakau, 1910, pp. 728-736. Reviewed in Zentralblatt fiir Biochemie und Biophysik, 1911, xi, p. 467. Adrenal Secretion on Muscular Fatigue AS fatigued muscle, even when applied to the solution in which the isolated muscle was contracting, was claimed by Dessy and Grandis, who studied the phenomenon in a salamander.? Further evidence to the same conclusion was offered in a discriminating paper by Panella. He found that in heterothermic animals the active prin- ciple of the adrenal glands notably reinforced striated muscle, pro- longing its ability to do work, and improving its contraction when fatigued. In homothermic animals the same effects were observed, but only after experimental procedures (anaesthesia, section of the bulb) had changed them to a condition resembling the heterothermic.*® The foregoing evidence indicates that decapsulation has a debili- _ tating effect on muscular power, and that injection of extracts of the capsules has an invigorating effect. It seemed possible, therefore, that increased secretion of the adrenal glands, whether from direct stimulation of the splanchnic nerves or as a reflex result of pain or the major emotions, might act as a dynamogenic factor in the performance of muscular work. With this possibility the present investigation was concerned. THE METHOD The general plan of the investigation consisted primarily in observing the effect of stimulating the splanchnic nerves, isolated from the spinal cord, on the contraction of a muscle whose nerve, also isolated from the spinal cord, was rhythmically and uniformly excited with break induction shocks. Thus only a blood connection existed between the splanchnic region and the muscle. Cats were used for most experiments, but results obtained with cats were con- firmed on rabbits and dogs. To produce anaesthesia, in the cats and rabbits, and at the same time to avoid the fluctuating effects of ether, urethane (2 gm. per kilo body-weight) was given by a stomach- tube. The animals were fastened back-downward, over an electric warming pad, to an animal holder. Care was taken to maintain the body temperature at its normal level throughout each experiment. The nerve-muscle preparation. — The muscle selected for record was usually the right tibialis anticus, though at times the right extensor 5 Dressy and Granpis: Archives italiennes de biologie, 1904, xli, p. 231. 6 PaNELLA: Archives italiennes de biologie, 1907, xlviii, p. 462. 46 W. B. Cannon and L. B. Nice communis of the digits was employed. The anterior tibial nerve was bared for about two centimetres, severed proximally, and set in a Sherrington shielded electrode around which the skin was fastened by spring clips. By a small slit in the skin the tendon of the muscle was uncovered, and after being tightly ligatured with strong thread, was separated from its insertion. Thus a nerve-muscle prepara- tion was made which was still connected with its proper blood supply. The preparation was firmly fixed to the animal holder by thongs looped around the hock and the foot, i.e., on either side of the slit through which the tendon emerged. The ligature tied to the tendon was passed over a pulley and down to a pivoted steel bar which bore a writing point. Both the pulley and this steel writing lever were supported in a rigid tripod. In the earliest experiments the contracting muscle was made to lift weights (125 to 175 gm.), and was sometimes “loaded” with these weights; but in all the later observations the muscle pulled against a spring. In most instances the muscle was afterloaded, but by raising the muscle clamp the tension developed in the muscle at rest was made nearly equal to the pull of the spring at its shortest length. The “support,” therefore, was little more than a constant base for relaxa- tion. The pull of the spring as the muscle began to lift the lever away from the support was in most of the experiments 110 gm., with an increase of 10 gm. as the writing point was raised 4.5 mm. The magnification of the lever was 3.8. The stimuli delivered to the anterior tibial nerve were, in most experiments, single break shocks of a value barely maximal when applied to the fresh preparation. The rate of stimulation varied between 60 and 300 per minute, but was uniform in any single obser- vation. A rate which was found generally serviceable was 180 per minute. In a few experiments a regularly repeated tetanizing current was used. Since the anterior tibial nerve contains fibres affecting blood- vessels, as well as motor fibres for skeletal muscle, the possibility had to be considered that stimuli applied to it might disturb the blood supply of the nerve-muscle preparation. Vasoconstriction would be likely to produce the most serious disturbance. The observations of Bowditch and Warren, that vasodilator rather than vasoconstrictor effects are produced by single induction shocks repeated at intervals Adrenal Secretion on Muscular Fatigue 47 of not more than five per second,’ reassured us as to the danger of diminishing the blood supply, for the rate of stimulation in our ex- periments never exceeded five per second and was usually two or three. Furthermore in using these different rates we have never noted any result which could reasonably be attributed to a diminished circulation. The splanchnic preparation. — The splanchnic nerves were stimu- lated in various ways. At first only the left splanchnics in the abdo- men were prepared. The nerves, separated from the spinal cord, were drawn into a Sherrington shielded electrode, which was attached by threads to the body wall. A rubber tube connected the elec- trode with the exterior of the body, and separated the wires from the abdominal viscera. The placing of this electrode, however, required so much time and, in spite of great care, was so likely to result in harmful pulling on the nerves, that some other device became necessary. The form of electrode which was found most satisfactory was that Ficure 1. The shielded electrode used in splanchnic stimulation. For description see text. illustrated in Fig. r. It was made of a round rod of hard wood, bevel- led to a point at one end, and grooved on the two sides. Into the grooves were pressed insulated wires ending in platinum hooks, which projected beyond the bevelled surface. Around the rod was placed a rubber tube which was cut out so as to leave the hooks uncovered when the tube was slipped downward. In applying the electrode the left splanchnic nerves were first freed from their surroundings and tightly ligatured as central as possible. By means of strong compression the conductivity of the nerves was destroyed proximal to the ligature. The electrode was now fixed in place by thrusting the sharp end into the muscles of the back. This was so done as to bring the platinum hooks a few milli- metres above the nerves. With a small seeker the nerves were next gently lifted over the hooks, and then the rubber tube was slipped downward until it came in contact with the body wall. Absorbent 7 Bowpitcu and WARREN: Journal of physiology, 1886, vii, p. 438. 48 W. B. Cannon and L. B. Nice cotton was packed about the lower end of the electrode, to take up any fluid that might appear; and finally the belly wall was closed with spring clips. The rubber tube served to keep the platinum hooks from contact with the muscles of the back and the movable viscera, while still permitting access to the nerves which were to be stimulated. This stimulating apparatus could be quickly applied, and, once in place, needed no further attention. In some of the latest experiments both splanchnic nerves were stimulated in the thorax. The rubber-covered electrode proved quite as serviceable there as in the abdomen. The current delivered to the splanchnic nerves was a tetanizing current of such strength that no effects of spreading were noticeable. Frcure 2. Upper record, contraction of the tibialis anticus, 80 times a minute, lifting a weight of 125 gm. Lower record, stimulation of the left splanchnic nerves, two minutes. Time, half minutes. That splanchnic stimulation causes secretion of the adrenal glands has been proved in many different ways which need not be recounted here. On this assumption the present investigation was undertaken. THE EFFECTS OF STIMULATING THE SPLANCHNIC NERVES The effect on contraction of fatigued muscle, which can often be obtained by stimulating the left splanchnic nerves, is shown in Fig. 2. In this instance the muscle was afterloaded, and while con- tracting lifted a weight of 125 gm. The rate of stimulation was 80 per minute. The muscle record shows a brief initial rise, followed by a drop, and that in turn by another prolonged rise. The maximum height Adrenal Secretion on Muscular Fatigue 49 of the record is 13.5 mm., an increase of 6 mm. over the height recorded before splanchnic stimulation. Thus the muscle was performing for a short period 80 per cent more work than before splanchnic stimu- lation, and for a considerably longer period exhibited an intermediate betterment of its efficiency. feane first rise in the muscle record.— The brief first elevation in the muscle record when registered simul- taneously with blood press- ure, is observed to occur at the same time with the sharp initial rise in the blood-press- ure curve (see Fig. 3). The first sharp rise in blood press- ure is due to contraction of FicurE 3. Top record, blood pressure with the vessels in the splanchnic membrane manometer. Middle record, contractions of tibialis anticus loaded with 125 gm. and stimulated 80 times a minute. the alimentary canal is re- Bottom record, splanchnic stimulation (two minutes). Time, half minutes. area, for it does not appear if moved, or if the coeliac axis and the superior and inferior mesenteric arteries are ligatured. The betterment of the muscular contraction is probably due directly to Ficure 4. (Four-fifths the original size.) Top record, blood pressure with membrane manometer. Middle record, contractions of tibialis anticus against a spring. Bottom record, stimulation of left splanchnics. At a both adrenal veins were clipped, at b the clips were removed. the better blood supply resulting from the increased pressure, for if the adrenal veins are clipped, and the splanchnic nerves are stimu- lated, the blood pressure rises as before and at the same time there may be registered a higher contraction of the muscle (see Fig. 4). 50 W. B. Cannon and L. B. Nice 2. The prolonged rise of the muscle record. — As Fig. 3 shows, the initial quick uplift in the blood-pressure record is quickly checked by a drop. This rapid drop does not appear in Fig. 4, a record made when the adrenal veins were obstructed. The difference between Figs. 3 and 4 in this respect agrees with Elliott’s observation of a similar difference in blood-pressure records before and after excision of the adrenal glands. And as Elliott,’ and as Cannon and Lyman ® have shown, this sharp drop after the first rise, and also the subse- quent elevation of blood pressure, are the consequences of liberation of adrenal secretion into the circulation. Fig. 3 demonstrates that the prolonged rise of the muscle record begins soon after this char- acteristic drop in blood pressure. In Fig. 4 removal of the clips from the adrenal veins after the splanchnics had been stimulated occasioned a slight, but distinct improvement in the muscular contraction. As in the experiments of Young and Lehmann, in which the adrenal veins were tied for a time and then released, the release of the blood which had been pent in these veins was quickly followed by a rise of blood pressure. The volume of blood thus restored to circulation was too slight to account for the rise of pressure. In conjunction with the evidence*that splanchnic stimulation calls forth adrenal secretion," the rise may reasonably be attributed to that secretion. The fact should be noted, however, that in this instance the prolonged improvement in muscular contraction did not appear until the adrenal secretion had been admitted to the general circulation. Figs. 2 and 3, and Fig. 4 illustrate two types of Improvement in the activity of fatigued muscle in response to adrenal secretion. Many variations on these types were noted in the course of the investi- gation. The improvement varied in degree as indicated by increased height of the record. In some instances the height of contraction was doubled — a betterment by roo per cent; in other instances the con- traction after splanchnic stimulation was only a small fraction higher than that preceding the stimulation; and in still other instances there * Exuiotr: Journal of physiology, 1912, xliv, p. 403. 9 CANNON and Lyman: this Journal, 1913, xxxi, p. 376. 0 ‘Younc and LEHMANN: Journal of physiology, 1908, xxxvii, p. liv. ! See AsHER: Zeitschrift fiir Biologie, 1912, lviii, p. 303; Extiorr: Journal of physiology, Loc. cit., p. 390. Adrenal Secretion on Muscular Fatigue 51 was no betterment whatever. Never in our experience, were the augmented contractions equal to the original contractions of the fresh muscle. The improvement also varied in degree as indicated by persis- tence of effect. In some instances the muscle returned to its former working level within four or five minutes after splanchnic stimula- tion ceased (see Fig. 2); in other cases the muscle continued work- ing with greater efficiency for fifteen or twenty minutes after the stimulation.” The question now arises, does the adrenalin liberated by splanchnic stimulation act itself, specifically, to improve the muscular contrac- tion, or does it produce the improvement by increasing the blood pressure and thereby increasing the blood flow through the laboring muscle? And further, since splanchnic stimulation results in an augmentation of the sugar content of the blood,‘ might not the greater ability of the muscle be due to a greater supply of this source of muscular energy? And in the cases in which no improvement occurred what was the reason for the failure? All these questions must be considered. THe CAUSE OF THE PROLONGED RISE IN THE MuscLeE RECORD The association of the first rise in the muscle record with an increase of blood pressure shows that the factor of blood supply is capable in itself of restoring to some degree the efficiency of a fatigued muscle. 2 “Tonus waves,” similar to those described by Storey (this Journal, 1904, xii, p. 83), have been repeatedly observed by us. They had interesting relations to the discharge of adrenalin into the blood. If the waves were not present, splanchnic stimulation would often cause an augmented contraction in which the waves were present. Or if the waves were already present, splanchnic stimula- tion would commonly result in their being more rapid (see Fig. 6, A and B, also Fig. 7). 13 Tt is assumed in this enquiry that vessels supplying active muscles would be actively dilated (See KAUFMANN: Archives de physiologie, 1892, xxiv, Pp. 283), and would, therefore, in case of a general increase of blood pressure, deliver a larger volume of blood to the area they supply. 144 MacLeop: this Journal, 1907, xix, p. 405, also for other references to the literature. 52 W. B. Cannon and L. B. Nice In order to differentiate between a possible specific action of adrenal secretion as an antidote to fatigue, and the effect which the secretion would have by increasing blood pressure, various methods were employed to keep the blood pressure constant during stimulation of the splanchnic nerves. Bayliss’s compensator !° proved too slow to— be effective. The pressure was maintained at a uniform level in some Ficure 5. Top record, blood pressure with mercury manometer; between the arrows the pressure was kept from rising by compression of heart. Middle record, contractions of tibialis anticus, 180 per minute, against a spring. Bottom record (zero of blood pressure) shows stimulation of left splanch- nics. Time, half minutes. instances, however, by compression of the heart through the walls of the thorax. In other instances a loop of strong thread was passed through a small opening in the belly wall and around the abdominal aorta just before its forking into the iliacs. By more or less tension on the thread the pressure in the arteries of the legs could be regulated at will. This pressure was registered by means of a manometer attached to the left femoral artery. As soon as the pressure showed a '5 See BAytiss: Handbuch der physiologischen Methodik, 1911, ii, Abtheilung Adrenal Secretion on Muscular Fatigue 53 tendency to rise the loop was pulled upon, and the inflow to the arteries of the leg thereby kept from increasing; as a tendency to fall mani- fested itself the pull on the loop was lessened. The rédle of increased blood pressure when adrenal secretion is liberated. — In Fig. 5 is presented the record (from a decerebrate cat) of a fatigued muscle contracting 180 times per minute, and reduced to a very low degree of activity when splanchnic stimulation was started. The stimulation was continued for two minutes. During the first minute blood pressure was prevented from rising by compression of the heart, and in that time no betterment of the contraction appeared. As soon as compression of the heart ceased, however, the blood pressure promptly rose from approximately 48 mm. of mercury to 110 mm., and simultaneously the height of the muscular contraction increased about six-fold. It is noteworthy that although the blood pressure gradually fell to its former level the muscle did not return to its former inefficiency. Merely because the blood supply had been better, probably because depressive metabolites had been thus washed away more effectively, and possibly because fresh sources of energy had been brought to the muscle in larger amount, the muscle continued to show a greater ability. When the blood pressure was restored to its former level, the splanchnic nerves were again stimulated — this time for one minute. The blood pressure was kept down in this instance for one and three- fourths minutes by compression of the heart. Only slight tendency to higher contractions was manifested by the muscle during this period. And again only when the heart was permitted to fill and empty in a normal manner, and the pressure consequently rose, was there a considerable betterment of the contraction. In these instances splanchnic stimulation undoubtedly liberated adrenalin, for the blood pressure remained elevated for fully seven minutes after the first stimulation ceased — a much longer time than is required for the adjustment of the circulation after compression of the thorax, but a time corresponding to the duration of effects of adrenal secretion. In spite of this the height of the muscle record failed to increase in any remarkable degree so long as the blood pressure was prevented from rising. The adrenal secretion, if it improves the contraction of fatigued muscle in a specific manner, 54 W. B. Cannon and L. B. Nice seems to have in that respect an influence much less important than that which it exercises in bettering the blood supply. Does adrenalin have any specific effect on muscular fatigue? — Although in the instance represented in Fig. 5, and in some other instances when blood pressure was prevented from rising, no clear evidence of remarkable recovery from fatigue was noted after splanchnic stimulation, we have been unable to prove that adrenalin is without any specific effect on fatigued muscle. In Fig. 6 A, for example, a fatigue record of uniform height was changed to a rhythm of higher contractions when the splanchnics FIGURE 6. (Two-thirds the original size.) A, top record, blood pressure in left femoral artery with mercury manometer. Middle record, contractions of the extensor communis against a spring. Bottom record, stimulation of left splanchnics, and time in half minutes. 3B, the same, five minutes after A. From the beginning of splanchnic stimulation to the end of the record in A and B, rise of blood pressure prevented by pull on the aorta. were stimulated and the blood pressure in the legs prevented from rising by a pull on the aorta. And in Fig. 6 B, taken five minutes after A, the slow rhythm of A was changed by stimulation of the splanchnics to a more rapid rhythm of slightly higher contractions. That adrenalin was secreted in these cases 1s shown by the fall of blood pressure shortly after stimulation of the splanchnics was begun. It is noteworthy that the first oscillation in A does not start with the beginning of stimulation, but is coincident with the first indication of the fall of pressure. Similar results are obtained if, instead of arousing adrenal secre- tion, adrenalin is injected. In Fig. 7, 2 c.c. of adrenalin (1: 100,000) were injected intravenously during the period indicated on the time line. The blood pressure was prevented from rising by pulling on Adrenal Secretion on Muscular Fatigue wn wn the thread looped round the aorta. There was a typical fall of pres- sure after the injection. Accompanying it was a distinct increase in the height of the muscular contractions. Changes in blood pres- sure in the legs almost exactly the same as those following the injec- tion of adrenalin could be produced by pulling on the aorta, but there resulted no alteration in the height of the fatigue record. The rise in that record shown in Fig. 7, there- fore, is not due to diminished blood supply.!® Also it is not due, there- fore, to the introduction of sodium bicarbonate from the manometer connection into the circulation. The drop in blood pressure following injection of adrenalin, or as a consequence of adrenal secre- tion, is due to vasodilation.” It might be supposed that, because of vasodilation, and in spite of a general fall of pressure, the blood supply to the muscle would be improved; and therefore, even in Figure 7. Top record, blood pressure, the case represented in Figs. 6 and mercury manometer. Middle record, 7, the higher contraction should ae of UES EOS 180 times per minute, against a spring. be ascribed to better circulation. Bottom record, injection of 2 c.c. Against this supposition is the ob- adrenalin (1: 100,000), and time in : : half minutes. Rise of blood pres- servation that when the arteries sure prevented by pull on the aorta. are deprived of their central in- nervation, as was the case with the arteries supplying the con- tracting muscle, adrenalin causes not a dilation but a constriction of the vessels.'8 And even if adrenalin did not cause vasoconstriction in this region, it could hardly produce much further dilation, for, as already noted, the vascular nerves had been cut and furthermore 16 LEE’s suggestion (this Journal, 1907, xviii, p. 272) that increase of carbon dioxide increases the height of muscular contraction makes the effect of lessened blood supply worthy of consideration. 17 See CANNON and LyMAN: Loc. cit., p. 384. 18 See CANNON and LyMAN: Loc. cit., p. 387. 56 W. B. Cannon and L. B. Nice were being stimulated at a rate favorable to relaxation (see p. 47). It seems probable, therefore, that adrenalin can itself act specifically in a manner not yet explained, to improve the contraction of fatigued muscle. This conclusion is in harmony with the observation of Dessy and Grandis, previously cited, that adrenal extract betters contraction when added to a solution in which the muscle is submerged.!® Consideration of the cases in which no improvement was ob- served. — Comparison of the records reproduced in Fig. 5 and in Fig. 7 shows that in all probability the part played by adrenalin itself in directly augmenting the power of fatigued muscle is much less than the part which it plays by improving the blood supply. The astonishing increase in the power of the muscle shown in Fig. 5, however, occurred in an animal with a blood pressure of only 48 mm. of mercury, a pressure which was more than doubled by splanchnic stimulation. In the early experiments of this investigation the sole object of recording blood pressure was to note the relations between changes of pressure and the variations in muscular contraction, or the results on muscular contraction when the adrenals were stimulated and the pressure kept under control. In these early experiments, unfortu- nately, because of the reasons just given, the actual height of the blood pressure was not recorded. The marked improvement of muscular contraction in some instances and the slight improvement or lack of improvement in others (cf. Figs. 2 and 7) in which the conditions seemed the same, led to an enquiry regarding the occasion for the difference. The fact soon appeared that although the slight improvement which we have attributed to adrenalin directly (see Fig. 7) can be manifested even if the blood pressure is as high as 140 or 150 mm. of mercury, such improvement as is shown in Figs. 3 and 5 does not appear unless there is a low blood pressure. And if the pressure is down as low as 50 mm. of mercury, splanchnic stimulation or injection of a small dose of adrenalin will cause the pressure to increase greatly, and a striking betterment of muscular work results. The effects of adrenal ‘9 We have been unable to confirm this observation by similar use of frog muscles. *0 The exact relations between variations of blood pressure and muscular efficiency are now being investigated in this laboratory by Mr. C. M. GruBEr. Adrenal Secretion on Muscular Fatigue 57 secretion, therefore, seem to be of two sorts; (1) a direct specific effect which benefits a fatigued muscle even when blood pressure is high, and (2) an indirect effect due to better circulation, and (within limits which are as yet undetermined) more striking the lower the blood pressure. Elimination of hyperglycaemia as a cause of the higher con- traction. — In the course of these experiments we repeatedly found sugar in the urine—a result probably due to hyperglycaemia from splanchnic stimulation. Recently Wilenko* has declared that the ability of the organism to burn sugar is lessened by adrenalin. It may be, however, that because of the artificial conditions of his experi- ments, their pertinence, when applied to the natural hyperglycaemia and adrenalinaemia of splanchnic origin, is questionable. Whether the adrenal secretion liberated with sugar when the splanchnics are stimulated does or does not similarly check the utilization of the sugar, data are at hand to prove that the hyperglycaemia is not essential to improved muscular contraction as described above. The sugar which makes the hyperglycaemia following splanchnic stimulation comes from the liver. The liver can be almost wholly removed, with- out disturbing the blood flow in the inferior vena cava, and yet splanchnic stimulation causes the typical rise in the muscle curve. DISCUSSION OF RESULTS It is noteworthy that the best results of adrenalin on fatigued muscle reported by previous observers were obtained from studies on cold-blooded animals. In these animals the circulation is maintained normally by an arterial pressure about one-third that of warm-blooded animals. Injection of adrenalin.in an amount which would not shut off the blood supply would, by greatly raising the arterial pressure, markedly increase the circulation of blood in the active muscle. In short the conditions in cold-blooded animals are quite like those in the pithed mammal with an arterial pressure of about 50 mm. of mercury (see Fig. 5). Under these conditions the improved circula- tion causes a striking recovery from fatigue. That marked results 21 WILENKO: Biochemische Zeitschrift, 1912, xlii, p. 49; Zentralblatt fir Physiologie, 1913, xxvi, p. 1050. 58 W. B. Cannon and L. B. Nice of adrenalin on fatigue are observed in warm-blooded animals only when they are deeply anaesthetized or are deprived of the medulla, was claimed by Panella. He apparently believed that in normal mammalian conditions adrenalin has little effect because quickly destroyed, whereas in the cold-blooded animals, and in mammals whose respiratory, circulatory and thermogenic states are made similar to the cold-blooded by anaesthesia or pithing, the contrary is true.22. In accordance with our observations on the effects of blood pressure on fatigued muscle, we would explain Panella’s results not as he has done but as due to two factors. First, the efficiency of the muscle, when blood pressure is low, follows the ups and downs of the pressure much more directly than when the pressure is high (see p. 56). And second, a given dose of adrenalin raises a low blood pressure in atonic vessels, whereas it may lower the pressure or fail to cause a marked rise in vessels tonically contracted. The improvement of circulation is capable of explaining, therefore, the main results obtained in cold-blooded animals and in pithed mammals. Oliver and Schafer reported more effective contractions in mus- cles removed from the body after adrenal extract had been injected. As shown in Fig. 5, however, the fact that the circulation had been improved results in continued greater efficiency of the contract- ing muscle. Oliver and Schifer’s observation may reasonably be accounted for on this basis. How the improvement in muscular contraction, after adrenal secretion is evoked or after adrenalin is injected, can be explained, is not clear. The results above reported show that the improvement, though not great, is distinct, and that it apparently does not result from better circulation. According to Panella * adrenalin has an effect antidotal to curare, and, injected either mixed with or follow- ing curare, is capable of preventing the complete immobility which the curare alone would produce. This experiment points to an action of adrenalin in the region of transfer of influence from the nerve to the muscle. Radwdnska*‘ also reported finding that adre- nalin has a more favorable action on fatigued muscle if the muscle is being stimulated through its nerve than if stimulated directly, and % PANELLA: Loc: cit., p. 462: *3 PANELLA: Archives italiennes de biologie, 1907, xlvii, p. 30. 24 RADWANSKA: Loc. cit. Adrenal Secretion on Muscular Fatigue 59 he drew the inference that its beneficial effect is on the nerve endings. It seems quite possible therefore that the improved contraction of fatigued muscle after splanchnic stimulation, when rise of arterial pressure is prevented, is the consequence of a facilitation of the passage of impulses into the fatigued muscle. The original purpose of this investigation was to learn whether the increased adrenal secretion accompanying major emotional states and pain ®° might act as a dynamogenic factor in the perform- ance of muscular work. Apart from the increase of arterial pressure in conditions of low pressure, the change wrought by adrenal secre- tion on the efficiency of muscle is too slight to account for the feats of strength which are performed in times of great excitement. The main source of power under these conditions is probably to be found in an immensely augmented activity of the nervous system. The observations here recorded, however, indicate that adrenalin may operate favorably in making more effective the nervous impulses delivered to fatigued muscles. SUMMARY If the tibialis anticus muscle, stimulated through its isolated nerve, is writing a fatigue curve, excitation of the left splanchnic nerves, also isolated, usually produces an increased height of contraction in the muscle record. (See Figs. 2 and 3.) Since splanchnic stimu- lation discharges adrenal secretion, the question is raised as to the effect of adrenal secretion on skeletal muscle. The betterment of action of the fatigued muscle is mainly due to the increased blood flow resulting from splanchnic stimulation, and is more marked the lower the blood pressure when the splanchnics are excited. (See Fig. 5.) The betterment of the muscular con- traction may long outlast the change in the circulation. Probably most previously reported effects of adrenalin on skeletal muscle (observed in cold-blooded animals with low blood pressure) should be attributed to the change in circulation and not to a specific action of adrenalin. If, by pull on the aorta or by compression of the thorax, blood 2% See CANNON and DE LA Paz: this Journal, 1911, xxvili, p. 64; CANNON and Hoskins: this Journal, 1911, xxix, p. 274. 60 W. B. Cannon and L. B. Nice pressure in the hind legs is prevented from rising, splanchnic stimu- lation still causes a slight but distinct rise in the height of contraction of the fatigued muscle. This betterment may appear in rhythm or altered rhythm. (See Fig. 6.) Its initial appearance coincides with evidence of adrenal secretion. It is produced also when adrenalin in weak solution (1: 100,000) is given intravenously. (See Fig. 7.) The improvement of muscular contraction which apparently results from adrenal secretion (when the blood pressure is controlled) is too slight to account for the increased muscular power observed during excitement. Fatigued muscles may, however, be thus pre- pared, by secretion of the adrenal glands, for better response to the demands of powerful nervous discharges. THE RECEPTIVE RELAXATION OF THE COLON By HENRY LYMAN [From the Laboratory of Physiology in the Harvard Medical School] HAT the cardiac sphincter is relaxed as food is started toward it in the oesophagus was shown by Kronecker and Meltzer in 1883.1 In 1g11, Cannon and Lieb found that if the stomach is tonically contracted when food is swallowed, the tonus is momentarily abolished by vagus impulses, at a time when the swallowed bolus would be delivered by the oesophagus.” Thus muscles which would otherwise be opposed in action are made to cooperate reciprocally. A similar relation was proved by Joseph and Meltzer to exist between the stomach and intestine, — inhibition of contractions of the duo- denum in the rabbit occur coincident with peristalsis of the pyloric portion of the stomach.* Might not the same mutual relation be present between the ileum and proximal colon? Cannon noted in his first observations of the movements of the intestines that “as food is nearing the ileocolic valve the large intestine is usually quiet and relaxed,” and that contraction near the valve disappears “just previous to the entrance of the food.’’* Since the characteristic activity of the proximal colon is anastalsis, large and small intestine would be set in direct opposition if that activity continued while the small intestine discharged material through the ileocolic valve. The relation between these neighboring parts of the alimentary canal seemed worthy of further study. In the present study the intestines were observed directly. The animal (cat )was anaesthetized with urethane (2 gm. per kilo body- weight), and in occasional instances when the corneal reflex returned ether was used in addition. After a tracheal cannula was _intro- 1 KRONECKER and Me ttzer: Archiv fiir Physiologie, 1883, Supplement Band, p. 358. 2 CANNON and Lies: this Journal, rg11, xxvii, p. xiil. 3 JosePH and MEtrTzer: this Journal, 1ort, xxvii, p. Xxxi. 4 CANNON: this Journal, 1902, vi, p. 267. 62 Henry Lyman duced, the spinal cord was pithed from the sacrum to the lower thoracic region (to remove the inhibitory effect of splanchnic influ- ences), and the opening in the skin closed with sutures. Eserine salicylate (gr. ¢;) was given subcutaneously, and, if necessary, the dose was repeated in two hours. Half an hour after the lower cord was pithed the abdomen was opened, bleeding points were clamped or tied off, and the animal then placed in a bath of physiological salt solution at 38° C. A glass tube with a lumen of 8 mm. was tied into the rectum so that defecation might occur without spoiling the bath. To excite movements of the small intestine when food was not present in it warm starch paste, colored with methylene blue and rendered more stimulating by the addition of yellow soap, was injected through the wall by means of a large hollow needle. If natural digestion was in process the gut reacted well to the presence of the paste-soap mixture, but if the tract was empty the injected material was quite without effect.» The mixture was not introduced through the wall of the colon because injury to this part of the tract seemed to affect unfavorably its motions. If digestion was not in process when the abdomen was opened, I5 ¢.c. 79 HCl was poured into the stomach through a tube in the oesophagus, and this was followed by 100 c.c. of warm milk. Natural gastric peristalsis would then begin, the contents would be discharged, and the course of the food along the intestinal canal could be clearly observed. From two and a half to three hours were usually taken for material to reach the ileocolic junction. In one case, however (a cat with diarrhoea), the entire process was complete, and the whole tract, including the colon, was empty in an hour and twenty-four minutes.® Observations on the relations of activity in the ileum and in the colon were as follows: ° Cf. Macnus: Archiv fiir die gesammte Physiologie, 1904, cil, p. 130. ® Activity of the upper part of the gastro-intestinal canal, when the abdomen is opened, stimulates the large gut to empty itself. In this process the first change is the drawing down of the distal part of the large gut into the pelvis. Then a strong katastaltic wave, starting in the caecum and usually traversing only the proximal third of the colon pushes material into the distal two thirds where katastalsis is the usual activity. After one or two such waves from the caecum defecation occurs if that is possible. Cf. CANNoN: The mechanical factors of digestion. London and New York, 1o11, p. 161. - i The Receptive Relaxation 63 When material was being driven through the ileocolic sphincter by peristalsis in the ileum, the colon, which a moment before had been in tonic contraction and exhibiting anastalsis, became motion- less and quite relaxed. As soon as the process was finished and the ileum was again quiet, anastaltic waves again appeared in the colon (see Fig. 1, A, Band C). In fourteen cats which were studied in this manner this reciprocal relation between the activities of ileum and colon was repeatedly noted.’ In one case only was there an exception; the activities of the colon in this instance consisted in strong contractions directed towards Ficure 1. Photographs of the intestines, taken with flash-light. A. Anastalsis on the colon at 12: 22: 27. Ileum relaxed. Time recorded by submerged watch. B. At 12: 25: 28 colonic anastalsis stopped asa peristaltic wave in the ileum is pushing material into the colon. Note the pallor of the ileum. C. At 12: 26: 35. Strong anastalsis on the colon again, and inactivity of ileum. defecation, alternating with vigorous anastalsis, and no material was pushed onward from the ileum. The rectal tube was afterwards found to be plugged. When the contents of the colon were gaseous, and when the colon had very little solid or semisolid material in it, receptive relaxation did not always occur. The colon was not inhibited by mere presence of food in the lower ileum, for segmentation was often noted in the small gut close to the 7 Destruction of the spinal cord did not abolish the tone of the ileocolic sphincter in the animals observed in this investigation, for the anastaltic waves never forced material from the colon into the ileum. ‘This result does not agree with Elliott’s observations on the rat (Journal of physiology, 1904, xxvi, p. 166), but his animals were examined several days after the lower cord had been destroyed. 64 Henry Lyman sphincter while active anastalsis continued in the colon. Moreover, when the small gut was clamped next the sphincter with rubber- tipped forceps, and the lower ileum distended with starch paste, ana- staltic waves kept running over the proximal part of the large gut without interruption. And when a cotton swab, coated with vaseline, was pushed up to the sphincter no inhibition of the activities of the colon resulted; but when the swab was forced through the sphincter the colon at once relaxed to become active again as soon as the swab was withdrawn. This effect could be repeated several times. Since the receptive relaxation of the colon occurred in the absence of nervous connections with the spinal cord, the mechanism control- ling it is local, probably as the relation of the pyloric part of the stomach to the duodenum is local. It is another instance of the reciprocal innervation of opposed muscles. REMARKS ON THE ORIGIN OF THE PHRENIC NERVE Dyin RABBIT, CAT, AND DOG By ABBY H. TURNER [Fom the Laboratory of Comparative Physiology in ithe Harvard Medical School] N an experimental study! of crossed respiration it was necessary to sever and to stimulate the phrenic nerve in the neck of the rabbit, cat, and dog. Complete section and adequate stimulation present some difficulty because variations in the origin of the nerve leave the observer sometimes in doubt whether he is dealing with the whole nerve or only part of it. Systematic dissections were therefore made at Dr. Porter’s suggestion, to learn the frequency and char- acter of these variations and to determine the best place for cutting the nerve and for stimulating its central end. THE RABBIT In making the dissection from the ventral side of the rabbit’s neck the ventral rami of the fourth, fifth, and sixth cervical nerves are found beneath the external jugular vein and the sterno-mastoid muscle. The ventral rami of the seventh and eighth cervical and the first thoracic nerves appear well beneath the pectoral muscles and the union of the external jugular, cephalic, transverse-scapular, and axil- lary veins. Since the purpose of the dissection was physiological the points of origin of the phrenic roots and the site of their union to form the main phrenic stem will be mentioned as they would appear were the dissection made in the living animal without injury to the nerves, not as they might be found to be after a final separation of all the minute connective tissue strands. Different individuals and the two sides of a single animal may vary in the number of phrenic roots and in their place of union. 1 Porter, W. T., and Assy H. Turner: To be published in the next issue of this Journal, June 1, 1913. 66 A. H. Turner There are usually three roots (Fig. 1), one each from the fourth, fifth, and sixth cervical nerves, but there may be two or four. When only two roots were found they were from the fourth and the fifth cervical nerves. The root from the fourth nerve is slender and leaves the ven- tral ramus at or before its appearance from the deep neck muscles. It passes backward over the fifth nerve and may go as far as the eighth before being joined by other roots, although it usually unites with the fifth root be- tween the fifth and sixth nerves. The fifth phrenic root leaves the fifth nerve with the ansa uniting the fifth and sixth nerves, from which it is wont to sepa- rate to join the fourth root nearer the sixth than the fifth nerve. This fifth root is often though not always the largest Hae So phrenic component. The sixth phrenic to < root presents the greatest variation as it may be single or double, long or short. Ficure 1. The right phrenic nerve in a rabbit with fourth, fifth, and “long” sixth root. The central ends of five cervical nerves are marked 4, 5, 6, 7, When it leaves the sixth cervical near the latter’s place of appearance be- tween the muscles it will be called and 8, respectively; (1) marks the first thoracic nerve. The subdivisions of the nerves are incompletely indicated. PA, phrenic nerve; Ax, axillary vein; EJ, external jugular vein; M, manubrium; R, car- tilage of first rib. “short” because in that case it typi- cally unites at once with the other roots. When it leaves the sixth cervical a cen- timetre or more farther out it will be called “long” because in that case its course is usually a _ loop, laterally extended, across the seventh nerve, and its union with the other phrenic roots is as far or farther back than the seventh nerve. In some cases both short and long roots were found. E Any union of the phrenic roots further back than the sixth cervical presents difficulty to the operator because of the large overlying veins. The variation in origin and course of the roots makes it essential that all roots be carefully identified to insure complete section or stimula- tion. In all cases, except in the few instances where only two roots occur, the length of nerve between the final union of phrenic roots and the nerve’s entrance into the thorax is short, rarely if ever more Phrenic Nerve in the Rabbit, Cat, and Dog 67 than 1.5 cm. and frequently only a few millimetres. In stimulating the nerve, therefore, the danger of escape of current to the nearby brachial plexus is obvious and a pure phrenic effect is rendered less sure. As a result of these dissections it seems advisable to use the nerve in the thorax rather than in the neck whenever possible. If opening the thorax is undesirable, the safest place to identify the phrenic is under the axillary vein as close as possible to the entrance of the nerve into the thorax. Tables I and II afford a survey of the variations in the rabbit. TABLE I ORIGIN OF PHRENIC NERVES IN THIRTY-THREE RABBITS Cervical nerves from which nerve originates |Right Left Atheoth woth (Shorbroot)) =. 5 4. 5. . : 11 9 4th, 5th, 6th (long root) Ee eae MUST! «4%, 15 /11 (+ 1?) 4th, 5th, 6th (both short and long roots) .. 2 |4(4+1?) aia. Suilet. 2. SE cy ieee em eae at ee 5 5 ANiln., Gila: (Gil Dy aeeiae eee ae eile gees raul eae 1 4th, 5th, 6th (twolong roots) . . . . . me 1 THE CAT Seven dissections of the origin of the phrenic in the cat were made. In all except one case the origin was from the fifth and sixth cervical nerves. In the one exception, a left nerve showed also a small root from the fourth cervical. In ten of the fourteen nerves the roots united at about the seventh cervical nerve, somewhat beneath the large veins, but furnished a piece of nerve anterior to the thorax long enough for safe stimulation after it was carefully freed from the neighboring brachial plexus. In one case the two roots united well down inside the thorax opposite the second rib, and in another case opposite the third rib, but in both these instances the roots were parallel and adjacent. In two cases however, the fifth root ran 68 A. H. Turner TABLE II Praces oF UNION oF Roots OF PHRENIC NERVES IN THIRTY-THREE RABBITS 4th root joins 5th between Sth and 6th cervical 4th root joins 5th at or near 6th cervical 4th root joins 5th between 7th and 8th cervical 4th root joins 5th and 6th roots at 6th cervical or anterior to 7th cervical 4th root joins 5th and 6th roots at 7th cervical or beyond 4th and 5th roots join 6th root at 6th cervical or anterior to HED KCervical es ee ee eee ee 2 4th and 5th roots join 6th root at 7th cervical or beyond . | 138 (+17) 4th, 5th, and 6th roots unite at 6th cervical . . . . . 1 4th, 5th, and 6th roots unite at 7thcervical . . . . . chal 4th, 5th, and 6th roots unite at 8th cervical 5th root joins 6th root at or near 6th cervical . 5th root joins 6th root at 7th cervical 5th and short 6th roots join 4th and long 6th roots at 7th cervical Full face figures call attention to instances where a union of roots occurs at or posterior to the 7th cervical nerve. Total, 42 (+ 3 ?). dorsal to the subclavian vein as usual while the sixth root was ventral to it, a modification unexpected and difficult for the operator. The union of the two roots was within the thorax in these cases. THE Doc Two dissections only were made. All four nerves took their origin from the fifth, sixth, and seventh cervical nerves. The place of final union varied from the level of the seventh nerve to that of the first Phrenic Nerve in the Rabbit, Cat, and Dog 69 rib within the thorax, and in all cases careful exploration beneath the large veins in the neck would have been necessary to insure the use of all roots of the nerve anterior to the thorax. CONCLUSION As a result of these dissections it is advised that the phrenic nerve whenever possible be severed or stimulated in the thorax rather than in the neck. ON THE RELATION OF PULSE PRESSURE TO RENAL SECRETION By ROBERT A. GESELL [From the Physiological Laboratory of the Washington University] BSERVATIONS concerning the relation of blood pressure to the activity of the kidneys are numerous, but the relation of pulse pressure to renal secretion has but rarely been noted. Erlanger and Hooker! in their observation of blood pressure in man noted a relation which was as follows: 1. As a rule the amount of urine secreted varied directly with the magnitude of the pulse pressure. ; 2. In a case of orthostatic albuminuria the amount of albumin in the urine varied inversely with the magnitude of the pulse pressure. 3. The amounts of urea, chlorides and phosphates secreted in the urine varied directly with the magnitude of the pulse pressure. More recently Hooker? has again investigated this problem. By means of a specially devised pump he studied the effects of variation of the magnitude of the pulse pressure upon the perfused, isolated kidney of the dog. He obtained the following results: rt. With a constant mean perfusion pressure the amount of urinary filtrate varied directly as the magnitude of the pulse pressure. 2. With a constant mean perfusion pressure the amount of protein in the urinary filtrate varied inversely as the magnitude of the pulse pressure. 3. With a constant mean perfusion pressure the rate of blood flow through the organs varied directly as the magnitude of the pulse pressure. 1 ERLANGER and Hooker: Johns Hopkins Hospital reports, 1904, xii, p. 346. ® HOOKER: This journal, 1910, xxvii, p. 24; Hooker: Archives of internal medicine, 1910, v, p. 491; HOOKER, HEGEMAN and ZARTMAN: This journal, 1900, XXiil, p. Xi. Relation of Pulse Pressure to Renal Secretion 71 Unfortunately the conditions in man, owing to the difficulty of controlling the pulse pressure and of obtaining at the same time accurate data concerning mean blood pressure and volume flow through the kidneys, do not allow of a finer study of the relation of pulse pressure to renal activity. The method employed by Hooker offers rather great technical difficulties, subjects the kidneys to abnormal conditions, and intro- duces two variable factors at one time,—the rate of blood flow through the kidneys varying directly with the magnitude of the pulse pressure. A method by which the pulse pressure can be altered at will in the animal is of course the ideal method. Such a method was used in the present research. The principle of air compression was em- ployed. An air chamber under mean blood pressure was connected indirectly with one or both renal arteries and the magnitude of the pulse pressure controlled by varying the size of the air chamber. Various modifications of this general procedure were employed. Each will be considered in detail in its proper place. Dogs were used in all the experiments. They were anaesthetized with morphine and ether. The kidneys were left intact and were not manipulated. THE EFFECTS OF THE AIR CHAMBER UPON PULSE PRESSURE The pulse curve represents a series of pressure changes lying between diastolic and systolic pressure occurring during each cardiac cycle and may be altered in three ways by the method used for diminu- tion of the pulse pressure. There may be alterations in the time rela- tions of pressure changes, diminution of the suddenness of pressure changes, and diminution of the magnitude of pressure changes. In this research we are studying then the effects of changing three variable factors at one time. It is of interest to determine the rela- tive importance of these factors, if possible, and therefore attention is called to them at this point. Evidence will be brought out to show that probably the magnitude and suddenness of pressure changes are of importance and therefore when future reference is made to the effects of diminution or change of pulse pressure the suddenness of pressure changes should always be kept in mind. Robert A. Gesell ~I iS) THE RELATION OF PULSE PRESSURE TO MEAN BLooD PRESSURE AND VOLUME FLOW oF BLooD Numerous investigators? have noted the beneficial effect of an intermittent perfusion pressure upon isolated organs not only for the maintenance of the normal condition of the tissues but also for the rate of perfusion through the tissue. Since the normal condition of tissues and volume flow of blood as well as the mean blood pressure are important factors in secretion, the relation of altered pulse pres- sure to these factors was studied in a few preliminary experiments. A dog was prepared on a warm table. Its temperature was maintained at the normal level throughout the experiment. A means of injecting blood at a constant pressure. was arranged. The abdomen was opened and the gastro-intestinal tract removed in order to gain free access to the renal arteries and veins. By an arrangement shown in Fig. 1 the pulse pressure was eliminated in one kidney without altering the pulse pressure in the other. Cannula A was connected with the inferior mesenteric artery H, cannula B with the lower end of the abdominal aorta K, and A and B connected by a tube with two side branches F and J. Branch F was connected with the air chamber J, and communication between the two broken or established by pinch cock G. A manometer to record the blood pressure in the left renal artery was connected to the tube J. The whole system was filled with Ringer’s solution to the exclusion of all air bubbles. The tension of the air in J was increased to mean blood pressure. Ligature Fie. 1. 3 HooKER: Loc. cil.; SOLLMANN: This journal, 1905, xiil, p. 241; BRODIE: Journal of physiology, 1903, xxix, p. 267; HAMEL: Zeitschrift fiir Biologie, 18809, xxv, p. 274; HOFFMANN: Archiv. fiir die gssammte Physiologie, 1903, C, p. 242. Relation of Pulse Pressure to Renal Secretion 73 E, which lies on the aorta between the left and right renal arteries, was tied, thereby shunting the blood for the left renal artery out of its normal course, through the inferior mesenteric artery, through the tubes up the abdominal aorta to the left renal artery. The blood for the right kidney followed its normal course. By this arrangement it was possible to eliminate the pulse pressure in the left kidney without altering blood pressure conditions in the right kidney. The volume flow of blood from the right and left kidneys was measured directly by means of two tipping buckets. The arrangement of cannulae is shown in Fig. rt. One cannula Z was connected with the inferior TABLE I Mean blood Volume flow of pressure in blood in c.c. mm. Hg. per minute Right or left kidney Period no. PAPe | eee 100 100 88 85 vena cava directly above and another cannula directly below the renal veins. Ligature P, which lies on the inferior vena cava between the renal veins was tied and the blood from each kidney was led into separate tipping buckets Q. In this procedure the circulation of the kidneys was not interrupted for a moment on inserting either the arterial or venous cannulae. The blood was collected from the tip- ping buckets, defibrinated, and continually replaced by other defibri- nated blood, through the external jugular vein. The mean blood - pressure was maintained at any desired level by regulating the rate of inflow of blood. The magnitude of the pulse pressure was per- fectly controlled by regulating the size of chamber J. Figs. 3, 5, and 6 show effects which the air chamber exerted on 74 . Robert A. Gesell the pulse pressure. The difference in effect is due largely to the size of the air chamber employed. Returning to the relation of pulse pressure to mean blood pressure and volume flow of blood; it was found with the arrangement shown in Fig. 1 that it was possible to diminish the pulse pressure in the left renal artery without diminishing the pulse pressure in the right renal artery. When this was done the mean blood pressure remained practically constant. The volume flow of blood from each renal vein showed practically no change. Some of the data obtained are tabu- lated on page 73. These results seem to be opposed to the results of numerous investi- gators, but the fact that the data so far collected were obtained from perfusion of isolated organs and tissues under more or less abnormal conditions probably accounts for the difference in findings. With the kidneys intact, diminution of pulse pressure over long periods of time had, in the present experiments, very little effect upon the kidneys. In some experiments no change was noted. THE RELATION OF ALTERED PULSE PRESSURE TO RENAL SECRETION AND FURTHER DATA CONCERNING MEAN BLOOD PRESSURE AND VOLUME FLOW OF BLOOD With the above important data at hand the additional study of the relation of pulse pressure to renal secretion was attempted by a simplified method. The pulse pressure was altered simultaneously in both kidneys and the outflow of blood from both kidneys measured by one tipping bucket. The dogs were prepared as previously described, but a new arrange- + re PP. wy em ee em ee Relation of Pulse Pressure te Renal Secretion 75 ment shown in Fig. 2 for varying the magnitude of the pulse pressure was employed. A large cannula B was inserted into the abdominal aorta directly below the renal arteries. Blood pressure was recorded by one or both manometers G from side tube EZ. B was connected to a large air chamber which consists of a glass tube J and a rubber tube Z one inch in diameter and six feet long. The pressure in this chamber was raised to mean blood pressure by means of a rubber bulb K. The amount of diminution of the pulse pressure was regu- lated by adjusting the size of the chamber. The blood pulsates between the dotted lines MN and OP, and there the pulse pressure ELE oa “}-——}-- 4} — } ——--4-—- ai Oe Bee Ba sha ban Ane Bhs | ir — aaah WD. iain i ak Pk bd RA P| Da eh bear Ti Spe pretn AB. 4C. FicurE 3. About one half the original size. 45. Period of normal pulse pressure. 4c. Period of diminished pulse pressure. B.F. Volume flow of blood from both kidneys recorded, by tipping buckets. 125 cc. per minute in periods 4) and 4c. M. Mean blood pressure recorded by mercury manometer connected with tube £. 97 mm. Hg during periods 4b and 4c. #. Pulse pressure recorded by Hiirthle manometer connected with tube &. TJ. Time in seconds. is diminished. By recording blood pressures from E, D, and Q respectively it was found that the degree of elimination of pulse pressure in a rough way varied inversely with the distance from the point of elimination. The changes of magnitude of pulse pressure occurring in the renal arteries amount to about half those recorded by the Hiirthle manometer G. The blood pressure records are therefore used only as an index of the altered condition of pulse pressure in the renal arteries. As in the preliminary experiments important data were obtained 76 Robert A. Gesell concerning the relation of the pulse pressure to mean blood pressure and volume flow of blood through the kidneys. Fig. 3 shows that the volume flow of blood and the mean blood pressure were not changed by a diminution of pulse pressure by the method employed. The mean blood pressure was recorded with a mercury manometer and the magnitude of the pulse pressure by a Hiirthle manometer — both connected at E. On decreasing the magnitude of pulse pressure the volume flow of blood (125 c.c. per minute) and the mean blood pressure (97 mm. Hg) remained abso- lutely constant. Similar results were repeatedly obtained and are tabulated in Table II which is of interest in that it shows that a fall of mean blood pressure occurring with a diminution of pulse pressure may be accompanied by a slight increase in volume flow of blood. TABLE II Mean blood pressure Volume flow of blood Period no. in mm. Hg from both renal veins in c.c. per minute 1 ee Ba Pe 126 The uniformity of results leaves no doubt concerning the rela- tion of altered pulse pressure to mean blood pressure and volume flow of blood. Therefore if a change in renal secretion accompanies altered blood pressure that change must be ascribed to some specific effect of pulse pressure itself, whatever that may be. Fig. 4 shows a diminution of urine flow accompanying a diminu- tion of pulse pressure, even though the mean blood pressure and the volume flow of blood remain unchanged. Unfortunately the blood pressure was recorded from the carotid artery by a mercury manome- Relation of Pulse Pressure to Renal Secretion ~lI ae | ter and consequently does not show any pulse pressure changes. But previous experiments warrant us in assuming that the pulse pressure diminished on connecting the air chamber at the point indicated by the arrow head. The mean blood pressure remained at 122 mm. Hg throughout. The volume flow of blood (156 c.c. per minute) was unchanged by decreased pulse pressure. The urine flow was markedly decreased. This decrease must have been caused by the influence of pulse pressure itself. FURTHER DATA FROM SIMPLIFIED METHOD CONCERNING THE RELATION OF ALTERED PULSE PRESSURE TO THE RATE OF RENAL SECRETION It was desirable to obtain for analysis samples of urine over long periods of varying conditions of pulse pressure. This seemed impossi- u" i inl Mi It I2 Ficure 4. About one half the original size. x11. Period of normal pulse pressure. 12. Period of diminished pulse pressure. R. U. Urine flow in drops from right kidaey. ZL. U. Urine flow in drops from left kidney. M. Blood pressure in carotid artery registered by a mercury manometer. 122 mm. Hg during periods 1rand 12. B.F. Volume flow of blood recorded by tipping buckets. 156 cc. per minute during periods 11 and 12. ble because of the difficulties in the preceding method in maintaining constant vascular conditions. The procedure was therefore sim- plified still more by leaving the gastro-intestinal tract intact. The blood was no longer defibrinated but the glass portion of the air cham- ber partially filled with a dilute solution of hirudin. Since all the findings of the previous methods concerning the relation of pulse S 5 pressure to volume flow of blood and to mean blood pressure were constant it seemed justifiable to omit direct measurement of volume flow. But to be doubly assured that the same relations would hold Co 7 Robert A. Gesell under the new conditions the velocity flow of blood was measured in the inferior vena cava and used as an index to the volume flow through the kidneys. A thermo-electric method devised by Dr. Erlanger was employed. A delicate thermo-electric junction in cir- cuit with a d’Arsonval galvanometer was placed in the inferior vena cava at the base of the heart. Equal quantities of Ringer’s solution at room temperature were injected into the inferior vena cava directly below the renal veins and the time between the injection and the deflection of the galvanometer was used as an index to the velocity flow of blood. Numerous observations were made upon three dogs but no appreciable change in rate of blood flow was found under vary- Lt eo] Fe Jel J Ba Ep Eee Sfawae rela koe lead ee tal eb tet eo ot H. Ah : ie abi aH aoe LO ae aaa T. GJ 7 HH nH srt r HH CR eee RiE Es CSS SOR ERIE EEREM LBL OR OASIS LASS SSI IR IR SEES OSE L ees ALI RIREEIALEGALELELEAIRIATAARAELEEAATA EA I 2 3 4 FicurEe 5. Four sevenths the original size. LZ. U. Urine flow in drops from left kidney. (The right kidney was not secreting). H. Blood pressure recorded by Hiirthle manometer connected with tube £, showing effects of connecting the air chamber to the aorta. 1 and 3 normal pulse pressure of 60 mm. Hg. 2 and 3 diminished pulse pressure of 25 mm. Hg indicating a pulse pressure of 45 mm. Hg in the renal arteries. YZ. Time in seconds. ing conditions of pulse pressure. Therefore in the future experi- ments no determinations of blood flow were made, on the assumption that if velocity changes accompanied altered conditions of pulse pressure, they were too small to obscure effects of pulse pressure itself upon renal secretion. To produce diuresis a mixture of one part of 4 per cent sodium sulphate and three parts of Ringer’s solution was slowly injected at a constant pressure throughout the experiment. Figs. 5-8 show in general that the secretion of urine was stopped or diminished by a decreased pulse pressure; that with nor- mal pulse pressure there was a subsequent recovery. But the sudden- ness and the amount of diminution and the rate of recovery varied with different animals and with the same animal at different times. Relation of Pulse Pressure to Renal Secretion 79 A copious flow of urine may be abruptly stopped by a diminution of pulse pressure, as shown in Fig. 5. There was a mean blood pres- sure of 70 mm. Hg. A normal pulse pressure of 60 mm. Hg was reduced to 25 mm. Hg at tube £, which indicates a pulse pressure of approximately 45 mm. Hg in the renal arteries. This relatively small change in pulse pressure was sufficient to quickly stop the urine flow and hold it in check until the kidneys were again subjected to normal pulse pressure. Then recovery was prompt. The secretion of urine may only gradually be decreased by a diminu- tion of pulse pressure as shown in Fig. 6. In this case we have a slightly different state of affairs. There was a relatively higher mean 0 L. U. PEATE RETA EE TET PEAT ETT TTT ETE TET TA I 2 3 4 Ficure. 6. About four sevenths the original size. L. U. Urine flow in drops from left kidney. (The right kidney was not secreting). H. Blood pressure recorded by Hiirthle manometer connected with tube E, showing effects of connecting the air chamber to the aorta. 1 and 3 normal pulse pressure. 2 and 4 diminished pulse pressure. TJ. Time in seconds. pressure and a relatively greater decrease in pulse pressure. The secretion was only gradually diminished, not stopped by a diminu- tion of pulse pressure. There was a rapid recovery with normal pulse pressure. Fig. 7 shows the prolonged effect on secretion of a short period of diminished pulse pressure. An abundant flow of urine was stopped completely in twelve seconds. Recovery was very gradual and was not complete at the end of two and a half minutes. In other cases a short period of diminished pulse pressure stopped the secretion as long as an hour. This is of theoretical importance. It points out the deleterious effect of a constant pressure as well as the beneficial effects of a pulsatile pressure. The influence of pulse pressure upon renal secretion may be masked by conditions tending to increase the rate of secretion. For 80 Robert A. Gesell PTET eee ttt ~ ~ = a — = — =_— = a= = os zm = I = = eo = BS = = _— 3 = = ~~ D BS t instance in one experiment the rate of secretion at the beginning of the experiment was 16 and 18 drops re- spectively from the right and left kidney. Two hours later the rate was 41 and 24 drops respectively. Dur- ing the gradually increased rate a diminution of pulse pressure had no noticeable effect upon renal secretion. Later on, however, when the flow of urine had be- come constant the effect of decreased pressure, al- though slight, was demon- strable. _ In general, as shown in Figs. 4-7, the rate of secretion varied directly with the magnitude of the pulse pressure. In marked contrast to this are the results shown in Fig. 8, which indicate that in addition to magnitude of pulse pressure the sudden- ness of pressure changes may be a very important factor in renal secretion. It was found in certain instances that on connect- ing the air chamber with the aorta, the magnitude of the pulse pressure was unchanged, slightly dimin- ished or even increased Ficure 7. R. U. Urine flow in ‘ d co drops from right kidney. L. U. z = Urine flow iri drops from left kidney. H. Blood pressure recorded by Hiirthle manometer connected with tube EZ. 1 and 3. Periods of normal pulse pressure. 2. Period of diminished pulse pressure. J. Time in seconds. Relation of Pulse Pressure to Renal Secretion 81 and yet in every case a copious secretion was abruptly stopped, and held in check until the kidneys were again subjected to normal pulse pressure. In a specific instance: During normal pulse pressure there was a rapid flow of urine. Connecting the air chamber probably re- duced the pulse pressure 1 or 2 mm. Hg in the renal arteries, but raised the mean pressure a few millimetres. The secretion of urine was stopped by some influence of the air chamber. It was held in check for over three minutes. Recovery of normal secretion was prompt on subjecting the kidneys to normal pulse pressure. Of still greater interest are the results shown in Fig. 8, in which the magnitude of the pulse pressure was increased 15 mm. Hg A | B | c | D | E Ficure 8. Four sevenths the original size. Z.U. Urine flow in drops from left kid- ney. H. Blood pressure recorded by Hiirthle manometer connected with £. a, c, e. Periods of normal pulse pressure. 0, d. Periods of altered (increased) magni- tude of pulse pressure produced by connecting the air chamber with the aorta. on connecting the air chamber to the aorta. Whenever this occurred a rapid flow of urine was suddenly stopped and held in check until normal pulse pressure was again allowed to act upon the kidneys. It will be noted that although the magnitude of the pulse pressure may be unchanged, the form of the pulse curve is materially altered. The normal pulse curve is composed of sudden pressure changes. There is a sudden rise of pressure followed by a sudden fall in pressure which is abruptly checked and momentarily held in check at the di- crotic notch, there-giving way to another sudden fall. Compare this with the pulse curve obtained by the Hiirthle manometer when the air chamber is connected with the aorta. The most marked differ- ences are the relatively gradual rise and fall of the limbs, the rounded apex and especially the absence of the dicrotic notch. In other words, 82 Robert A. Gesell compared with the abrupt and rugged normal pulse it is a smooth and swinging pulse. The shape of the pulse curve, the suddenness of pressure changes,— vascular shocks may be very important factors in secretion. Their significance will be considered farther on. THE RELATION OF PULSE PRESSURE TO QUALITATIVE CHANGES IN THE URINE Urine for analysis was collected in four experiments during which no appreciable changes of mean blood pressure accompanied manipu- lation of the pulse pressure. The urine was collected during ten- minute periods; a period of normal pulse pressure alternating with a period of altered pulse pressure. The amounts of chlorides, total nitrogen, urea nitrogen, and albumin eliminated were determined. For determination of chlorides the Harvey* modification of the Volhard method was used; for total and urea nitrogen Folin’s® microchemical method; for albumin the heat and acid test, using the amount of precipitate as an index to the amount of albumin present. Tables III, IV, and V give data obtained from analyses of urine from the four experiments mentioned. In Table III A the rate of flow of urine was not registered by a drop recorder. The urine was caught in test tubes. The flow during period 2 (normal pulse pressure) was found to be 15 per cent faster than during periods 1 and 3 (altered pulse pressure). The amount of urea nitrogen eliminated per cubic centimeter of urine was prac- tically the same in all the periods. But on account of the greater elimination of urine during period 2, the absolute amount of urea nitrogen eliminated during the period of normal pulse pressure was greater than during the periods of diminished pulse pressure. The amount of chlorides eliminated during the period of normal pressure was both relatively and absolutely greater than during the periods of diminished pulse pressure. The amounts of albumin eliminated during periods 1 and 3 appeared to be in excess to that eliminated in period 2. Table III B shows analyses of urine gathered in 4 ten minute periods in another experiment. Unfortunately the amount of urea 4 EMERSON: Clinical diagnosis, 3rd edition, 1911, p. 137. 5 FoLIn: Journal of biological chemistry, 1912, xi. Relation of Pulse Pressure to Renal Secretion 83 and total nitrogen eliminated per cubic centimeter of urine gradually diminished as the experiment progressed. This may illustrate the deleterious effect of diminished pulse pressure upon renal epithelium, but was disconcerting in interpreting the results, in that it obscured the momentary effects of pulse pressure changes. Table III B therefore shows no perceptible relation between pulse pressure and TABLE III Chlorides Percentage in G..per | decrease or 5c.c. of | increase of urine chloride Urea N. in G. | Percentage per c.c. of decrease of urine urea N. Pulse Albumin pressure Number trace + trace trace + trace trace + trace trace + the elimination of urea. In contradistinction to the progressive decreased elimination of urea, the chlorides show a progressive in- creased elimination. Yet the effects of diminished pulse pressure during periods 2 and 4 change this progressive tendency of increased elimination to actual decreased elimination amounting to 30 and 10 per cent respectively. Compare this with period 3 (normal pulse pressure) during which there was a percentage increase of chloride elimination amounting to 64.5 per cent. Albumin was eliminated during all periods but to a slightly greater extent during periods of diminished pulse pressure (2 and 4). 84 Robert A. Gesell Table IV shows analyses of 8 samples of urine from another experi- ment. Samples 4, 5,6 and 7 were collected during the first part of the experiment and samples 18, 19, 20 and 21 two hours later. Again as in the preceding experiment, the elimination of nitrogen progressively diminished, the greatest percentage decrease occurring at the beginning of the experiment. The momentary effects of varied pulse pressure on nitrogen elimination therefore were again masked. TABLE IV Mean pressure in mm. Hg chlorides Total N. in G. per c.c. of Percentage increase in Rate of urine flow from Percentage decrease of Chlorides in G. per c.c. Rk. & L. kidney oO ~ | Pulse pressure in mm. Hg oO ~—“- SE Ae me eird ~The ~I S (=) S Tt=) a on _—_ = MIO On ors ——s —“— Pi PR PR oe — ——. As in Table III B the chlorides showed a tendency of progressive increased elimination, marked during periods of normal pulse pressure. The elimination of chlorides increased only during periods of normal pulse pressure. During periods of diminished pulse pressure it remained the same as in the preceding period of normal pulse pressure. TABLE V aqnutur sad sdosp = jo Jaquinu osei0ay a = Ay oy teh BSS GS We GSS Ne ON t= Ww iW -_ _ _ —_ bl ba | — _ aynur iad sdoip jo Joquinu aSRIVAYV aynurut 19d sdorp 3 : : as jo Jaquinu a8viaay a” eee DB eee 13:3 a Ay aynurut rad sdoap ae aren en GP jo taquinu asei0Ay SSE py AY aynurut rad sdoip jO Joquinu osRIDAY 86 Robert A. Gesell Although the experiment shows no actual decrease of chlorides in the urine eliminated during the periods of diminished pulse pressure, it shows that a gradual tendency to increased elimination of chlorides can be effectually checked by subjecting the kidneys to diminished pulse pressure. Tables V and VI give data from the last successful experiment. The urine was collected over 5 periods — 4 periods of diminished pulse pressure and 1 period of normal pulse pressure. The pulse pressure was diminished slightly and to the same extent in periods 3 and 4, was normal during period 5 and again diminished to the same extent during periods 6 and 7. Table V gives the number of drops of urine secreted per minute throughout the experiment. It shows very beautifully the gradual development of a deleterious action of diminished pulse pressure during periods 3 and 4, and 6 and 7, also the gradual recovery from this effect during period 5 — of normal pulse pressure. Table VI gives the chemical analyses of the samples of urine collected during the five periods. In this experiment the tendency to progressive decreased elimination of nitrogen was not so marked as in the two preceding experiments. The relation of pulse pressure to elimination of nitrogen is therefore very clearly shown. The percentage decrease of elimination of urea nitrogen during period 3 is unknown, but judging from data of other experiments it probably was larger than the following decrease during period 4. The same blood pressure conditions prevailed during periods 3 and 4. The TABLE VI Percentage ; Percentage ° Urea N. in G. 8€) Total N.inG.| Percentage : aun euc veil] Gecrease ; Chlorides per c.c. urine | per c.c. urine | decrease of urea N. total N. 0.0398 0.036 0.033 0.0227 0.0218 Relation of Pulse Pressure to Renal Secretion 87 percentage decrease of urea nitrogen elimination during period 4 was 9-57. During period 5 the pulse pressure was normal. The percen- tage decrease of urea nitrogen elimination was only 8.3. Had the urine been collected only during the latter half of period 5 the decrease probably would have been much less. During the following period (6) of diminished pulse pressure there was a very marked decrease of elimination of urea amounting to 31.2 per cent. In the following period (7) during which the same blood pressure condition prevailed, there was a decrease of only 3.8 per cent. The marked changes in elimination of urea occurred during periods in which the pulse pres- sure was altered. The relation of elimination of total nitrogen to pulse pressure as the table shows was in general similar to that observed for urea nitrogen. In this experiment the urine was free from chlorides and albumin. This total absence or diminution of chlorides has been noticed by others °, 7, 8, 9, ° especially when sodium sulphate was used as a diuretic. THEORETICAL CONSIDERATIONS AND DISCUSSION OF RESULTS The nutrition of an organ is of utmost significance for its proper functioning. The metabolism and gaseous exchange in the kidney is very great and for that reason the volume flow of blood through the kidneys must be considered as an important factor in secretion of urine. Preliminary experiments, however, showed no change in volume flow of blood accompanying pulse pressure changes as affected by the methods herein described. But the mere fact that the volume flow of blood is not changed does not necessarily rule out a deleterious nutritive effect of diminished pulse pressure. The gaseous exchange in the kidneys may be diminished by a diminished pulse pressure as indicated by the works of Fleishel v. Marxow."' He showed by experiment that the state of a gas in 6 Bropre: Harvey lecture on renal activity, 1909-1910. 7 SOLLMANN: This journal, 1902, vili, p. 155. 8 MaGnus: Archiv fiir experimentelle Pathologie und Pharmakologie, 1900, xliv, p. 68. 9 CusHNy: Journal of physiology, 1902, xxvii, p. 4209. 10 THOMPSON : Journal of physiology, 1900, xxv, p. 487. 11 FLEISHEL v. Marxow: Beitrige zur Physiologie, zu Ludwig gewidmet, 1887, p. 29. Quoted by Erlanger and Hooker, /oc. cit. p. 368. 88 Robert A. Gesell solution is markedly changed by shaking; that gas after agitation in a solvent is more readily given up than if agitation were omitted. He thinks that the gas is no longer in true solution, but in a state of suspension and therefore more readily given up by the solvent. He thinks that the shocks to which the blood is subjected plays an important part in exchange of gases. The massaging action of the pulse should also have a beneficial effect on promoting a freer flow of lymph and an increased streaming of protoplasm. Such massage as results from pulse pressure may be beneficial in still another way. Kahlenberg 2 has shown the importance in dialysis of stirring the solution in contact with the osmotic membrane. Only by stirring can the maximum osmotic pressure be obtained; but more important, the process of osmosis is materially hastened by bringing fresh solution of stronger concentration in contact with the membrane. It seemed, therefore, that massage might be of consider- able importance, not only in bringing fresh solution in contact with the renal cells but also in promoting diffusion of the solute through the cell itself. If osmosis is simply a matter of solubility and diffusion of the solute, and provided the renal cellular protoplasm has no marked activity of its own a pulsatile pressure would be of great theoretical importance. The massaging action produced by a smooth swinging pulse as described in Fig. 8 does not seem to be the fundamental factor producing changes in secretion accompanying alterations of pulse pressure. Whether the massage is not active enough to assist in promoting diffusion or whether the lack of sudden pressure changes brings about conditions which might counteract the beneficial effects of massage is a question. This will be discussed later on under filtration and molecular aggregates. Brodie !* emphasizes the importance of the glomerulus as an organ of propulsion, assisting in overcoming the resistance of passage of urine through the tubules. The present experiments give no direct evidence against or for that theory. In some cases complete oblitera- tion of the pulse pressure had barely any effect upon the rate of urine flow, while in other cases increasing the magnitude of the pulse pres- 12 KAHLENBERG: Journal of physical chemistry, 1906, x, no. 3. 18 BropiE: Loc. cit. Relation of Pulse Pressure to Renal Secretion 89 sure stopped the flow. While propulsion of the urine may be of im- portance over long periods of time, these experiments indicate that we must look elsewhere for the marked specific action of pulsation on the activity of the kidneys. The glomerulus has always been looked upon as a favorable site for filtration, and since filtration is nothing more than the mechanical process by which molecules or particles of matter are forced through the interspaces of other molecules or particles of matter it seems very likely that the normal pulsatile pressure might be more efficient than a constant pressure; not only because the pulsatile pressure reaches a higher level, but also on account of the relative behavior of small and large particles to sharp light taps. The larger tend to remain stationary, the smaller to take on the velocity of the impact. Take for example a vessel, the bottom of which is porous enough to allow the passage of fine grains of sand, and fill that vessel with a mixture of fine and coarse grains of sand. Exerting a constant pressure on this vessel above will have no effect on the passage of sand through the pores, but if constant pressure is replaced by sharp, light taps the results are entirely different. Not only are the finer granules at the membrane assisted by agitation in their passage through the membrane, but all the granules are rearranged — the finer granules lying near the membrane and the coarser near the sur- face. The finer granules, by means of a greater relative velocity imparted to them than to the heavier granules possessing relatively greater inertia make their way to the bottom of the jar; while the coarser granules remain in position or are forced upwards by the smaller granules. A similar process may occur within the cell in which the organized cellular structure represents the coarser granules with greater inertia and the urinary constituents (water, salts, urea, etc.) not directly combined with the cellular structure represent the finer granules. If this process occurs it can readily be seen that the magnitude of the pulse pressure is not the important factor in filtration, but rather the abruptness of pressure changes — slight sudden vascular shocks —may be of greater significance. Some records seem to support this view. For instance the experiments in which the magnitude of ‘pulse pressure was increased when the air chamber was connected with the abdominal aorta. In these cases the flow of urine was gO Robert A. Gesell copious during the period in which the pulse pressure was normal, but stopped abruptly when the pulse pressure was increased. Attention has been called to the form of the pulse curve at this time, the lack of any secondary waves, and the relative slowness of pressure changes. In these cases of increased pulse pressure the air chamber worked perfectly in taking up all sudden impacts, and trans- ~ forming them into a smooth swinging pulse. In some of the experi- ments the air chamber did not work as perfectly in these respects. Even though the pulse pressure was successfully reduced to a rela- tively small magnitude the dicrotic notch was always in evidence. In these cases, diminution of pulse pressure was not as effective in slowing secretion as in other cases where the dicrotic notch was more successfully eliminated. The effects of mechanical shock upon living cells have received considerable attention from Meltzer.1t He considers mechanical shock as a fundamental factor in the activity of living protoplasm and that every kind of protoplasm or cell has an agitation of optimum intensity for its growth. Of interest is Shacklee’s and Meltzer’s” work on the destructive action of shaking upon proteolytic enzymes S;, in which they find that pepsin, rennin and trypsin are completely destroyed by shaking. Even the churning action of the stomach on pepsin enclosed in rubber cots is sufficient to destroy the efficiency of pepsin 4o per cent. Abderhalden and Guggenheim " have shown the destructive action of shaking upon tyrosinase. Mathews “ found that the most careful transfer of starfish eggs produced sufficient mechanical shock to cause parthenogenesis. Mrs. Andrews !® working on the choana flagellata found that agitation produced distinct changes in the viscidity of the protoplasm. That the slightest tap on the cover slip, covering the organisms under observation, caused a distinct rigidity of the collar. That mechanical shock is an important factor in the activity of 14 Mettzer: Zeitschrift fiir Biologie, 1894, xii, p. 464. Johns Hopkins Hos- pital reports, 1900, ix, p. 135. This journal, 1903, ix, p. 245. 18 SHAKLEE and MELTZER: This journal, 1910, xxv, p. 81. 16 ABDERHALDEN and GUGGENHEIM: Zeitschrift fiir physiologische Chemie, 1908, liv, p. 352. ; 17 MATHEWS: This journal, 1901, vi, p. 142. 18 Mrs. ANDREWS: Journal of morphology (supplement 1897), xii, pp. 492-498. Relation of Pulse Pressure to Renal Secretion QI _ protoplasm has been sufficiently demonstrated; but the question arises what is the specific effect of mechanical shock? Is it a general effect upon the whole protoplasm of the cell favoring or retarding metabolic processes, or is it due to some phenomenon favoring or retarding a free exchange of material between the cell and bathing medium as indicated by these experiments? The relation of pulse pressure to renal secretion must be: con- sidered from two points of view: the beneficial effect of a pulsatile pressure and the deleterious effect of a non-pulsatile pressure. In some cases when the kidneys were subjected to diminished pulse pressure for only a short time the secretion was stopped for an hour. The question arises — is this prolonged after-effect due to a deleterious action on the renal protoplasm itself or to a clogging of protoplasmic interspaces hindering the normal passage of uri- nary constituents through the cells? The work of Ramsden and Winkelbach seems to be of significance in this connection. Ramsden" has shown the effect of shaking upon a solution of egg albumin. A clear solution becomes turbid with the production of fine coagulated strands of albumin which are no longer soluble in the medium. More recently 7° he has extended his experiments to other solutions and suspensions. He found “that quite apart from evaporation solid highly viscous coatings are spon- taneously and more or less rapidly formed upon the free surfaces of all proteid solutions; that similar coatings of solid or highly viscous matter occurs on the free surfaces of a large number of non-proteid colloid solutions and fine suspensions, and of a few apparently crystal- loid solutions, and that they are formed also at the interfaces of solu- tions which without being of high viscosity are capable of persistent emulsion.” Ramsden found that “by simple mechanical means adapted to produce heaping up of surface membranes, large masses of solids (mechanical surface aggregates) can be separated out from all proteid solutions and from a large number of colloid solutions and suspensions.” Winkelbach *! demonstrated the formation of molecular aggre- 19 RAMSDEN: Mann’s Chemistry of protein, p. 273. 20 RAMSDEN: Proceedings of the Royal Society of London, 1904, Lxxii, 21 WINKELBACH: Zeitschrift fiir angewandte Chemie, 1906, p. 1953. Quoted from Freundlich: Kapillarchemie, p. 444. 92 Robert A. Gesell gates on shaking a solution of gelatine, egg albumin, and other solutes with benzene or benzin. If under diminished pulse pressure a thick viscous layer can form at the interfaces of the renal cells and the bathing medium, as it does at the interfaces of certain solutions, secretion might be markedly varied by subjecting the cells to a pulsatile or constant pressure. By the shaking action of the normal pulse the albumin in the viscous layer might be heaped up into mechanical surface aggregates and kept in coarse enough suspension to prevent its passage through the cells, and at the same time continually break up the viscous layer and allow a freer entrance of urinary constituents into the cells. On the contrary by allowing the formation of a viscous layer by diminishing the pulse pressure the albumin might be kept in fine enough suspension to enter and pass through the cells into the tubules — finally clogging the protoplasmic interspaces, thereby preventing filtration. The rate of recovery of urine flow after a period of de- creased pulse pressure may depend on the rate of resolution of albumin which has bodily entered the cell. Ramsden points out that the failure of proteids and other colloids in solution to pass through a fine filter without considerable loss is largely due to the formation of surface membranes and mechanical coagule upon the air, grease, and other suitable surfaces in the pores of the filter. With the formation of these membranes the rate of flow through the filter is decreased. Why if this phenomenon occurs in a simple filter might it not be still more pronounced in living cells, especially designed to prevent the passage of highly organized substances such as albumin, from the blood? From the foregoing it would seem that the relation of pulse pres- sure to the elimination of urine has a practical therapeutic bearing. Drugs which increase the pulse pressure without markedly lowering the blood pressure or producing undue constriction of the renal vessels theoretically should be good diuretics. Among such drugs are strophanthus and digitalis. Their marked diuretic effect has long been noted. Concerning their mode of action various sugges- tions have been made. But the effects of pulse pressure itself, which is altered by the administration of these drugs, apparently has not been taken into account. Judging from the marked changes in secretion produced experimentally by altering the pulse pressure it Relation of Pulse Pressure to Renal Secretion 03 _ would seem that the increased pulse pressure per se produced by digitalis and strophanthus may account largely or the diuresis produced. SUMMARY 1. A method has been described by which the pulse pressure in the intact animal can be altered without materially changing the mean blood pressure or volume flow of blood through the kidneys. 2. Employing this method pulse pressure has proved to have a specific effect of its own upon renal secretion. 3. It was found that normal pulse pressure exerted a beneficial effect upon secretion of urine. 4. Constant or diminished pulse pressure produced by the method described had a deleterious effect upon the activity of the kidneys. 5. The amount of urine eliminated, as a rule, varied directly with the magnitude of the pulse pressure. 6. A few exceptions were noted which suggest that in addition to magnitude of pulse pressure, the suddenness of pressure changes, vascular shocks, may be an important factor in the secretion of urine. 7. The amounts of chlorides, urea, and total nitrogen eliminated, as a rule varied directly with the magnitude of the pulse pressure. 8. In two experiments in which albumin occurred in the urine, the amount eliminated varied inversely with the magnitude of pulse pressure. 9. Various theories concerning the specific action of pulse pressure on renal activity are discussed. 10. The practical therapeutic bearing of this problem relative to the diuretic action of such substances as digitalis and strophanthin are pointed out. It is my pleasure to acknowledge here the suggestions and assist- ance Doctor Erlanger rendered me in this work. I also wish to thank Doctor Shaffer for the use of his laboratory for making chemical analyses. THE American Journal of Physiology VOL. XXXII JUNE 2, 1913 NO. II DIRECT AND CROSSED RESPIRATION UPON STIMULA- mon OF THE PHRENIC, THE SCIATIC, AND THE BRACHIAL NERVES! By W. f., PORTER ann ABBY H. TURNER [From the Department of Comparative Physiology in the Harvard Medical School] N 1895 it was discovered that the respiratory impulse descends from the bulb to the phrenic nuclei in the lateral column of the spinal cord.” At the level of the phrenic cells, the path divides. The greater part of the respiratory fibres remain on the side of their origin; the lesser part cross to the phrenic cells of the opposite side. When the lateral column is severed between the bulb and the phrenic nuclei, the diaphragm ceases to contract on the side of the hemisec- tion. The arrest of that half of the diaphragm is not momentary, but permanent, for the diaphragm has been found passive in rabbits kept alive for twenty-four days after the operation.’ Yet the phrenic cells are not inhibited by the hemisection, for if the phrenic nerve upon the opposite or active side be severed, the diaphragm on the passive side (the side of the hemisection) at once begins to contract. From these observations it was concluded that the crossed path carried impulses which ordinarily did not rise to the threshold value 1 An account of this research was given to the American Physiological Society, December 28, 1911. This Journal, 1912, xxix, p. xxxi. 2 W. T. Porter: The Journal of physiology, 1895, xvii, p. 455. 3 W. T. PorTER and W. MuuHLBeErc: this Journal, 1900, iv, p. 334. 96 W. T. Porter and Abby H. Turner necessary to discharge a motor impulse from the phrenic cells of that crossed side. But when the phrenic nerve on the direct side was cut, the impulses taking the crossed path to the opposite side were increased and crossed respiration became possible. This conclusion has recently been called in question* on the ground that the stimulation of the central end of the phrenic nerve in the cat causes reflex contraction of both sides of the diaphragm, from which it is argued that the phenomenon described above is due to the mechanical stimulation of the phrenic nerve by its section. The afferent impulse thus set up might pass to the phrenic cells of the passive (the hemisected) side and there call forth an efferent impulse which would descend the phrenic nerve of the passive side and cause that half of the diaphragm to contract. In this event, crossed respiration would be due to a bulbar or purely spinal reflex, and the evidence for a division of the bulbo-spinal respiratory tract into a direct and a crossing portion would be destroyed. - We purpose in this communication to show (1) that in the rabbit, the animal used by Dr. Porter, but upon which Messrs. Deason and Robb made no observations, the stimulation of the central end of the phrenic nerve calls forth no respiratory reflex whatever; and (2) that in the cat, whose phrenic nerve has long been known to carry afferent fibres, the freezing of the phrenic nerve upon the crossed (uninjured) side causes the diaphragm on the direct (the hemisected) side to resume its contractions, as in the rabbit, although the inter- ruption of the physiological continuity of a nerve by freezing does not stimulate the nerve We shall also present observations upon the alleged frequency of bilateral contractions of the diaphragm after hemisection. * METHOD In these experiments rabbits and cats were used. The animals were etherized and tracheotomized and preparations were then made to record the contractions of the diaphragm. It is here that the unwary observer may come to grief. It may be stated positively that accurate observation in this field requires special precautions 4 DEASON and Ross: this Journal, 1911, xxviii, p. 57. Direct and Crossed Respiration 97 for the section and stimulation of the phrenic nerve and demands the direct inspection of the diaphragm. The Section and Stimulation of the Phrenic Nerve. — The sec- tion of the phrenic nerve in the neck is frequently fallacious because the nerve often receives a branch from the brachial nerves posterior to the point of section. It was for this reason that Porter advocated grasping the nerve near the first rib and pulling it out of the chest.® By this procedure it is reasonably but not absolutely certain that the lowest root of the phrenic nerve will be torn across. The following protocol is instructive. Experiment May 12, 1911. In an anaesthetized rabbit the spinal cord was hemisected. The diaphragm of that side ceased to contract. Puiling out the phrenic nerve of the opposite side near the sixth nerve was followed by bilateral contractions of the diaphragm. When the nerve was found under the subclavian vein and pulled out there, the ordinary one-sided crossed breathing at once appeared. Pulling out the nerve from the neck is safe only when followed by a positive — not a negative — observation; e.g., if after this supposed destruction of the nerve only the opposite side of the diaphragm contract, the phrenic has been torn across distal to its lowest com- ponent; but if both sides of the diaphragm contract, this last com- ponent has escaped and both sides of the muscle are still connected with the spinal cord. The truth of these remarks will be acknowl- edged on reading Miss Turner’s study in the preceding number of this journal.® In view of these dangers we have in this investigation severed the phrenic nerve within the thorax. It is even more necessary to use the intrathoracic rather than the cervical portion of the phrenic nerve for electrical stimulation. A glance at Fig. 1 in Miss Turner’s paper’ will show how very short is the portion of the nerve accessible to stimulation in the neck; at 5 W. T. Porter: The Journal of physiology, 1895, xvii, p. 466, and especially Experiment LXVIII, pp. 478-479. 6 Assy H. Turner: this Journal, 1913, xxxii, p. 66. 7 ABBY H. TurNER: Joc. cit., p. 66. 98 W.T. Porter and Abby H. Turner this point it is impossible to make sure that current has not escaped to the brachial nerves. The Observation of the Diaphragm.— The contractions of the two halves of the diaphragm cannot be determined accurately by recording the intrathoracic pressure. Respiration may be carried on by the muscles of the neck alone,* or by the intercostal muscles, or by the abdominal muscles. The part played by the intercostal and abdominal muscles is seen in the following experiment. Experiment April 20, 1911. In an adult cat anaesthetized with ether, the left half of the spinal cord was severed at the third cervical vertebra. The left half of the diaphragm at once ceased to contract. Artificial respiration was begun and the diaphragm was divided into two parts by a median incision extending from the sternum to the vena cava. The left half was connected to a recording lever. The right phrenic nerve was severed within the thorax. Crossed respiration immediately followed. The respiratory contractions of the intercostal muscles on the right side continued unimpaired. The artificial respiration was so regulated that the action of the diaphragm was uniform; there was no dyspnoea. On stimulation of the central end of the divided right phrenic nerve, and of the divided sciatic nerve, vigorous reflexes were obtained from the intercostal and the abdominal muscles. In another cat, traction upon the phrenic nerve within the thorax caused strong contractions of the intercostal muscles. It cannot be doubted that changes in the intrathoracic pressure caused by such contractions of the intercostal and the abdominal muscles may be attributed to the diaphragm when the observer depends merely upon a tambour connected with the trachea or the pleura. Nor can such errors be avoided by the inspection of the un- opened thorax. It is frequently impossible to say whether a dimin- ished excursion of the body wall upon one side is due to that side being dragged passively by contractions of the opposite side or is the result of a contraction feeble on one side and, by compensation, strong upon the other. Even when the abdomen is open and the under surface of the diaphragm viewed directly, experience is needed to avoid self- deception, so closely do passive movements simulate contractions. 8 W.T. Porter: Journal of physiology, 1895, xvii, p. 457. Direct and Crossed Respiration 99 Only when the artery which encircles the central tendon is seen to approach the wall of the thorax may the spectator be certain that the diaphragm on the suspected side actually does contract.° For these reasons we have always observed the exposed diaphragm. In some cases, the contractions were recorded by Head’s method, in which the diaphragm is reached through an opening in the linea alba, the median end of the ventral muscular slip fastened to the thoracic wall, the xiphoid cartilage severed from the sternum, and the con- tractions of the isolated portion of the diaphragm carried to a record- ing lever by a thread passed through a hook in the split cartilage. In other cases, the xiphoid cartilage and the contiguous part of the sternum were cut away and the diaphragm divided in the middle line back to the vena cava. The movements of either half could then be recorded through a lever connected by a thread to the central tendon. The abdominal organs were kept from interfering by broad, smooth, metal plates suitably curved and carefully fixed in place. This method has the advantage of using the main part of the diaphragm rather than the less vigorous ventral slip. Both methods are open to criticism in that the record is influenced by the movement of muscles other than the diaphragm, particularly by general body reflexes. These are apt to cause a change of level in the sensitive recording lever, due to altered tensions in the thread connecting it to the dia- phragm. It is usually easy, however, to distinguish in the record such alterations from those due to changes in the frequency. and extent of the contractions of the diaphragm itself. THE STIMULATION OF THE CENTRAL END OF THE PHRENIC NERVE IN THE RABBIT During Normal, Direct or Uncrossed, Respiration. — In three rabbits the central end of the cut phrenic nerve of one side was stimu- lated with spinal cord and medulla intact, while a record was taken of the movements of the diaphragm of the opposite side. No reflex change in the rhythm or amplitude of the diaphragm’s contractions was found. A typical protocol follows. ® Additional precautions are given by Porter and Muhlberg, Joc. cit., p. 338. TOO W. T. Porter and Abby H. Turner Experiment March 29, t911. A rabbit was etherized and tracheotomized. The contractions of the left half of the diaphragm were recorded by a lever attached to the central tendon. The right phrenic nerve was cut anterior to the diaphragm and the central end stimulated. The secondary coil of the Harvard inductorium was at 4 cm. (a strong stimulus; with the secondary coil at 12 cm. the current was still perceptible to the tongue). No reflex was observed. To show the sufficient irritability of the animal the right sciatic nerve was pre- pared and the central end stimulated with the secondary coil at 13 cm. A strong reflex contraction of the diaphragm was observed. Ficure 1. Two thirds the original size. From the experiment of March 29, 1911. The contractions of the left half of the diaphragm of a rabbit recorded by a lever attached to the central tendon. The rabbit had received j45 grain of strychnine in the external jugular vein. The electro-magnetic signal (second line) records three successive stimu- lations of the central end of the phrenic nerve with a very strong current (the secondary coil at 4cm. There is no reflex.) But a very brief stimulation of the central end of the sciatic nerve with an exceedingly weak current (13 cm.) caused violent reflex con- tractions. The lowest curve marks two second intervals. As no reflex contraction of the diaphragm was obtained by stimu- lation of the central end of the phrenic nerve under normal experi- mental conditions, it was determined to increase the activity of the respiratory centre by dyspnoea. Experiment March 23, 1911. In an animal prepared as in the preceding experiment, the artificial respiration was stopped and the central end of the phrenic nerve stimulated during the progressive asphyctic increase of the contractions of the diaphragm. The progressive Direct and Crossed Respiration IOI change in respiration in the dyspnoeic animal was not influenced by the phrenic stimulation. The phrenic nerve was stimulated also in a rabbit whose irritability had been heightened by strychnine, as follows. Experiment March 29, tg1r. As noted above, the contractions of the left half of the diaphragm of a rabbit were recorded by a lever attached to the central tendon of the diaphragm. One hundredth grain of strychnine in sodium chloride solution was given by the external jugular vein. Apparently spontaneous spasms occurred at intervals and the increase in the irritability of the centres was also shown by the response to sciatic stimulation. But the stimulation of the phrenic nerve caused no reflex (Fig. 1). These experiments support the conclusion that in normal (direct) respiration, the stimulation of the phrenic nerve does not cause a reflex contraction of the diaphragm. Phrenic stimulation during crossed respiration should now be examined. During Crossed Respiration. — In nine rabbits the spinal cord was hemisected between the calamus scriptorius and the phrenic nuclei FicurE 2. Two thirds the original size. From the experiment of March 21, 1911. The contractions of the left ventral slip of the diaphragm (Head’s method) in a rabbit with the spinal cord hemisected on the left side at the third vertebra. Prolonged stimulation of the central end of the right phrenic nerve with a strong induction cur- rent (7 cm.) produced no change in the contractions of the diaphragm. The lowest line marks two second intervals. and paralysis of the diaphragm on the operated side observed to exist. The phrenic nerve of the opposite side was then severed and 102 W.T. Porter and Abby H. Turner crossed respiration resulted. The contractions of the diaphragm were recorded by Head’s method or by a lever attached to the central tendon. No reflex change in the rhythm or amplitude of the con- tractions of the diaphragm took place on phrenic stimulation. Typical protocols follow. Experiment March 21, tg1r. An etherized rabbit was tracheotomized and the left half of the cord was severed at the third vertebra. After section of the right phrenic nerve the contractions of the left ventral slip of the diaphragm were recorded by Head’s method. Stimulation of the central end of the right phrenic nerve cut within the thorax caused no change in the contractions of the diaphragm. Experiment March 25, t911. The left half of the spinal cord in a rabbit anaesthetized with ether was severed about the third vertebra. Aifter section of the right phrenic nerve, the contractions of the left half of the diaphragm were recorded by a lever attached to the central tendon. The strong stimulation of the central end of the right phrenic nerve cut near the diaphragm produced no reflex action but the very weak stimulation of the right brachial nerves caused strong reflexes. Experiment March 27, torr. In an etherized rabbit the cord was hemi- sected on the left side at the third vertebra and the right phrenic nerve was cut just anterior to the diaphragm. A lever was attached to the central tendon on the left side of the diaphragm. The right crural nerve was now cut and the central end weakly stimulated. The crossed respiration of the left half of the diaphragm was for some moments greatly increased, and during this excitement the central end of the right phrenic nerve was strongly stimulated, but without effect on the diaphragm. Experiment March 30, t911. In an etherized rabbit whose irritability had been increased by strychnine, the abdomen was opened and the dia- phragm observed directly while the central end of the right phrenic nerve was strongly stimulated. The crossed respiration due to the left hemisection of the spinal cord was not affected by this stimulation. In none of our experiments on crossed respiration in the rabbit did the preparation of the phrenic nerve in the thorax or traction there by pulling the nerve over a smooth glass hook cause bilateral diaphragmatic contractions. There is therefore no evidence of afferent fibres in the phrenic nerve of the rabbit, the stimulation of which affects the movements Direct and Crossed Respiration 103 of the diaphragm. Nor were reflexes in answer to phrenic stimulation observed in any other part of the animal. The phrenic nerve in the rabbit is apparently a purely motor nerve. THE STIMULATION OF THE CENTRAL END OF THE PHRENIC NERVE IN THE CAT Three experiments were made on cats to demonstrate again the existence of afferent fibres in the phrenic nerve of this animal. A protocol follows. Experiment April 20, rg1r. In an adult cat, anaesthetized with ether, the spinal cord was hemisected on the left side at the third vertebra, and a lever attached to the left half of the diaphragm, which had been separated from the right half as far as the vena cava. Cutting the right phrenic nerve in the thorax was followed by crossed respiration. The artificial respiration was so regulated that the contractions of the left half of the diaphragm were uniform; there was no dyspnoea. On stimulating the central end of the right phrenic nerve, the frequency and force of the contractions of the opposite half of the diaphragm were clearly altered. A similar reflex action on the diaphragm fol- lowed the stimulation of the central end of the sciatic nerve. The threshold value for the sciatic reflex was much lower than that for the phrenic reflex. The fact that the stimulus required for reflexes through the phrenic nerve is so much stronger than that for the sciatic dt brachial nerves is in itself evidence that crossed respiration can hardly be due to the much weaker stimulus of section; but this possibility may be com- pletely excluded by severing the phrenic nerve physiologically by a method that cannot stimulate the nerve. CROSSED RESPIRATION IN THE CAT FOLLOWING THE FREEZING OF THE PHRENIC NERVE In December, 1911, the spinal cord of an anaesthetized cat was hemisected on the left side at the third vertebra and the thorax opened. Only the right side of the diaphragm contracted. The right phrenic nerve was carefully freed from underlying tissues in its course through 104 W. T. Porter and Abby H. Turner the thorax and placed upon a small metal tube connected with a reservoir of liquid carbon dioxide. On admitting the carbon dioxide, frost formed on the tube and the nerve was frozen at the point of contact. The right half of the diaphragm ceased to contract, while on the left, or hemisected side, the diaphragm contracted vigorously. Crossed respiration therefore cannot be due to the mechanical stimulation of the phrenic nerve by section. THE ALLEGED FREQUENCY OF BILATERAL CONTRACTIONS OF THE DIAPHRAGM AFTER HEMISECTION OF THE SPINAL CORD The earlier students in this field noted many instances ” in which hemisection of the spinal cord between the bulb and the phrenic nuclei was followed apparently by contractions of both sides of the diaphragm. It is probable that almost all these observations were erroneous. The difficulty of determining whether both halves of the diaphragm are actually contracting is not generally appreciated. Moreover, no one would believe without much experience how diffi- cult it is to sever completely one half the spinal cord. It is never absolutely safe to trust to an ordinary cross-section, for the respira- tory fibres are found in the lateral column which lies so far within the arch of the vertebrae that it is very difficult to reach all the fibres with a knife. The use of a wire or other blunt instrument raises always the doubt whether the fibres were sufficiently crushed. Under these conditions, the only safe method is to hemisect the spinal cord by making two incisions about three mm. apart and removing the piece between. If this piece with its lateral column intact can be laid upon the table all doubt is at an end. When these precautions are observed, it will be found.that the adult animals in which both halves of the diaphragm contract after hemisection are so few in number that they may be classed reasonably as anomalies. Thus in twenty-nine rabbits and dogs W. T. Porter noted only two with bilateral respiration; in thirteen rabbits and two cats hemi- 10 This literature is cited by W. T. Porter in the Journal of physiology, 1895, Xvii, p. 462. rath i Direct and Crossed Respiration 105 sected by Porter and Muhlberg there was not a single instance of bilateral contractions; nor have we observed one case in the present series of thirteen rabbits and three cats. Crossed respiration was indeed apparently present in two animals. In one of these (March 24, to11) the lateral column of the cord was found to be only partly severed, so that the normal uncrossed pathway may not have been interrupted. In the second case (May 12) on pulling out the phrenic nerve near the sixth nerve bilateral breathing appeared after hemi- section. When the nerve was found under the subclavian vein and pulled out there, the ordinary one-sided crossed breathing at once appeared. In this instance it seems possible in view of the variety in the phrenic nerve that the first section was incomplete and that sufficient stimulus was given to the adjacent brachials to cause the bilateral breathing. In no case did the preparation of the phrenic nerve in the thorax or traction there by pulling the nerve over a smooth glass hook cause bilateral diaphragmatic respiration, though strong contractions of the expiratory muscles were noted on stimulating the parietal peritoneum and pleura. In an animal in which the cord was hemisected and which was poisoned by strychnine, it was noted that the strong contractions of the diaphragm in the strychnine spasms involved only one-half the muscle, the paralyzed side remaining passive throughout. The phrenics here were untouched. In two cats on which observations were made, neither dyspnoea nor the stimulation of the central end of either sciatic resulted in bilateral breathing after hemisection of the cord and consequent unilateral paralysis of the diaphragm. The preparation of the opposite phrenic nerve in the thorax was also without effect as was traction on the prepared nerve in the thorax. The reflex to the sciatic stimulus was in general strong and while the paralyzed side of the diaphragm was inactive, the active side changed its rhythm in response to phrenic traction. On cutting the prepared phrenic nerve, crossed respiration occurred at once in one case, and after slight sciatic stimulation in the other. The good condition of the latter animal was indicated by the continuance of crossed respiration unimpaired for three hours. All our data, therefore, point unmistakably to the conclusion that hemisection of the cord arrests the contractions of the homo- 106 W. T. Porter and Abby H. Turner lateral half of the diaphragm, with exceptions so infrequent as to be anomalous. SUMMARY 1. In the study of crossed respiration, the section and stimula- tion of the phrenic nerve within the chest and the direct inspection of the diaphragm are of great advantage. 2. The accurate stimulation of the central end of the phrenic nerve in the rabbit does not cause contraction of the diaphragm or other reflex movements. 3. In the cat, reflex contractions of the diaphragm may follow the stimulation of the phrenic nerve. | 4. In the cat, a strong stimulus is required to call forth a reflex with the phrenic nerve, while in the same individual a very weak stimulus to the sciatic or the brachial nerves will cause reflex contrac- tions of the diaphragm. 5. Hemisection of the spinal cord between the bulb and the phrenic nuclei stops the contractions of the diaphragm on the same side, but these contractions are at once resumed when the opposite phrenic nerve is severed by freezing. A mechanical stimulus therefore . cannot be the cause of the crossed respiration. CARBON DIOXIDE PRODUCTION FROM NERVE FIBRES WHEN RESTING AND WHEN STIMULATED; A CONTRIBUTION TO THE CHEMICAL BASIS OF IRRITABILITY}! By SHIRO TASHIRO [From the Department of Biochemistry and Pharmacology, the University of Chicago, and the Marine Biological Laboratory, Woods Hole, Mass.] INTRODUCTION HERE have been two theories of the nature of conduction — one upheld among others by Hermann, that it was a prop- agated chemical change; the other, at present the dominant view, that it is a propagated physical change. In 1901 Professor Mathews suggested ? that it was in the nature of a coagulative wave propagated along the fibre; this coagulation of the nerve colloids leading either directly or indirectly to the electrical disturbance accompanying the impulse. At the time, there was no evidence of chemical change in the nerve fibre, and its indefatiga- bility seemed to point to an absence of metabolism. Certain facts were known, however, which were difficult to reconcile with this phys- ical theory. Darwin had observed that in Drosera,*? conduction occurred only if the protoplasm had oxygen; and Mathews # observed that salts would not stimulate a nerve, or, at any rate, their power of stimulation was much reduced if the nerve remained in the body for a time after death, or if the nerve were brought into the salt solution in an atmosphere of hydrogen. This clearly indicated a dependence of the irritability on oxygen. 1 The preliminary report of these investigations was given in part in Bio- chemical section of Eighth International Congress for applied chemistry, Sep- tember, 1912. See original communications, Eighth International Congress of applied chemistry, xxvi, p. 163. See also this Journal 1913, xxxi, p. xxii. * Mathews Century Magazine, 1902, pp. 783-792; Science, 1902, xl, p. 492. 3 Insectivorous Plants, p. 57. 4 Unpublished observations. 108 Shiro Tashiro This fact lead to a search for evidence of the chemical nature of irritability and in a number of papers °® it was clearly pointed out that the anaesthetics were probably acting directly in a chemical manner instead of indirectly, by affecting permeability, and that probably the anaesthetics acted by uniting with the protoplasm where O» usually took hold. This view was strengthened by the temperature coeffi- cient of conduction, which is nearly that of a chemical reaction; by the importance of Os for artificial parthenogenesis; and by many other facts some of which have recently been collected by Haberlandt, Buijtendijk and others. Although it has been established by repeated demonstrations, that the nerve does not fatigue under ordinary conditions, as measured by the method used in muscular studies, yet Frohlich © observed that the nerve undergoes certain changes by long activity. Gotch and Burch discovered’ in 1889 that if two stimuli are successively set up within <$9 of a second, only one negative variation is produced. This critical interval, or refractory period, is found to be altered by temperature changes, by drugs, asphyxiation, and anaesthetics.$ Thus by prolonging the refractory period by partial anaesthesia, Fréh- lich easily demonstrated that with a frequency of stimulation less than this normal refractory period, stimulation of the attached muscle no longer occurred. He interprets this as a phenomenon of fatiga- bility of the nerve. Théner’s ° observation seems to lead to a similar interpretation, for he found recently that fatigability is less effec- tive when the refractory period is shortened by high temperature. There seems, then, to be fatigue in the nerve, but it cannot be measured by an ordinary scale. After the complete failure of the chemical detection of CO, and 5 A. P. Matuews: Biological bulletin, 1904-5, viii, p. 333; this Journal, 1904, xl, p. 455; zbid., 1905, xiv, p. 203; Biological Studies by the pupils of William Sedgwick, 1906, p. 81; Journal of pharmacology and experimental therapeutics, 1911, ii, p. 234. 6 Frouticu: Zeitschrift fiir allgemeine Physiologie, 1903-4, ili, p. 445. Ibid., Pp. 75- 7 Gotcu and Burcu: Journal of physiology, 1899, xxiv, p. 410. ® See Tait and Gunn, Quarterly journal of experimental physiology, 1908, i, p. 191; Tart, ibid., 1909, ii, p. 157. ° THONER: Zeitschrift fiir allgemeine Physiologie, 1908, viii, p. 530; eee Carbon Dioxide From Nerve Fibres 109 acids in the excited nerve, Waller still believes that it must give off CO, when stimulated. In 1896, he showed, with an electro-physio- logical method, that among other reagents, COs, in minute quanti- ties, increased the excitability of the isolated nerve of the frog, and that the normal nerve, when excited, also increased its activity.” From this he ingeniously formed the hypothesis that every activity in the nerve fibre must be associated with CO, production. That there may be CO, production in the nerve, but too small to be measured by ordinary methods, is shown by the following calcu- lations: A frog (Rana temporaria) gives off 0.355 gram of CO, per kilogram per hour at 19 — 20° C." A small piece of the nerve fibre of the same animal, say 1 cm. in length, will weigh in the neigh- borhood of 1o milligrams. Now, if the mass of the nerve respires at the rate of the whole animal, it would give off about 0.0000007 grams of CO, during ten minutes. This calculation at once suggested that the lack of positive evidence of metabolism in the nerve fibre was not at all conclusive that such metabolism did not occur, in view of the limitation of the methods for the estimation of COs. It was evidently necessary to devise methods for the detection of very minute quantities of CO.. Thus at Professor Mathews’ suggestion a new method for COs, analysis was first devised, and then, under his direction, I have undertaken to go back once more to the question of CO, production in the nerve fibre during the passage of a nerve impulse. To study the nature of metabolism involved in a tissue, one should at least determine the oxygen consumption and the carbon dioxide production. Inasmuch, as the present problem, however, is concerned only with direct evidence for the existence of metabolism in the nerve fibre, I have attempted to measure CO: production only, for it is true that the lack of oxygen consumption may not necessarily indicate the absence of chemical changes, while the pro- duction of CO, will surely prove the presence of metabolism. Further- more, as CO, production is the only sure universal expression of the respiratory activity in anaerobic and aerobic plant and animal tissue in normal condition, the inquiry of CO, production in an excited nerve will not only concern the problem of the nature of the nerve impulse 10 WALLER: Croonian lecture, Philosophical transactions, London, 1896. Taken from Pott’s figures. See figures in Table ix, p. 129. IIo Shiro Tashiro itself, but may, also, aid in forming a fundamental conception of the tissue respiratory mechanism. In this way, if the protoplasmic irri- tability has a direct connection with the cellular respiration, then our idea of the general nature of the pharmodynamics of many reagents on a living tissue may be essentially modified. METHODS AND MATERIALS Two new apparati were constructed which will detect CO, in as small quantities as one ten-millionth of a gram and estimate it with quantitative accuracy. The detailed method has been described in a separate article.’ Preliminary experiments with these new apparati showed that the sciatic nerves of dogs gave too large quantities of CO, for my method so that I was compelled to use a smaller nerve of a cold-blooded animal for quantitative estimation. For exact measurements of CO; - production, I have used only two kinds of nerve, although I have used a large variety of nerves in qualitative experiments. For a non- medullated nerve fibre, Prof. G. H. Parker * was so kind as to sug- gest to me that I use the nerve trunk of the claws of the spider crab (Labinia Caniliculata) which is a bundle of mixed sensory and motor fibres. The frog, whose sciatic was used as a representative for medullated nerve, was exclusively Rana pipiens, obtained from Indiana. As my apparati in the present form cannot be used for a muscle nerve preparation nor for the normal nerve in situ, the use of an isolated nerve could not be avoided. Experimental factors thus intro- duced should be carefully considered before we interpret the observa- tion as a normal metabolism. This serious objection, however, can be overlooked, as far as our fundamental question of different metabolic activities before and after a stimulation is concerned, for Waller ™ has demonstrated that the presence of excitability in an isolated nerve persists as long as nineteen hours provided that the electrical changes correctly represent the state of excitability. Although 2 See pp. 137-145. 18 For this and other suggestions, I am under great obligation to Dr. Parker. 14 WALLER: 1806, Brain, xix, p. 53. Carbon Dioxide From Nerve Fibres rT Herzen claims that under certain conditions of local narcosis the nerve fibre may give an action current without any muscular con- traction (Wedenshi and Boruttau both deny this), and Ellinson ™® recently demonstrated by the use of cinchonamine hydrochloride the absence of negative variations without abolishing the excitability of the nerve, yet evidences are now abundant to indicate that the action current is a normal physiological phenomenon in uninjured tissue expressing the simultaneous activity resulting in a corre- sponding change in the peripheral organ. These facts, therefore, must be taken as showing that as long as a negative variation remains, the nerve is probably excitable; and that the phenomena observed in the isolated nerve could be regarded as identical with that of a nor- mal nerve as far as the passage of a nerve impulse in an isolated nerve fibre is concerned. CO, PRODUCTION FROM RESTING NERVE In this study of the metabolism of the resting nerve, particular care was taken to select those fibres which were free from nerve cells. The work of several investigators! seems to indicate that tissue oxidation is primarily concerned with the cell nucleus. Inasmuch as the respiration in the central nervous system is certain * and the blood supply to fibres is seemingly scanty, the notion persists among certain biologists that a nerve fibre should not respire since it has no nucleus. In order to test the correctness of such an idea, I have studied quantitatively the output of CO, from various lengths of nerve which are known to be free from nerve cells.” Here is the result: 15 ELLINSON: Journal of physiology, 1o11, xlii, p. i. 16 For further details, see. GotcH and Horstey: Philosophical transactions of the Royal Society, 1891, clxxii, p. 514; BERNSTEIN: Archiv fiir die gesammte Physiologie, 18098, Ixxiii, p. 376; Retr and McDonatp: Journal of physiology, 1898-9, xxili, p. 100; Lrewanpowsky: Archiv fiir die gesammte Physiologie, 1808, lxxiii, p. 288; Atcock and SEEMANN, ibid., 1905, cviii, p. 426. 17 See Spitzer: Archiv fiir die gesammte Physiologie, 1897, lxvii, p. 615; M. Nusspaun: Archiv fiir mikroskopische Anatomie, 1886, xxvi, p. 485; R. S. Lit11E: This Journal, 1902, vii, p. 412. 18 L. Hitt: Quoted from Hulliburton’s Chemistry of nerve and muscle, p. 79. 19 Tn this connection, I wish to express my indebtediiess to Prof. H. H. Donald- son for his kind advice. I12 Shiro Tashiro Non-Medullated Nerve Fibre. — (The nerve of the spider crab, and apparatus 2 for the qualitative, and apparatus 1, for the quanti- tative, estimations were used.) When I place the nerve of a spider crab in the right chamber and no nerve in the left, and watch for the deposit of barium carbonate, the drop on the right will soon be coated with the white precipitate, but no precipitate whatever is visible with a lens in the left. CQO: is thus shown to be produced by this resting nerve. Now, by interchanging the nerve from the right to the left, no nerve being in the right, we can convince ourselves of the correct- ness of this conclusion, by eliminating any technical error which might produce the different results in different chambers. The rate at which the precipitate appears and the quantity of the precipitate, depends on the size of the nerve. In fact, CO, production from the resting nerve of the spider crab is found to be proportional to its weight, other things being equal, and is constant: For 10 milligrams per ten minutes it gives 6.7 X 10-7 grams at 15 — 16°C. The quantitative determination of this amount is made in the following manner: The claws of the crab are carefully removed, and, by gently cracking them, the long fibre of the nerve trunk is easily isolated. After removing the last drops of the water by a filter paper, the nerve, with the aid of glass chop sticks, is carefully placed on the glass plate,” and quickly weighed. The glass plate with the nerve is now hung on the platinum hooks in the respiratory chamber A, and then the chamber sealed with mercury. The analytic chamber is now filled with mercury in the manner described elsewhere,” and then the apparatus is washed by CO, free air as usual. The time when the barium hydroxide is introduced to the cup in chamber B is recorded, and the stop-cock between the two chambers is closed. When at — the end of ten minutes the drop at cut F is perfectly clear, having not a single granule of the precipitate visible to a lens, thus insuring that the air is absolutely free from CO, then a known portion of the gas from the respiratory chamber is introduced into the chamber below in which the clear drop of barium hydroxide has been exposed, and it is determined whether or not the amount of the gas taken contains 20 The weight of this plate is known so that the weight of the nerve can be determined very quickly. See p. 120. ai See pp. 138-139. Carbon Dioxide From Nerve Fibres 113 enough CO, to give the precipitate in ten minutes. If it does, a fresh nerve is prepared and a less volume of the gas is withdrawn; if it does not, a larger volume should be taken till the precipitate appears within ten minutes. (See footnote, page 140.) In this way, by repeated experiments with several fresh nerves, a minimum volume of the gas for a known weight of the nerve which gives a precipitate is determined. This minimum volume should contain exactly a definite quantity of CO, — namely 1.0 X 1077 gram.” In this way, since we know the original volume of the respiratory chamber from which this minimum volume is withdrawn, and since we know the quantity of CO. contained in this volume, it is easily calculated, how much CO, is produced by the nerve during the known period. It should be understood that in determining the minimum volume of gas taken from the respiratory chamber, a series of experi- ments were conducted in order to calculate both the minimum volume which just gives the precipitate and the maximum volume which does not give the the precipitate for a known weight of the nerve for a known period of respiration In the tables following, columns 8 and g refer to these volumes calculated from experiments. Table I, gives the result for a non-medullated nerve. Medullated Nerve Fibre. — For the quantitative estimation of CO, production from the medullated nerve I have taken a frog’s sciatic, using apparatus 2. The results given in Table II, obtained by similiar methods, show that each ten milligrams of the frog’s sciatic nerve gives off 5.5 X 10-7 grams for the first ten minutes. A large quantity of nerves were tested and it was determined whether or not all resting nerves give off COy. Asa result, I found no exception in any of them. The following varieties of nerves were examined: 1. Motor NERVE: Occulo-motor nerve of the skate. (Raza Ocallata.) . SENSORY NERVE: Olfactory nerve of the same. (Raia Ocallata.) 3. MEDULLATED NERVE: Sciatic nerve of the dog, frog, turtle, mouse; optic nerve of the skate. (Both Raia Ocallata and Raia Erinecia.) 4. NoN-MEDULLATED NERVES: Nerves of the spider crab; olfactory nerve oftheskate. (Raia Ocallata.) 5. 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NERVE OF VERTEBRATE: Nerves of frog, dog, mouse, squiteague (cynoscion Regalis), and skate. (Both Raia Ocallata and Raia Erinecia.) 7. NERVE OF WARM-BLOODED ANIMAtsS: Those of dog, mouse and rabbits. 8. NERVE oF CoLpD-BLOOoDED ANIMALS: Frog, squiteague (cynoscion Regalis) and skate. (Both Raia Ocallata and Raia Erinecia.) From this I have concluded that isolated nerves of all animals give off COs. It remains, now, to consider whether this CO, is the product of normal respiratory activity or due to disintegration of the dead tissue. Is THE CO, GIVEN OFF PRODUCED BY LIVING PROCESSES? Comparison of Dead and Living Nerves. — In the first place, it was thought that if CO. was due to normal metabolism of a living nerve, its production should be diminished when the nerve was killed. The following result (Table IIT) is self explanatory. TABLE III COMPARISON BETWEEN NORMAL AND KILLED (BY STEAM) NERVES OF SPIDER CRAB 1 2 3 4 5 6 7 Tempera- ture of room jnerve in mg. Weight of | c.c. of gas taken from respiratory chamber Duration of minutes ‘respiration: Ppt. of Ba(COs) after ten minutes 40 (killed) 5 10 40 (killed) : 10 16 (normal) : 10 16 (killed) : 12 16 (normal) | : 10 Comparison of Anaesthetized and Non-Anaesthetized Nerves. — It is naturally feared, however, that the killing experiment itself may not prove that CO, production is necessarily due to the living mechan- ism, for high temperature may drive off CO, produced already by the process of tissue disintegration, just as the CO» diffused out from a wet thread saturated with the gas, the rate of diffusion being a func- tion of temperature. Thus anaesthesia was tried, although we should Carbon Dioxide From Nerve Fibres LLY expect at the outset that if ether had no direct affect on the respira- tory process, as some physiologists believe, then the negative results would not at all interfere with my contention. The fact is, however, that either an isolated nerve directly treated with ether vapor or urathane, or the nerve isolated from a deeply anaesthetized frog gave a much less quantity of CO, than the normal nerve isolated from a normal frog whose heart has been cut away for a period of time equal to that of etherization. Anaesthetics, then, diminish CO, production from an isolated nerve fibre. These experiments are being continued quantitatively. CO, Production of Isolated Nerve at Successive Time Intervals. —It was also thought that if COz production was due to bacterial decomposition, although it is highly improbable for such a fresh tissue, we may expect that either killing by steam or treating with TABLE IV SHOWING DECREASED CO: PRropucTION By LONG-STANDING (FROG’s ScrAtTIc) il 2 3 4 Minimum c.c. Total CO: pro- emperature)/Time elapsed) necessary to give |duced from nerv of room after isolation! | calculated for | of 10 mg. for 10 10 mgs. 10 minutes minutes immediately DIEG.G: So < LOCO; 1 hour 7.08 c.c. 2.1 X 10-7g. CO, 2 hours 10.8 c.c. 1.4 X 10-7g. CO, 5.5 hours 12° SiGsc: 1.1 X 107g. COz 7 hours 15:3 c-c: 9 X 10-7g. COz 10.5 hours 2NOvercs .6 X 107g. CO2 26 hours a Ce 1.6 X 10-7g. COz 27.4 hours 8.1 X 10-7g. COz 1 The gradual increase at this point should be noted (after 26 hours, it is clear that bacterial decomposition sets in). ether would check the CO, production, and that the results observed above may not necessarily prove that CO2 production from the isolated nerve fibre is due to a respiratory process. Hence a number of the nerves were isolated from several frogs of the same size and sex, and 118 Shiro Tashiro were left in Ringer’s solution, and then the rate of the gas production is determined with the different nerves removed at successive inter- vals of time from the Ringer’s solution for twenty-five hours. The interesting results given in Table IV not only show that CO: from the fresh nerve is not due to bacterial decomposition, but it also indicates that when such abnormal decomposition sets in, the output of gas takes asudden jump. This Table further shows that the vital process by which COs is produced gradually slows up as the tissue approaches death, indicating that the decrease of CO: production is parallel to the decrease of irritability of the nerve. Increase of CO: on Stimulation. The most convincing evi- dence of all that CO. is formed by a vital process is the fact that a stimulated nerve gives off more CO, (Part II) indicating the presence of normal metabolism in the living nerve which is accelerated when the nerve is stimulated. Thus we may safely conclude here that like any other tissue or organs, the nerve, too, respires whether it has a nucleus or not, and that the rate of CO. production is pro- portionate to its weight, other things being equal. CO, PRODUCTION FROM STIMULATED NERVE We have now come to our main inquiry, namely, is there any chemical basis for irritability? Just what relation exists between nervous activity and chemical changes is the question that a biologist should consider before he attempts to build any conception of the real dynamics of living matter. For it is the phenomena of excita- bility in the nerve fibre that has stood so long in the path of under- standing protoplasmic irritability in general. As for the brain, it is now established that certain chemical changes are involved during stimulation and that definite chemical changes are associated with pathological cases either in its chemical composition * or in the for- mation of abnormal metabolites.2* Aside from the confused facts concerning histological changes in the ganglion cells of fatigued ani- mals, Hill has observed, using Ehrlich’s method of methylene blue % Kocu and Mann: Archiv of neurology and psychiatry, 1909, iv, p. 44. *4 Drxson: Journal of physiology, 1899-1900, xxv, p. 63; CROFTAN: American journal of the medical sciences, 1902, p. 150. Carbon Dioxide From Nerve Fibres 119 for the determination of the rate of oxidation, that a spot of cerebral surface, if stimulated, loses its blue color owing to the using up of the oxygen.” In case of the nerve fibre, however, we have already seen that no direct evidence has ever been presented to show any chemical changes connected with its activity, although there has been some indirect evidence. As considered before, the failure of the direct detection of CO, from the stimulated nerve must be due to the lack of a delicate method. Thus using the new method we have already demonstrated that a resting nerve gives off CO2, and will now attempt to prove that nerves give off more CO, when stimulated.” Electrical Stimulation of non-Medullated Nerve. — Owing to the scope of delicacy of the new method, which is sensitive to as small a quantity as 1.0 X 10°’ gram (an amount corresponding to the CO, contained in % cc. of pure air), the utmost caution must be taken to prevent any complication which may result in formation or absorption of minute quantities of CO. After I had found by experiment that there is no appreciable increase of CO, due to the direct electrical decomposition in the nerve when stimulated by a weak induction current and that several other forms of stimulation qualitatively confirmed the results obtained by the electrical stimulation, I have naturally employed the induction current as a stimulant in all my experiments on the quantitative estimation of CO: production from the stimulated nerve.” As Table V shows, the stimulated non-medullated nerve fibre of the spider crab gives off 16. X 10-7 grams of CO: for 10 milligrams of 2 Firiy: loc. cit. 26 Professor Carlson has very kindly called my attention to a recent publica- tion from the Physiologisch Laboratorium der Utrechtsche Hoogeschool, in which Buijtendijk reports that certain head nerves of fishes take up more O2 when electrically stimulated. He could not, however, find any increase of O2 con- sumption in the sciatic of the frog. Also see: Koninklijk Akademie van Wetenschappen, Amsterdam, afd, xix, pp. 615-621. Haberlandt also recently reports (Archiv fiir Physiologie, 1911, p. 419) that the resting nerve takes up of Os, 41.7 — 33-4 cmm. at 19° — 24° per gram per hour. When this nerve is excited, intake of O: is increased. Since the respira- tory quotient of the stimulated nerve is equal to that of the resting, he con- cludes that when the nerve is excited, it must give off more COz. He does not, however, indicate how much CO: is produced by stimulation. 27 Use of non-polarizable electrodes was impossible for my apparatus, for the presence of foreign liquid in the chamber interferes with CO: estimation. As 120 Shiro Tashiro nerve for ten minutes, while a fresh resting nerve gave only 6.7 by ro~’ grams for the same units. The details of the methods are as follows: The nerve of the claw of the spider crab is isolated as before. A comparative estimation was made first. Two pieces of the nerve of equal weights and length were placed separately on the two glass plates, each nerve being laid across the electrodes of the plate, in the manner shown in Figure 1. In this way either nerve can be stimulated at will. These glass plates are hung by their wires upon the platinum wires fused into the side of the apparatus, these wires being con- nected in turn with the induction coil. Under this condition, when both nerves are not stimulated, the amounts of the precipitate are equal in both chambers. However, when one of the nerves is elec- C Ficure. 1. Glass weighing plate. A.B. Platinum wire fused in the rear of the glass plate, with hooks. C. The nerve which is stimulated at D. G. The plate proper. I have the other piece of the same glass out of which this plate is made. This piece of glass is weighed exactly equal to this weighing plate, so that any wet tissue can be weighed very quickly. In order to make results more accu- rate, no attempt was made to weigh closer than 3 milligram. trically stimulated (the distance between the primary and secondary coils was always more than 1o cm. using a red dry battery, the current being barely perceptible on the tongue), not only does the precipitate appear sooner in the chamber in which the excited nerve is placed, but also the quantity of the carbonate is much greater. To test whether the increase of CO: production from the stimu- lated nerve is due to the direct decomposing influence of the current, or to the increase of metabolism produced by the passage of a nerve long as we are not concerned with the electrical changes in the nerve, the use of platinum electrodes instead, is not a great objection, provided that the current is weak enough not to decompose the tissue directly, and that the duration of stimulation is not very long. Carbon Dioxide From Nerve Fibres 121 impulse, the following experiments were performed. If we assume that the condition under which an electrical decomposition takes place is the same both in the living and the dead nerve, then if the increased CO, is due to the current itself, we should expect that when a killed nerve is stimulated by a current, it ought to increase COs production just as much. When I placed two nerves killed by steam in each chamber, and stimulated only one of them, the stimulated nerve did not give any more CO, than the unstimulated, using the same strength of current employed in the other experiments. In the next place, it was thought that if the increase of CO, is due to direct electrical decomposition, not limited to the point of contact with the electrodes, we ought to get a proportional increase of CO, by altering the distances through which the current directly passes. The fact was, however, that we could produce an increase of CO, production by stimulating with electrodes 2 mm. apart as well as by 15 mm. apart. Increase of COs, therefore, is due to nervous excita- tion and not to the direct influence of the electric current itself. With this consideration, I have proceeded to make a quantitative estimation of CO, from the stimulated nerve in the manner described before. The results are shown in Table V. Electrical Stimulation of Medullated Nerve. — With apparatus 2, the output of CO, from the excited sciatic nerve of the frog has been quantitatively estimated. As shown below, to mgs. of the sciatic nerve gives off 14.2 X 10-7 grams of CO: during ten minutes stimula- tion while the resting nerve of the same animal gave off 5.5 x 1077 grams for the same units. Mechanical Stimulation. —- We have now established the fact that when a nerve is stimulated by an electrical stimulus, it gives off more CO,. In order to prove more conclusively that this CO2 production is due to the passage of a nerve impulse, I have employed several other means which are known to have definite influence on excita- bility of the nerve. So far, the use of these methods has been confined to qualitative experiments, but the results are a sufficient confirmation of the observations made by electrical stimulation. I cite them here as a preliminary report. Since the ordinary method for mechanical stimulation cannot be applied directly to the nerve in my apparatus in its present form, I used a different method, namely, crushing the nerve. 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Fletcher reports that injured muscle gives off more CO, than the normal. Later he and Hopkins * discovered that muscle, under a similar condition, is richer in lactic acid. Dr. Mathews has observed a similar activity in crushed eggs of Arbacia. Quite accidently, I have discovered that a fresh nerve, too, when crushed with the rough edge of a glass rod gives off more CQ». This increase of gas production from the injured nerve, I take to be due to mechanical stimulation. To test.this hypothesis, I rendered the nerve unexcitable by means of ether and 0.2 m. solution of KCl, which is known to abolish excitability of a nerve.” Under these conditions, I observed no increase of gas production when the nerve is crushed. Therefore, the metabolism existing in. the living nerve must be accelerated by this stimulation when it is injured. This interpretation, however, is not accordant with that of Fletcher and Hopkins, on muscle. In studies of lactic acid formation in muscle, they found that lactic acid is spontaneously developed, under anae- robic condition, in excised muscle, and that fatigue due to contractions of excised muscle is accompanied by an increase of lactic acid. In an atmosphere of Os, there is no survival development of lactic acid for long periods after excision. From a fatigued muscle, placed in Oz, there is a disappearance of lactic acid already formed. But this disappearance of lactic acid, due to oxygen, does not occur, or is masked, at supraphysiological temperature (e. g., at 30). Now traumatic injury to an irritable muscle too produces a rapid develop- ment of acid. Since, however, in this case the disappearance of lactic acid due to O» does not occur, they conclude that one essential condi- tion for this effect of oxygen appears to be the maintenance of the normal architecture of the muscle. Thus they contend that the increase of the lactic acid by mechanical injury is not due to stimula- tion, but must be due to tissue destruction. They, however, did not determine, as far as I know, how much the output of CO, is affected by treating the injured tissue with Or. 28 FLETCHER: Journal of physiology, 1898-9, xxiii, p. 37. 29 FLETCHER and Hopkins: ibid., 1906-7, xxxv, pp. 261, 288. 30 MATHEWS: This Journal, 1904, xi, p. 463. Carbon Dioxide From Nerve Fibres 125 Unless it is proven that CO: production from the injured muscle is quantitatively equivalent to lactic acid formed, their interpretation cannot be applied to the injured nerve, for in the case of the “plateau” of the survival muscle respiration, when in complete loss of irritability, the lactic acid yield remains stationary, Hill calculated that the CO, production corresponds to the amount liberated from the carbonate of the tissue by the lactic acid formed.*! Furthermore, if their interpretation is applied to the nerve, the fact that etherized nerves or nerves rendered unexcitable by KCl do not increase CO, output when crushed, cannot be explained. The fact that only excitable nerves when injured increase their CO» pro- duction, is a sufficient proof that some sort of stimulation is applied to the nerve when crushed, the tissue destruction, no doubt, following afterward. The increase of CO: production on crushing the living nerve and its absence on crushing the anaesthesized nerve is the point that I want to emphasize here in order to confirm my results obtained by electrical stimulation. I may add here that a perfectly parallel increase of CO, by crushing has been observed in dry seeds, including wheat, wild oats, Lincoln oats, Swedish select oats, leaves of Japanese ivy, and spinal cords of rabbit.*? Chemical Stimulation. The study of the nature of chemical stimulation has been so thoroughly made * that at first it was thought that chemical reagents would be ideal as stimuli. It was soon discovered, however, that the presence of minute quantities of a foreign liquid is such a disturbing factor that stimula- tion by salt solutions could not be used for quantitative experiments. With a qualitative analysis, however, I found a variety of evidences which show that the nerve stimulated chemically gives off more COs, and that the nerve rendered less excitable by reagents decreases CO» production. When each sciatic nerve of a frog is isolated and one is left in the normal saline in one case, and in the body of the frog in the other, for the same length of time, and then transferred to the two chambers of the apparatus, if the quantities of the precipitate are compared, it is found that the nerve which has been in normal saline gives more CO.. 31 Hitt: Journal of physiology, 1912, xliv, p. 481. 32 Fuller discussion of these will appear in a subsequent paper. 33 MATHEWS: This Journal, 1904, xl, p. 455; 1905, XIV, p. 203. 126 Shiro Tashiro It is known that normal saline stimulates frog’s sciatic nerves. The different rates at which COs: is produced from the different nerves treated by various concentrations of KCl is equally instructive. It is known that when a nerve is placed in a molecular solution of KCl, a stimulation takes place for a considerable time. Then it finally becomes unexcitable,** whereas, .2 m. KCl solution abolishes nervous excitability in a short time without primary stimulation. The CO, production follows exactly analogous to this. The nerve treated with the stronger solution gives more CO: than that of a weaker solution. This was true even after both nerves became unexcitable, showing that the nerve must be giving off more CO, while being stimulated by the stronger solution. Although my quantitative data are not complete at this stage, this preliminary statement is sufficient to show that the nerve chemically stimulated gives off more CO:. It may be added in passing that the different solubility of CO: in the different concentrations of these salts solutions cannot explain these results solely by a physical interpretation, for there is not enough difference in the solubility of CO: in dilute equimolecular solutions of KCl, and NaCl, whose effect on CO, production is so divergent, the former salt diminishing, the latter increasing it. Heat Stimulation. — It may be recalled in Table I that high tem- perature increases the output of CO. from the resting nerve. A respiratory process should increase proportionally to the temperature. Raising of temperature, however, not only increases the rate of res- piration, but also (particularly by sudden changes of it) stimulates the nerve. A very interesting fact is observed in connection with the killing of the nerve. When the nerve is killed gradually by a slow increase of temperature, it gives off more CO, than when killed suddenly, the determination being made after both are killed. CO, production from the dead nerve under this condition must be due to the diffusion of the gas which was formed previously, just as Fletcher’s dead muscle is charged with CO» gas. The different outputs of CO between slowly killed and suddenly killed nerves cannot be accounted for unless we assume that in one case, CO: is produced more while being killed than in the other. Whether such increase of CO: produc- tion, however, was due to the acceleration of normal respiration by the slowly increasing temperature, or due to direct stimulation caused 34 MatuHews: Joc. cit. | / Carbon Dioxide From Nerve Fibres 1297 by heat, or due to both, cannot be decided here unless we consider the relation between excitation and tissue respiration.*® It is hoped that we may have a better understanding of this matter when we study the temperature coefficient of normal respiration of the nerve. At present, we are satisfied to state only that there is a strong evidence to support the conclusion that heat, too, increases CO, production from the nerve. DISCUSSION OF THE RESULTS Comparison of Metabolism of Non-Medullated and Medullated Nerve. — Although it appears ridiculous to attach any significance to the marked similarity in the magnitudes of CO, production from non- medullated and medullated nerves, the temptation is irresistible to comment on the high output of CO, from the non-medullated nerve fibre. Let us study the Table following (Table VIII), in which a summarized comparison is given. TABLE VIII Rate of Nerve CO, from CO, from increase resting nerve stimulated nerve of CO, Non-medullated (spider crab) 6. Medullated (frog) | 5 | 16. X 107 g. (14° — 16°) |2.4 time 07 g. (15° — 16° ) 07 g. ( 14.2 X 107g. (20° — 22°) |2.6 “ -7 g. 19° — 20° ) | Since I have found that injury increases the CO, production from the nerve, the values I have obtained from cut, or isolated, fresh resting nerves, such as I had to use, may be somewhat greater than the output of normal uninjured nerves would be. But since Alcock has shown that a non-medullated nerve gives a higher electrical response, both in the negative variation and the injury current, the COs increase due to the cut alone will probably be greater in case of the non-medullated nerve than in that of the medullated one. That means that the value of the CO, production for the resting uninjured, 35 See p. 134. 38 Arcock: Proceedings of the Royal Society, 1904, Ixxiii, p. 166. 128 Shiro Tashiro non-medullated nerve should be reduced more from the figures found for the isolated nerve, than that of the medullated one. In other words, by lowering 6.7 X 10-7 gram which is the value for resting, non- medullated, isolated nerves, the rate of increase of CO» by stimula- tion in the uninjured nerve would become higher than 2.4 times, and probably higher than 2.6 times, which is the rate for the medullated nerve. This greater effect in the non-medullated nerve is what we should expect if our present conception that conduction is in the axis cylinder only, is correct. Before any accurate comparison of the increase of CO: production on stimulation of non-medullated and medullated nerves can be made it will be necessary, however, to determine how much of the CO: from the resting nerve is due to injury alone. Before we consider this point seriously, also, we should deter- mine the metabolic activities of greater numbers of nerves of different animals. Such an investigation is at present useless until we deter- mine more quantitatively the relation between CO, production and the various strengths of stimulation and the degree of excitability. If any uniformity of CO, output in respect to anatomical varia- tions is discovered, light may be thrown on the function of the medullary sheath and other differentiations. However insignificant these results may be as far as the similar rates of the gas production of these two nerves is concerned, it should be strongly emphasized that technical error plays no part in these determinations. Inasmuch, as we are dealing with such an extremely small amount of the gas, it is quite natural for those who are not familiar with my apparati to suspect, by a hasty inspection of my results, that the small differences I found under different metabolic conditions may be due to mere experimental variations. For this reason, particular attention is called to a detailed description of the quantitative method I used, especially the footnote on page 144, where I have cited a series of determinations of unknown quantities of CO, in testing my apparati. I may repeat here that my experi- ments with the spider crab and the winter skate were done at Woods Hole*®’ during the summer of tro11, while those with the frog were done in Chicago during the winter of 1912. Under these different conditions, I have not only used the different sizes of nerves, but also 37 T take great pleasure in acknowledging my indebtedness for the kind accom- modation offered me by Drs. Lillie and Drew at Woods Hole. Carbon Dioxide From Nerve Fibres 129 experimented with two different apparati, the respiratory chambers of which have had entirely different capacities.** Comparison between the Metabolism of Resting Nerves and that of Other Tissues. — To compare the rate of metabolism of the nerve with that of other tissues is a matter of no great physiological value on account of great variations which do not affect equally the rate of CO, production Simply to give a better picture of the scope of nervous metabolism, however, let us make the following comparison: Since there is no exact determinations made on either the other organs, or the whole animal, in the case of the spider crab, I have quoted those of the nearest crustacea of which data are available. (Table IX). TABLE IX CO: per Kg. per hour Temperature | Determined by! Animals Crustacea (whole animal) Jolyet and Regnaut Cray fish (A stacus) SWoll lS Crab (Cancer pagurus) 89.9 c.c. obster (Homarus vulgaris). . . . 54.4 c.c. erve of spider crab (Labinia canit- liculata) DAQIGG: - Tashiro Frog: (Rana esculenta) (whole animal) . .082 gms.| Schultz (Rana temporaria) (whole animal) 2355128 - Pott (Rana pipiens) (sciatic nerve) .| 33 Tashiro (Rana temporaria *) (isolated) muscle) : Fletcher ‘Regnaut and Reiset Pettenkoffer and Voit Speck 1 All the figures are quoted from Schifer’s Text Book of Physiology i, pp. 702, 707 and 708, except that of the isolated muscle which I calculated from Fletcher (loc. cit.). Fletcher fails to state the weight of a leg, but gives the value .2 c.c. for one-half hour. Hill believes that if we take each leg 6 g. in average, the value will not be far from the truth. 2 Fletcher fails to state the species of the frog, but it is inferred from Hill’s paper. 38 See the last columns of Table I and Table II. 130 Shiro Tashiro Active Nerves. — That the nerve increases its CO. production approximately 2.5 times when stimulated, is in accordance with our conception of the metabolism of other acting organs. Just how much increase of CO, takes place during functional activity of an organ or organisms depends on conditions as well as on habits of different organs and animals. Pettenkofer and Voit ®* report that a man (weighing 70 kgs.) gives off when working 0.76 grams per kg. per hour, while resting only .56 gram. Barcroft*® found that the submaxillary gland when stimulated by the chorda tympani gives off 3-7 times more CO, than the resting gland. In the case of contracting muscle, the results are very contradictory. Hermann * found that the contract- ing muscle gave off 9.3 per cent of CO» (by volume) while the resting one, only 1.4 per cent. Tissot ** and other workers also found a similar increase of CO, from contracting muscle. Minot,* working with Ludwig, maintains that there is no relation whatever between CO, production and muscle tetanus. L. Hill** and Fletcher * both con- firmed Minot’s work by finding no increase of CO2 production from muscular tetanus. According to Fletcher, the increase he found in CO, production from a contracting muscle in a closed vessel is due to the rigor. Under this condition, he believes, increased formation of lactic acid is responsible for liberating CO, already produced. In elther case, it is understood that functional activity in the muscle is accompanied by an increase of metabolic activity. It is difficult to compare this increase of metabolic activity of the muscle with that of the nerve unless we determine how much and what ! ind of metabol- ism takes place in contracting muscle. Respiration Quotient of the Nerve Fibre.— As quoted before Haberlandt found that a resting nerve consumes 41.7 to 83.4 cmm. O, for x gm. for an hour at 19° — 24°. Although he has not deter- mined chemically the production of COz he could easily read the respiration quotient by means of the index fluid. Thus he found 39 PETTENKOFER and Voir: Joc. cit. 40 Barcrorr: Ergebnisse der Physiologie, 1908, vii, p. 735. 41 HERMANN: Stoffwechsel der Muskeln, Hirschwald, Berlin, 1867. 4 Tissot: Archives de physiologie, 1894-5, (5) vii. p. 460. 43 Minot: Arbeiten aus der physiologischen Anstalt zu Leipzig, 1868, p. tI. _“ L. Hrrz: See Schifer’s Text Book of Physiology, 1898, 1, p. 911. 45 FLETCHER: Journal of physiology, 1898-9, xxiii, p. 68. Carbon Dioxide From Nerve Fibres 131 that the respiratory quotient of the resting and acting nerve is nearly unity. Since he found that O, consumption is increased when stimu- lated, and since the respiration quotient remains constant before and after the stimulation, he concluded that it must give off more CO2 when stimulated. It is very interesting to compare the O. consump- tion in this experment with the CO, production of mine.” Taking his lowest figure, because he worked in 19° — 24° and I in 19° — 20°, 41.7 cmm. of O, amount to .00007 cc. for ro milligrams for ten minutes. My figure of 5.5 X 10-7 grams for the same units may be translated to .00027 cc. of CO» (ignoring temperature and pressure ; CO. 00027 f ; correction). Therefore = = 3.8,the respiratory quotient. O, 00007 As Ihave not determined O, consumption of the nerve of Rana pipiens, this figure has no particular value, but the fact that the CO, produc- tion is comparatively higher than O2 consumption is a matter of considerable interest. One of the most important observations made by A. V. Hill * is the fact that he could not detect any rise of temperature in a frog’s nerve as measured by an apparatus which is sensitive to a change of one-millionth of a degree. From this, according to his calculation, he concludes that not more than one single oxygen molecule in every cube of nerve of dimension of 3.7 » can be used up by a single propa- gated nerve impulse. Therefore, he suggested that an impulse is not of irreversible chemical nature but a purely physical change. Although, I confess, my ignorance makes it impossible to interpret his valuable results from my observations, I may add that these two apparently irreconcilable facts may throw light on the true nature of nervous metabolism. Dr. Mathews has suggested that metabolism in the nerve may be something of the order of alcoholic fermentation, which is not a direct oxidation, and where heat production cannot be so large as CO» production, since the energy content of glucose is only a trifle higher than that of the alcohol produced. The compara- tively little heat production in the case of working glands is a matter of interest in this connection. At any rate we should not forget the 46 He used Rana esculenta, which, by the way, gives for the whole animal .082 g. CO» per kg. per hour at 17° according to Schultz. My frog was Rana pipiens. 47 Hii: Journal of physiology, 1912, xliii, p. 433. 132 Shiro Tashiro anatomical as well as the chemical differences between muscle and nerve. In this respect the ratio between CO: production and O, consumption from the nerve is suggestive. The extremely small intake of O»2 has another point of interest in relation to the general nature of irritability. It has been repeatedly reported that a nerve can remain excitable several hours in an oxygen- free atmosphere, although there is no doubt its excitability diminishes, yet there is a considerable amount of evidence to show that oxygen is very closely associated with the state of excitability. To har- monize these two facts, the oxygen-storage hypothesis has been suggested, by which the exhaustion is attributed to complete consump- tion of the stored oxygen and that excitability is restored when atmospheric oxygen is readmitted. Without committing ourselves to this hypothesis, I may add that according to Haberlandt’s figure, the resting nerve of 1o milligrams will consume only .0042 cc. O:2 in ten hours. If we take our figure and assume that one volume of oxygen was necessary to produce one volume of CO, (this assumption is made without any significance except to give a liberal estimate), the CO production would require about .or5 cc. of O» for ten hours. And if we assume again that activity will increase O. consumption in propor- tion of CO, production, then it means that the nerve when stimulated takes up only .o3 cc. of Oz, during ten hours stimulation. I am not aware, at present, of the existence of any method which will surely remove O: as completely as this from a large vessel; and this is a very liberal estimate. My experiences in rendering the air free from CO, encourages me to raise the question, How can one remove every trace of O, from a nerve fibre? Without having a correct criterion for an oxygen-free medium we cannot at present consider definitely any question of the relation of O, to irritability. CONCLUSION In spite of all the negative evidence against the presence of meta- bolism in the nerve fibre, we have established three important facts: namely, (1) A resting nerve gives off a definite quantity of carbon dioxide; (2) stimulation increases CO, production; and (3) CO, production from the resting nerve proportionally decreases as irri- Carbon Dioxide From Nerve Fibres 133 tability diminishes. These facts prove directly that the nerve con- tinuously undergoes chemical changes, and that nervous excitability is directly connected with a chemical phenomenon. There is still another question left, namely, Is there any direct relation between excitability and tissue respiration? To put this question more directly, we may ask: Does excitability depend on the respiratory process in the protoplasm? To answer these questions we must refer to two facts; namely the direct relat on between the rate of respiratory activity and the decrease of excitability; secondly, the influence of reagents on CO, production and their effects on the state of excitability. By the studies of CO, production by Fletcher * lactic acid forma- tion by Fletcher and Hopkins,” and heat evolution by A. V. Hill,°° it has been established that in isolated muscle, respiratory processes decrease when irritability diminishes. In the case of the nerve, as shown in Table 3, CO, production reaches this minimum when excitability approaches zero. These relations, however, do not show conclusively that the protoplasmic irritability depends on respiratory activity, for it is quite probable that the dying nerve may alter its physical condition as well, which according to the physical school, may consequently alter the state of excitability. That irritability is independent of the respiratory processes has been hitherto successfully contended in the case of the dry seed. The works of Horace Brown, Thiselton-Dyer * and others indicate that the dry seed can be kept alive at the conditions where no ordinary gaseous exchanges are possible. It is argued, therefore, that life is possible without any metabolic activity.°” While a definite poten- tiality for irritability may exist without any metabolic activity, yet that the irritability can persist without respiratory activity, or vice versa, is a matter by no means settled. In the case of ordinary air-dry seed, Waller could demonstrate the response of electrical changes when stimulated although the detection of CO, was impossi- 48 FLETCHER: Joc. cit. 49 FLETCHER and HopKINs: Joc. cit. By aa. ELGEL: boc. cit. 5 THISELTON-DYER: Proceedings of the Royal Society, 1897, lxii, p. 160; ibid., lxv, p. 361. 8 T am indebted to Professor Crocker for his kind suggestion as to botanical literature. 134. Shiro Tashiro sible. This failure, however, as he himself expected, was due to the lack of delicacy of the chemical methods for detecting CO». I ob- served, with my apparatus that even a single kernel of a dry seed gives off a definite quantity of CO, as long as it is alive. In ordinary condition not only a living dry seed gives off more CO, than the dead one, but also like the nerve, it always gives off more CO, when stimu- lated by mechanical injury. In the normal condition, therefore, we may safely conclude, there is always metabolic activity as long as the seed is irritable, and that in the different states of irritability, the respiratory activity is proportionately different. At present, therefore, we have no decided evidence which will prevent us from considering excitability as a function of respiration under ordinary conditions. This relation is more directly studied by the use of anaesthetics. I have already demonstrated that an etherized nerve gives off considerably less CO. than the normal. Such an etherized nerve will not give more CO, when it is crushed. This may be interpreted by some to mean that the etherized nerve may be already dead. This, however, is not the case. This objection, also, I have considered by studying the nerve treated with KCI. When the nerve is treated with .2 m KCl and then crushed, it does not give an increase of CO, production. Mathews has shown that while a .2 m. KCI solution renders the nerve unexcitabie, yet it will recover its excitability by being replaced into n/8NaCl. These two facts, therefore, support the idea that any agents that suppress excita- bility of the nerves also decrease the CO2 production and that COs production by crushing the nerve must be largely due to stimulation. This hypothesis is strikingly supported by similar observations on the dry seed. Etherized seeds give much less CO, and cannot be stimulated to give more CO, by crushing, while under normal con- ditions, crushing a seed always increases its CO. production. Quan- titative experiments in this direction will be given in another paper. These facts directly support Mathews’ hypothesis that substances which suppress irritability must act on the tissue respiration pri- marily. If such an hypothesis is correct, we can easily picture what is happening in the nerve fibre. Vernon considers that a tissue contains certain substances which can absorb oxygen from their sur- 88 VERNON: Journal of physiology, 1909-10, xxxix, p. 182. lt ae Carbon Dioxide From Nerve Fibres 135 roundings to form an organic peroxide, and by the help of a peroxidase can transer this to amino acid and carbohydrate molecules bound up in the tissue, just as H2 O2*! can oxidize, with the help of an activator, an acid of formula R. CHNH, COOH to COs, NH; and an aldehyde RCHO, and then oxidize this aldehyde to RCOOH and ultimately to CO, and H:.O. Poisons such as HNC, NaHSO; and NaF, which he found to decrease CO, production, temporarily paralyzed respiration, he thought, by uniting with aldehyde groups, while formaldehyde, acid and alkali temporarily paralyze CO, forming power of the tissue by destroying the peroxidase. The organic peroxide, though it can still affect some oxidation, cannot of itself carry it to the final CO, stage. Recovery of CO, forming power is due to the regeneration of the peroxidase. Although I doubt that such a process occurs in nervous respiration, the idea of two similar metabolic phenomena involved in the nervous metabolism is very helpful to understand the behavior of the nerve during continued activity. Most recently Tait discovered that a refractory period has two phases, absolute and relative.*® When he treated the sciatic nerve of a frog with yohimbine, the relative phase is greatly prolonged, while the absolute one is little affected, a result quite different from other common anaesthetics. Waller®® has already observed that protoveratrin slows up the positive variation of the nerve, while the negative variation is little in- fluenced. Waller contends that this drug does not alter cata- bolic change, but retards anabolic activity to a considerable degree. Since pharmocological action on animals of protoveratrin and yohimbine are very similar, Tait concludes that these drugs must attack the nerve in similar manner, and that a refractory period, toc, must consist of two phases corresponding to the catabolic and anabolic processes which Waller observed in the case of protovera- trinized nerves. Thus, he considers that his “ absolute phase”’ of the refractory period corresponds to negative variation or catabolic process of the nerve, and the “ relative” to the positive return or anabolic. Yohimbine, in other words, retards anabolic processes con- siderably, thus prolonging the refractory period, or increasing nerve & Dakin: Journal of biological chemistry, 1908, iv, pp. 63, 77, 81, 227. 8 Tart: Journal of physiology, 1912, xl, p. Xxxviil. 56 WALLER: Brain, 1900, xxiii, p. 21. 136 Shiro Tashiro fatigue easily. These considerations suggest very strongly that the absence of fatigability in the nerve as measured by the ordinary methods, is not a question of absence of metabolism, but merely the speed by which these two processes come to an equilibrium. Although we have an infinite number of facts still unexplainable, by our present knowledge of nerve physiology, we have established a few new facts around which we may build up some idea concerning this most essential phenomena of living matter, —i.e., irritability. As to the true nature of the nerve impulse, I can only confess my ignorance. SUMMARY t. All nerve fibres give off CO». The resting, isolated nerve of the spider crab produces 6.7 X 1to-7 gram per 10 milligrams per ten minutes. The frog’s sciatic 5.5 X 107’ grams. 2. When nerves are stimulated they give off more CO:. The nerve of the spider crab claw produces 16. X 10-7 gram when stimu- lated, the frog nerve 14.2 X 10’ grams. The rate of increase of CO, by stimulation amounts to about 2.5 times. 3. The CO, output of resting nerve is due to a vital active process. 4. Anaesthetics greatly reduce the carbon dioxide output of nerves and dry seeds. 5. Mechanical, thermal and chemical stimulation also increases the carbon dioxide output of nerves. 6. Single dry living seeds (oat, wheat, etc.) react in most par- ticulars similar to nerves as regards their irritability, relation to anaesthetics, mechanical stimulation and carbon dioxide outputs. 7. The general conclusion is drawn that irritability is directly dependent upon and connected with tissue respiration and is primarily a chemical process. These results strongly support the conception that conduction is of the nature of a propagated chemical change. To Prof. A. P. Mathews, under whose direction I have carried on these experiments, I express my appreciation and gratitude. For many suggestions, I am under obligation to Dr. F. C. Koch. a se) a) A NEW METHOD AND APPARATUS FOR THE ESTIMATION OF EXCEEDINGLY MINUTE QUANTITIES OF CARBON DIOXIDE! By SHIRO TASHIRO [From the Department of Biochemistry and Pharmacology, the University of Chicago, and the Marine Biological Laboratory, Woods Hole, Mass.] N connection with the study of the metabolism of the nerve fibre, I undertook, at the suggestion of Prof. A. P. Mathews, to work out a method for the detection of exceedingly minute quantities of carbon dioxide. Following a suggestion made by Dr. H. N. McCoy, a very simple method was devised, which I reported first to the Chicago Section of the American Chemical Society; * later in con- junction with Dr. McCoy, its further details were reported to the Analytic Section,’ of the Eighth International Congress of Applied Chemistry. The principle of the new method is as follows: 1. Exceedingly minute quantities of carbon dioxide can be precipi- tated as barium carbonate on the surface of a small drop of barium hydroxide solution. 2. When a drop of barium hydroxide is exposed to any sample of gas free from carbon dioxide, it remains perfectly clear, but when more than a quite definite minimum amount of carbon dioxide is intro- duced, a precipitate of carbonate appears, detectable with a lens. 3. By determining, therefore, the minimum volume of any given sample of a gas necessary to give the first visible formation of the precipitate, its carbon dioxide content can be estimated accurately, since this volume must contain just the known detectable amount of carbon dioxide. 1 One of these apparati was described at the biochemical section, Eighth International Congress of applied chemistry, September, 1912; see also, Journal of biochemistry, 1913, xiv, p. xli. 2 May 18, 1912. 3 Original Communication: Eighth International Congress of applied chemis- Ery, FOr2, 1, p. 361. 138 Shiro Tashiro I have constructed two apparati, based on this principle, which are especially adapted for the estimation of the output of carbon diox- ide for very small biological specimens. With these apparati, one cannot only detect easily a very small amount of gas, given off by a small dry seed, or a small piece of a frog’s sciatic nerve, but can also estimate it with considerable accuracy. The apparatus shown in Fig. 1 consists of two glass bulbs. The upper bulb A, is a_ respiratory chamber, having a capacity of about 15 c.c., which can be diminished to 9 c.c. by means of mercury. The lower bulb B is an analytic chamber with a volume of 25 c.c., which can be made to 5 c.c. by filling up with mercury. These two bulbs are con- nected with a capillary stop-cock D. The respiratory chamber is fitted with a tight glass stopper, R, which is connected to a three-way capillary stop-cock C. This glass stopper is so arranged that the chamber can be sealed by putting mercury above the stopper. The tubes are thick walled capillaries of about 1 mm. internal diameter, excepting upturned tubes inside the bulbs, which should be rather thin walled, especially at F and H, where it is widened to an internal diameter of about 2 mm. It is important that the glass of which these tubes are made should be of a quality not readily attacked by barium hydroxide. The details of the method of procedure are as follows: The apparatus is first cleaned and dried. The specimen is FIGURE 1 One-third the actual size. 4 The apparatus is made in such a way that it can be cleaned and dried in ten minutes without being taken apart. For this, the stop-cock D is closed and E and L are opened. The arm at L is connected to the suction pump. Then a little acidulated water is introduced through G. By closing E, and opening D and G, the excess of water is drained off. Then the process is repeated with dis- tilled water, alcohol, and alcohol ether. The last drying is completed by passing a current of air through G while D is closed. Apparatus For Estimating Carbon Dioxide 139 placed on a glass plate® and weighed. The glass plate is hung on n and m, which are electrodes fused into the side of the respiratory chamber A. The chamber is now closed with the stopper R and sealed with mercury. Through L, a connection is made with a pump °® and about 20 c.c. of mercury is introduced through G. Not too much mercury should be used; its surface should not be within 5 mm. of the cup F. Then wash the whole apparatus with carbon dioxide-free air,’ which is introduced through C, by successive evacuations. After the evacuation and washing out with pure air, which is repeated three or four times, the pressure inside of the bulbs is made equal to the atmospheric pressure by adjusting it at the nitro- meter in the usual fashion. Stop-cock E is then closed, and the space between E and L is evacuated so that the barium hydroxide can rush in, a process which is very advantageous to obtain a clear barium hydroxide solution. Then clear barium hydroxide solution isrunin through L. By opening E very slowly and carefully, the solu- tion is now introduced into the chamber so that a small drop stands up upon the upturned end of the capillary at F. Then the connection between the two chambers is closed by D. It is imperative that this drop of the solution should be perfectly clear at the start. If no deposit of barium carbonate forms on the surface of the drop within ten minutes,® a portion of the sample gas is drawn into B by with- drawing mercury through G and opening the stop-cock D. The volume of mercury withdrawn, which may be readily determined by volume, or more accurately by weight, gives the volume of the sample 5 The kind of glass plate used in connection with the nerve and small animals like Planaria is shown on p. 120, Fig. 1. (The first paper.) 6 The pump should be capable of giving a vacuum of at least 25 or 30 mm. of mercury. 7 Air cannot be freed completely from carbon dioxide by passing it through wash bottles. In my work, carbon dioxide-free air is prepared by shaking air with twenty per cent solution of sodium hydroxide in a tightly-stoppered carboy, fitted with suitable tubes. When this is to be used, it is driven into a nitrometer which is filled with less concentrated alkaline solution (a weak solution is used so that the chamber may not be too dry) by displacing it by running in a solution of sodium hydroxide. After each evacuation, this air is introduced from the nitro- meter into the chamber A through stop-cock C. 8 If no precipitate appears within ten minutes, it is a sure control that the apparatus is free from carbon dioxide. 140 Shiro Tashiro gas taken from the respiratory chamber, since the pressure in A and B is kept equal to the atmospheric during the transfer. One now watches the surface of the drop at F with a lens to see whether any formation of barium carbonate occurs within ten minutes. With this apparatus, I have repeatedly introduced accurately known quantities of carbon dioxide of very high dilution into B in the manner just described and as a result have found, with remarkable regularity, that 1.0 X 1077 gram of carbon dioxide is the minimum amount which will cause a formation of barium carbonate within a period of ten minutes. Smaller amounts of carbon dioxide give no visible results; while larger amounts give a deposit more rapidly, and appear in larger quantities. This minimum detectable amount I.o X 107 gram is about the amount which is contained in @ c.c. of natural air, in which we assume 3.0 parts of carbon dioxide in 10,000 by volume.® In order to determine the concentration of carbon dioxide in the respiratory chamber, one must first find, for the apparatus used, the minimum detectable amount of carbon dioxide. Then one finds, by trial,!° the minimum volume of gas necessary to give. the first visible formation of barium carbonate. This volume must, therefore, con- tain the known minimum detectable amount of carbon dioxide. From the ratio between this volume and the original volume of the respira- tory chamber, out of which this amount is withdrawn, the absolute ® Letrs and BLAKE: Proceedings of the Royal Dublin Society, 1899-03, ix, Pp: 107. 10 In the case of biological problems, when the specimen gives off carbon dioxide continuously, and sometimes at different rates, varying with the time, it is much simpler not to attempt to determine the minimum volume by a continuous trial with the same sample; but instead to repeat the experiments with a series of samples of known weights for a known time, and determine the minimum volumes which give the precipitates, and the maximum volumes which do not give the precipitates. In this way, it can easily be calculated what is the mini- mum volume which gives the precipitate for the given weight of the specimen for agiventime. Table I on page 114 will illustrate this more clearly. Another upturned cup H provided in the respiratory chamber A is used in case only the qualitative detection of COz is wanted. In sucha case, the perfectly clear barium hydroxide solution is introduced, after the necessary cleaning and washing, to the respiratory chamber, forming the usual drop at H instead of F. It should be noted that in case a smaller capacity is necessary for the respiratory chamber, the mercury is introduced by a pipette to the bottom of the chamber at K. Apparatus For Estimating Carbon Dioxide 141 quantity of carbon dioxide, given by the specimen, may be computed. At the suggestion of Dr. F. C. Koch, another apparatus was con- structed, which provides a control drop of the barium hydroxide solution, side by side with the other. The apparatus (Biometer) shown in Fig. 2, although it appears complex, is nothing more than apparatus 1, inclined go°, but each of its chambers is provided with a barium hydroxide cup d and f. It is made of glass consisting of two respi- ratory chambers, serving also as analytic chambers, connected by a three-way stop-cock L, the other arm of which is connected to one arm of another three-way stop-cock K. Each of the other two arms of stop-cock K is connected to a nitrometer W and X. The nitro- FicureE 2. Biometer, one-third actual size. The shaded portions of the apparatus indicate the rubber connection which is first coated by shellac, and then sealed with a special sealing wax. Some parts are also sealed with mercury. meter on the right, is connected toa carboy with air free of CO; and the other, on the left, to a similar reservoir with air free of CO: plus any gas which is desired as a medium for conducting the experi- ment. Chamber A is drawn to a capillary stop-cock C; chamber B is drawn to the three-way stop-cock G, one arm of which is con- nected with a mercury burette T, which is used for adjusting the pressure. Both of the chambers have a capacity of 20 to 25 c.c. and are provided with a pair of platinum electrodes n and m, and also with the glass stoppers S and R, which can be sealed as usual with mercury. The pump is connected through J, and the barium 142 Shiro Tashiro hydroxide solution is introduced through V to d and f, where drops are formed as before. As stated above, this apparatus can be used for the combined purposes of qualitative detection, quantitative estimation, and com- parative determination of the output of CO, from the various biolog- ical specimens. It has a decided advantage over the other in the fact that we have a control drop, side by side, under exactly the same conditions, and that the comparative estimation of CO, produced by different specimens can be made very easily and accurately. The de- tailed method of procedure is described under three different headings: (a) For the Qualitative Detection of Carbon Dioxide. — After the apparatus is cleaned and dried," a weighed tissue is placed on the glass plate and hung on n and m of the chamber A, and no tissue in the other chamber. After both chambers are closed with the stop- pers S and R and sealed with mercury, they are so filled with mercury that the remaining volumes in both chambers are now exactly the same. The chambers are now evacuated and washed with pure air. When evacuation and washing with pure air is complete, the pressure is made atmospheric, by adjusting with the nitrometer the connec- tion between A and B is then closed with stopcock L. If any CO: is given off by the tissue, the desposit of carbonate will soon appear on d, while in the control chamber the drop on f remains perfectly clear. In order to avoid any possible error of a technical nature this experiment is repeated by exchanging the chambers, now using chamber B for the respiratory chamber and the other A as a control. (b) For Comparative Estimation of CO, from Two Different Samples. By repeated quantitative experiments, it was found that the speed with which the first precipitate appears and the sizes of the deposits on the drops at d and f represent corresponding quanti- ties of carbon dioxide. Thus with remarkably simple means, we can determine simultaneously the comparative outputs of the gas from two different tissues or from the same tissues under different conditions. The method of procedure is best illustrated by the following example. Two pieces of the sciatic nerve are isolated from the same frog and exactly weighed. One piece is laid on one glass plate, and the other 1 This, too, can be cleaned and dried without being taken apart. See foot- note on p. 138. Apparatus For Estimating Carbon Dioxide 143 on the other plate in such a way that one part of the nerve lies across the electrodes of the glass plates as shown in Fig. 1, page 120. In this way, when the plates are hung on the electrodes n and m, any desired nerve can be stimulated with the induction current. These plates are now hung on the electrodes in each chamber, and the usual procedure is followed for the cleaning and the washing of the appara- tus to make it CO, free. After the connection between the two chambers is closed by means of stop-cock L, the nerve in chamber A is stimulated by the current. Then if one can watch over the surfaces of the drops carefully from the start, he finds the first deposit of the carbonate on cup d of chamber A in which the stimulated nerve is placed. Later, the total amount of the precipitates grows much larger in the case of this cup. This increased output of the carbon dioxide from the stimulated nerve, thus observed, can be duplicated by repeating the similar experiment, after exchanging the chambers, as usual. This comparative estimation can be more accurately made by exact quantitative measurement, the method for which the follow- ing will illustrate. (c) For Quantitative Measurement of Gas.— The detailed method is exactly analogous to that of apparatus 1. Here we use chamber B as the respiratory chamber and A as the analytic cham- ber. Barium hydroxide should be introduced into chamber A only at d, and the stop-cock F is always closed except at the time of wash- ing. The pressure should be adjusted by mercury burette T, or by the potash bulb of the nitrometer. In case the mercury burette is used, the remaining volume in the respiratory chamber should be recorded.!?. The introduction of a known amount of gas from the respiratory chamber B to the analytic chamber A is accomplished by withdrawing the mercury from C into a very narrow graduated cylinder, while the stop-cocks L G and H are opened. After a quick adjustment of the mercury burette to equalize the pressure, the stop- cock L is closed and the presence of carbonate is looked for exactly in the same manner as described in connection with the other appara- tus, determining the minimum volume that gives the precipitate for the known mass of tissue for a known time. 2 The bulbs are marked at the point where their capacity became 15 C. Cc. by introducing mercury. The variation of capacity can easily be read by noting the mercury burette. 144 Shiro Tashiro In summarizing, I may emphasize the following points: 1. Particular care must be taken to test the air-tightness of the apparatus. 2. Purifying the air must be done with greatest care, as this is essential. 3. The apparatus must be perfectly dry. 4. A weak suction pump cannot be compensated by frequency of washing. 5. As long as the ratio between the c.c. taken from the chamber and the original volume of the chamber is needed, it is most important to have the pressure in A and B equal to the atmos- pheric. If this is accomplished we can neglect any caution against pressure and temperature variations — a correction which is always necessary for ordinary methods of analysis of exceedingly minute quantities of any gas. In devising this method and in constructing this apparati, I am under great obligation to Professors McCoy and A. P. Mathews and to:Dr, BF C.. Koch: . In order to test the accuracy with which an estimate of concen- tration of carbon dioxide could be made, many determinations were carried out with samples of air which contained accurately known concentrations of carbon dioxide prepared by Dr. F. C. Koch. The experimenter did not learn the concentrations of the samples until after the analysis had been completed. In making up the test sam- ples, pure carbon dioxide, made by heating sodium bicarbonate was diluted with the carbon dioxide free air several times in succession, as illustrated by the following example: 5.5 c.c. of pure carbon diox- ide was diluted to 52.0 c.c. over mercury and thoroughly mixed; 5.5 c.c. of the first mixture was diluted to 52-0) 'c.c:} “an geemae the second was diluted to 50.7 c.c.; of this third mixture 5.6 c.c. was received from Dr. Koch. I diluted this a fourth time to 255.6 c.c. to form a mixture to be analyzed. The following observa- tions were made: 0.5 c.c. was introduced into the apparatus and pro- duced no precipitate in ter’ minutes; 0.5 c.c. more of the same sample, gave no precipitation in another interval of ten minutes; 0.5 c.c.more, a total of 1.5 c.c., was run into the bulb. In six minutes the first evidence of a precipitate appeared on the surface of the drop at d of apparatus 2 and in eight minutes was well developed. Since Apparatus For Estimating Carbon Dioxide 145 the amount of carbon dioxide required to give the precipitate is 1.0 X 1077 grams, this amount is contained in 1.5 c.c. of the sample or 1 c.c. contained 6.7 X 10° grams of carbon dioxide. The amount of carbon dioxide actually contained in the sample was em Oe 55 OK 7. OK 50 Bax 92 X. 50.7 255.0 In six such determinations, all made with samples the concentra- tion of which were unknown to the experimenter at the time of the analysis, the results given in the following table were obtained: c.c. = 6.2 X 10° grams. Volume of sample re- Weight of carbon dioxide in one c.c. quired to give a precipitate Found Taken 10) ee 1.0 X 10’¢. 0.92 < 107 g. ESE G:C! Py ATS 230+ X10 Ss) (OSC 1.82 X 107 g. S35 10708: tlds) (ere) 67 << 10-" g. 0.62 X 107 g. DDS COE AS X 10-7 g. 0.45 & 10-7 g. STUDIES ON THE PHYSICAL PROPERTIES OF PROTOPLASM I. THe PHYSICAL PROPERTIES OF THE PROTOPLASM OF CERTAIN ANIMAL AND PLANT CELLS By “G.” LSRITE [From the Hull Laboratories of Biochemistry and Pharmacology, University of Chicago] INTRODUCTION \ LTHOUGH tthe living substance of animal and plant cells was correctly interpreted by Dujardin and von Mohl in the second quarter of the nineteenth century, almost nothing is definitely known of the physical state of protoplasm. Properties described by such adjectives as glutinous, slimy and hyaline were recognized by the early microscopists, who were forced to study liv- ing cells and tissues. During the last fifty years an extensive literature has grown up on the subject of the structure of protoplasm. For the purposes of this paper, these investigations may be divided into two groups. The first group comprises those studies on the structure of protoplasm, made with the aid of fixatives. A large part of our knowledge of the morphology of the cells and tissues of animals and plants is the direct result of the development of fixing methods. The errors involved in the attempts to determine the true molar structure of protoplasm by the use of fixing reagents have been pointed out particularly by Flemming,! Berthold,? Schwarz,’ Fischer,* and Hardy.’ In this connection, it will suffice to state that Hardy’s cun- clusion that fixing reagents always cause structural changes in pro- 1 FLEMMING: Zellsubstanz, Kern und Zelltheilung, 1882, Leipzig. * BERTHOLD: Studien iiber Protoplasmamechanik, 1886, Leipzig. 3 ScHWARz: Cohn’s Beitrage zur Biologie der Pflanzen, 1887, v, p. 1. 4 FiscHER: Archiv fiir Entwicklungsmechanik, 1901, xiii, p. I. ®5 Harpy: Journal of physiology, 1899, xxiv, p. 158. Physical Properties of Protoplasm 147 toplasm that are frequently very different from the normal living substance, has never been refuted. Hence, at least, it does not seem that more than an approximation of the actual structure of pro- toplasm can be attained by the use of fixatives. Besides, this method is worthless as a means of investigating the physics of protoplasm. The papers that fall in the second group deal with the study of living cells. Strassburger,® Wilson,’ Foot and Strobell,’ Lundegardh?® and others have shown that many of the structural elements of the mitotic figure can be seen in living animal and plant cells. Numerous investigators have pointed out the presence of granules, vacuoles, and fibrils in various types of unfixed cells; but for the most part, the studies on living cells have been made for the purpose of decreas- ing the error due to the use of fixing reagents. Ali such investigations are open to several sources of error. Hardy '° writes that the process of dying produces structural changes in the cell substance, since coagulation appears to occur in all dying cells. Many cells are certainly quickly asphyxiated when mounted for microscopical examination in the usual manner. I have been able to overcome, largely, this source of error, by the use of an open- end moist chamber, that does not appear to interfere with normal respiration. A second source of error is due to the nature of the optical prin- ciples involved in microscopical vision. Many years since Abbé ™ demonstrated that the optical image is a diffraction pattern produced by the object and that under certain conditions the image may be quite different from the object. More recently, Porter,'* experi- menting under ordinary working conditions, has described a number of interesting examples of this sort. Porter !2 says, “Images were formed which were utterly false in their smaller details, and other images were profoundly modified by the presence of structure lying 6 STRASSBURGER: Zellbildung und Zelltheilung, Jena, 1880, iii. Auflage. 7 Witson: Journal of morphology, 1899, xv, Suppl. 8 Foor and STROBELL: American journal of anatomy, iv, p. 199. 9 LUNDEGARDH: Jahrbiicher fiir wissenschaftliche Botanik, li, p. 236. 10 Harpy: Journal of physiology, 1899, xxiv, p. 158. 1 Aspe: Archiv fiir mikroskopische Anatomie, 1874, ix, p. 413; Gesam- melte Abhandlungen, 1904, i, p. 45. 2 PorTER: Philosophical magazine, 1906, ii, p. 154. 148 G. L. Kite entirely beyond the focal plane.” Such facts should serve to make it evident that one can easily fall into error in interpreting the optical image of a living cell. Porter recommends “a working knowledge of the phenomena and laws of diffraction,” as a safeguard against this form of error. The third and by far the most important source of error is due to the peculiar and little known optical properties of living matter. The phenomena of reflection, refraction, absorption, dispersion, inter- ference, diffraction * and a scattering action on light ** are all exhib- ited by this substance, with the result that a correct interpretation of the image of a living cell is frequently impossible. Further- more, many cells are so opaque and turbid that the interior is not visible. Cloudiness or turbidity is almost a universal property of protoplasm and appears to be due chiefly to dispersion, refraction, diffraction, and the scattering action on light of the colloidal par- ticles which may be considered as the real structural units of all protoplasm. Globules, granules and cell walls frequently show diffraction halos that are difficult to interpret in undissected cells. The aim of this investigation is to determine the physical state and the molar structure of protoplasm. The methods are radically different from those heretofore used, and are believed to be adequate for this purpose. Dissection and vital staining are used to deter- mine the truthfulness of the optical image and the actual structure of cells. Unfortunately, the amount of the error involved in the employment of these methods depends entirely on the skill of the experimenter; but it is believed that the error becomes quite small with complete mastery of the methods. The structural changes that cells may undergo during the time 13 Excellent expositions of the principles of physical optics are given by: Woop, R. W: 1011, Physical Optics; DrupE, P.: 1912, Lehrbuch der Optik; PRESTON: 1901, Theory of Light. 144 Lord Rayleigh (Philosophical magazine, xli, p. 107) has pointed out that reflection and refraction have no application unless the surface of the disturbing body is larger than many square wave-lengths. The turbidity of protoplasmic sols, then, is due entirely to the scattering action on light of the minute aggre- gates of the disperse phase, while reflection, refraction, diffraction, dispersion and a scattering action on light are all seemingly involved in the production of turbidity by gels. Physical Properties of Protoplasm 149 required for their dissection is a possible source of error that may appear, at first sight, to be very difficult to control. Certainly many biologists hold the view that cells rapidly undergo important morphological changes following mechanical injury. With few exceptions it has not been found difficult to follow the structural changes that occur in cells that are being dissected; but the really remarkable fact is the marked slowness of such death changes as granulation, fragmentation and general coagulation, following mechanical injury. It seems best to limit this introductory paper to a description of selected types of widely different cells and in future publications to treat systematically, selected types of the principal phyla of animals and the chief groups of plants. The special literature bearing on this investigation will be dis- cussed in subsequent papers. A review of such well-known theories as those of Biitschli, Flemming and Altmann lies outside the province of this paper. METHODS AND MATERIAL The development of a really adequate method for the dissection of living cells, under the highest powers of the microscope, has made possible this study. The principles of this method are simple. The dissecting instrument is a glass needle that may measure less than one micron in diameter and is drawn on the end of a piece, of special Jena glass tubing about 200 mm. long and 4 mm. in diameter. The needle is held in a three-movement Barber pipette holder. The cell chosen for dissection is mounted in a hanging drop in an open-end moist chamber and held in place by water-glass surface tension, which can be varied at will. Both diffuse sunlight and artificial light are used as sources of illumination. For the latter a Nernst Glower has been found satis- factory, but all the light waves outside of 450 and 670 PH are cut out by the use of appropriate ray screens. The same means is used to remove enough of the orange and red rays to make the transmitted light perfectly white. Such light is composed of the waves that are least injurious to living cells. A special condenser” of a focal dis- 15 The condenser was made by E. Leitz & Co., Wetzlar. 150 G, 1. Kite stance of about 20 mm., a 2 mm. apochromatic objective, compensat- ing oculars, and a number of vital stains, are necessary additions. An open-end moist chamber 25x 60x15 mm. has been found satisfactory. The bottom is separated into three compartments by very small glass rods and water is placed in the end compartments. If water be put in the middle compartment it may decrease the efficiency of the condenser. The chamber is held in a mechanical stage and most of the dissections are made by quick movements of the chamber and therefore of the cell being dissected, the needle remaining fixed. The use of acetylene which can be burned in a glass micro-burner has greatly simplified the making of extremely fine needles. An acetylene flame that is so small that it is invisible in a well-lighted room can be kept alive. The more important of the vital stains used include methylene blue, new methylene blue N (Cassella Color Co.), new methylene blue GG (Cassella Color Co.), new methylene blue R (Cassella Color Co.), janus green (Metz & Co.), pyronin (Griibler), vusuvin (Griibler), toluidin blue (Griibler), neutral red (Griibler). , The chief structural components of a cell can usually be quickly brought out by using a large enough number of vital stains, thus effecting a great economy of time, when the dissection of the unstained cell is made. Barber’s ‘® isolation and intracellular injection methods, vari- ously modified, are frequently employed to supplement and control the data obtained by dissection. Nomenclature Employed.— The current nomenclature of de- scriptive physics, physical optics and colloidal chemistry will be employed. Such physical properties as solidity, tenacity, elasticity, hardness and viscosity have been determined for the cells, so far studied. In general, the term viscosity will be used to designate the degree of rigidity of protoplasmic structures, but such a structure as a vitelline membrane may be comparatively soft and yet have what must be considered as a high internal friction or viscosity. Elasticity is determined by transfixing a selected piece of a cell and stretching it and observing the power of resumption of the original form. Dis- 16 BARBER: University of Kansas Science Bulletin, 1907, iv, p. 3; Journal of infectious diseases, 1911, vili, p. 248; zbid., 1911, 1X, p. I17. Physical Properties of Protoplasm 151 section is the method employed for determining such properties as solidity, hardness and tenacity or cohesiveness. All physical proper- ties that have been enumerated are relative and it is hoped at a later time to increase the accuracy of description by the selection of arbi- trary standards. The usage of the terms employed in this paper is based on the dissection of many widely different types of animal and plant cells. Living matter occupies an intermediate position between true solids and true liquids and has many of the properties of both as well as properties peculiar to itself. It belongs to the class of colloids known as emulsoids and exists in either a gel (hydrogel) or a sol (hydrosol) state.” The term gel will be used to designate the amor- phous semi-solid state and sol the apparently homogeneous liquid state, of living substance. Protoplasmic sols usually appear as hazy homogeneous liquids on account of the very minute size of the protein aggregates that compose the solid phase. On the other hand pro- toplasmic gels are characterized by the large size of the particles of the solid phase which set to form the gel. Hence, living gels may exhibit either a homogeneous or heterogeneous molar structure. It should now be clear that the term homogeneous is used in a relative sense to describe the optical image and refers only to the molar structure that can be brought out by the usual microscopical powers and further that heterogeneity is the universal distinguishing characteristic of colloidal sols and gels. In this connection it may be noted that Pauli!’ states that the ‘‘unfixed”’ gel of gelatine is not structured in the sense of being composed of threads, networks, granules and vacuoles; it has the molar structure of a one-phase sys- tem, which is precisely what is meant by the term homogeneous as used in this paper; the molecular structure is unknown. The present unsettled state of the problem of phase relations of colloidal 17 For discussion of the classification of colloids see: Noyes, A: 1905, Journal of the American Chemical Society, 1905, xxvii, p. 85; OstTwaALp, Wo.: 1907, Zeitschrift fiir Chemie und Industrie der Kolloide, 1907, i, p. 291; PERRIN, J.: 1905, Journal de la chemie physique, iii, p. 50; FREUNDLICH and NEv- MANN, 1908, Kolloid Zeitschrift iii, p. 80; VoN WErMARN, P. P.: ibid., 1908, iii, p. 26. 18 Pauli: Der Kolloidale Zustand und die Vorgiinge in der lebendigen Sub- stanz, Braunschweig, 1902. 152 G. L. Kite solutions has been ably discussed in a recent paper by Hardy.’ It is usual to regard colloidal systems as consisting of two phases, a solid and a liquid, which have been termed by Wo. Ostwald ™ the dis- perse phase and the dispersion medium, respectively. For convenience of description arbitrary meanings will be given the terms microsome and globule; the former will be restricted to minute dense masses of gel, the latter to suspensions in protoplasm that show many of the physical properties of oil droplets and besides are usually free of protoplasm when dissected out of a cell. Most of the suspensions so far found in cells fall into one or the other of these groups, but intermediate forms have been observed. THe Ecc oF ASTERIAS The egg of Asterias is surrounded by a mass of either transparent or translucent jelly which is soft and somewhat elastic and glutin- ous; but it can be cut and torn to pieces and removed from the egg with little difficulty. Thirty-four and six-tenths microns is the average thickness of this jelly. This structure has a low viscosity for a gel and is therefore extremely dilute. On many eggs, the jelly has become turbid and undergone a change in refractive power and as a result is visible in the usual microscopical examination. The inner surface of the jelly envelope is closely applied to the outer surface of the vitelline membrane which is invisible except in eggs that have maturated. The vitellime membrane of the immature starfish egg is a transparent and invisible solid of about two microns in thickness. The physical properties of this structure are very definite since it exhibits extraordinarily high viscosity, elasticity and tenacity. A small piece can be drawn out into a mere thread and when freed the thread contracts to a more or less rounded mass. During maturation the vitelline membrane swells to two and three times its original thickness, undergoes a change in refractive index, and becomes quite cloudy and hence visible. In this state it is softer, more glutinous and less rigid. The inner surface of this 19 Harpy: Proceedings of the Royal Society, Series A, 1912, lxxxvi, p. 601. 20 OstWALD: Zeitschrift fiir chemie und Industrie der Kolloide, 1907, i, p. 291. Physical Properties of Protoplasm 153 -membrane is tightly glued to the surface of the cytoplasm, from which it can be dissected only with considerable difficulty. The misleading optical phenomena that are involved in a study of the cytoplasm are of great interest. ; It is usual for cytologists to consider the echinoderm egg a classi- cal example of the alveolar structure of protoplasm. No one can question the fact that beautiful round spaces with hazy, protoplasmic walls in which are embedded minute granules, can be seen in such eggs. Biitschli supposed these spaces to be filled with a watery fluid. What is the true structure of the cytoplasm of the egg of Asterias? Careful dissections give a clear-cut answer to this question. The cytoplasm is a quiet translucent gel of comparatively high viscosity; it can be drawn out into large strands, but is not cohesive and elastic enough to form small threads. It can be cut into small pieces with cofmparative ease. Fragments usually become spherical, though in some cases water is slowly taken up and the mass changes into the sol state. Minute granules measuring little more than one micron are scattered plentifully throughout the cytoplasmic gel. It has been found impossible to free these structures completely from the gel in which they are embedded. They are optically more dense and have a different refractive index from the surrounding living substance. A part of the total mass of cytoplasm is composed of what appears to be alveoli or spaces; but a careful dissection of such an alveolus reveals the presence of a globule that has many of the optical properties of an oil drop. Such a globule, freed from cyto- plasm, does not dissolve in sea water and in a light of low intensity exhibits the usual diffraction halo. The invisibility of liquid drop- lets of rather high viscosity when embedded in the cytoplasm might at first sight appear difficult to explain. This invisibility is due to the fact that the refractive index and dispersive power of the globules is very near that of sea water; also, the optical density of the cyto- plasm is evidently higher than that of the globule. No diffraction rings could be seen surrounding the globules when they were imbedded in cytoplasm. Centrifugal force dislodges the globules, proving them to be merely suspended in a living gel. The minute granules respond much less readily to centrifugal force. Besides they show optical properties — their index of refraction is certainly higher than that of the surrounding gel — that ally them to highly concentrated particles 154 G. L. Kite of the cytoplasmic gel. Yet it seems likely that all such structures as granules and globules must be considered as having separated out of the disperse phase and to be therefore of the nature of suspensions. The living cytoplasm, then, is an apparently homo- geneous and very viscous gel in which microsomes and globules are suspended. If the nucleus of the immature starfish egg be dissected out in sea water it undergoes no appreciable change. Dissection of the highly- translucent nuclear membrane shows this structure to be a very tough viscous solid, and, in fact, closely allied physically to the vitelline membrane and not at all the delicate structure of the conventional descriptions. With the exception of the nucleolus, the nuclear sub- stance is all in the sol state. The nuclear sol is apparently a homo- geneous liquid. The nucleolus is a small mass of quite rigid and cohesive granular gel that is suspended in the nuclear s@l. The polar body is a granular, elastic and highly viscous gel. In order to make it possible to observe the structural components of the starfish egg and of the eggs of other common marine inverte- brates, without having to use my tedious methods, vital staining was resorted to. The jelly envelope can be stained a beautiful light blue with dimethyl-safranin-azo-dimethyl-anilin; the vitelline membrane a very dark blue with isamin blue; the globules or droplets from yellow to orange with vusuvin; and the extremely small granules a slate blue with diethyl-safranin-azo-dimethyl-anilin. The dead or dying asterias egg shows remarkable morphological changes. The whole egg becomes. almost opaque. The cytoplasm separates into a large number of more or less rounded masses which still adhere to each other. Such masses vary greatly in size, some being as small as five microns in diameter. If the formation of such small masses be observed, one is easily misled into believing that fusion of the globules is occurring. Dissection of such a mass frees the original globules. The dead gel does not stick to a glass needle and can no longer be drawn out into strands; it has lost much of its - viscidity and cohesiveness. The nuclear fluid has set and the result- ing gel is more voluminous than was the nuclear fluid in the living egg. The nuclear membrane shows little change in its physical properties, while the nuclear gel is elastic and quite viscous and granular. The physical properties of the dead nuclear gel are very similar to those Physical Properties of Protoplasm 155 exhibited by the living cytoplasm. Small fragments of the dead nuclear gel do not go into solution when dissected out in sea water. AMEBA PROTEUS Small pieces of ectoplasm of proteus can be cut off in distilled water and show no change. ‘This living substance has a moderately high viscosity and cohesiveness; it does not stick to glass needles very readily and little difficulty is experienced in cutting it into pieces as small as the limit of microscopical visibility. Pieces of all sizes appear perfectly homogeneous. The cloudiness of the ectoplasmic gel is a well-known property. The inner three or four microns of the hya- line ectoplasm and particularly the interior of the outer end of small pseudopods, contain varying numbers of minute granules and glob- ules that may measure as much as four or five microns. If these gran- ules and globules are dissected out they do not go into solution. The globules show confusing diffraction rings; but, both globules and granules can be brought out by light staining with diethyl-safranin- azo-dimethyl-anilin. The endoplasm contains a large contractile vacuole in which the presence of protein has not been demonstrated, as yet, and numerous food vacuoles which contain either liquid or liquid and food masses. The same kind of granules and globules are found in the endoplasm as are found in the ectoplasm and the number of these structures varies in different animals. The sub- stance forming the walls of the vacuoles is of much higher viscosity and cohesiveness. The living endoplasmic substance is a very dilute and apparently homogeneous gel that possesses a remarkable affinity for water. The ectoplasm of ameba then is a quite concentrated gel while the interior is quite dilute and is continously changing its water- holding power in different regions. New methylene blue R and trypan blue are of great value in bringing out the globules, granules and vacuoles. The nuclear membrane is an extremely thin and moderately tough solid substance. It shows some elasticity and is quite viscous. The whole of the nuclear substance is a highly rigid and granular gel, the minutest pieces of which show no appreciable change when dissected out in distilled water. A slight elasticity and a definite 156 Gx Wiaie glutinicity are exhibited by this substance. There are variations in concentration of the nuclear gel that produce a characteristic but misleading optical image. The nucleus appears to contain an irregu- lar network with granules imbedded in it. The interstices of the network are very small luminous spots which have been misinter- preted to be vacuoles. Many dissections have shown that the granules are very concentrated masses of gel; the network irregularly disposed masses of a diluter gel; and the interstices or light spots the most dilute gel in the nucleus. The so-called network is a part of the nuclear gel that forms a concentration gradient; the interstices and granules may be considered constants connected by the grading network. It should be clearly understood that the network is not made up of definite threads of fibres but of irregular masses of hydrogel that are very dense immediately surrounding the granules, from which they grade into the dilute gel of the interstices. No free liquid was found in the nuclear substance. When the granules are in focus they appear gray and cloudy or opalescent; when out of focus as dark spots. They measure from less than one to about two microns in diameter. It seems that a part of the luminosity of the interstices of the network is due to diffraction and not simply to slight absorption of light by this portion of the nuclear substance. The structural details of the nucleus can be brought out with con- siderable vividness by staining with janus green (diethyl-safran—in azo-dimethyl-anilin). Slight cuts in the surface of proteus quickly close. Extensive cuts frequently cause an ameba to explode —in as short a time as two seconds nothing but the nucleus may remain. If the contrac- tile vacuole be cut and its liquid content caused to mix with the cytoplasm the Ameba is immediately destroyed with explosive vio- lence. A relatively large dose of distilled water and even $ to 1 molar cane sugar solution or one molar sodium chloride or potassium nitrate give a like result. It is not usually possible to produce more than a temporary vacuole with two molar cane sugar; a large dose of sugar of this concentration usually causes the appearance of granules, globules, fibrils and a hyaline appearance in any portion of the endo- plasm into which the injection is made. ‘The doses that were injected varied from about 270 cubic microns to 30,000 cubic microns. noe .e | Physical Properties of Protoplasm 157 A large number of indicators have been injected into the interior of proteus with the idea of determining a possible relation between an excess of H+ or OH ions and the extraordinary water-holding-power of the endoplasm. Azolitmin, sodium alizarin sulphonate, tropeolin ooo No. 1, methyl orange and congo red, dissolved in from $ to 3 molar cane sugar have been so far employed. A neutral to slightly alkaline reaction is shown by all the indicators. It seems probable then that the concomitant variation in water-holding-power of dif- ferent regions of the cytoplasm is the mechanism by which Ameba proteus moves and is associated with an excess of OH™ ions. A number of operations were performed on the ectoplasm of Ameba proteus for the purpose of determining the relation between movement and surface tension changes. The results of shallow and deep cuts in the ectoplasm have already been given. ‘The outer 5 to 7 microns of the pseudopods were cut away in some animals, and in others small doses of distilled water were injected into the ectoplasm. The removal of the outer end of a pseudopod was usually followed by rapid closure of the incision. The injection of distilled water into the ectoplasm had no noticeable effect on the formation of pseudopods. By means of such operations the rigid ectoplasm was either removed, for a short time, from a given area of the surface or at least greatly weakened; yet, no tendency to the formation of pseudopods was ever observed, in such weakened surface areas. These facts seem to justify the conclusion that surface-tension changes play a negligible role in the movement of Ameba proteus.. Furthermore, it may be recalled, that the outer surface of Ameba proteus is a semi-rigid solid of from 5 to 12 or more microns in thickness, and it has still to be shown, that the changes, in the tension of the surface film, that are commonly assumed to occur, can appreciably affect the underlying semi-rigid ectoplasm. The nutrient solution in which the amebae were grown was slightly alkaline in reaction. Proteus usually recovers from the large doses of neutral salts and sugar in much less than an hour, almost certainly by throwing them off. 158 Ga iktte PARAMECIUM The living substance of Paramecium is a soft, elastic and somewhat glutinous gel which can be drawn out into strands. It is filled with a large number of vacuoles of various sizes the walls of which are more dense than the surrounding gel. The surface layer is more viscous and cohesive than the interior. Small cuts usually close quickly, exten- sive deep cuts are either followed by a loss of cytoplasm or a rapid change of the whole cytoplasm into the sol state with almost explosive violence. If the fluid in the contractile vacuole be caused to mix with the cytoplasm a rapid change of this substance into the sol state results. Suspended in the living and apparently homogeneous and rather dilute gel are varying numbers of extremely small granules and small globules. Many of the granules are recently ingested bacteria. Neither the granules nor globules go into solution when dissected free from the cytoplasm. The food masses are granular gels of rather high viscosity. The optical properties of the meganucleus render its study ex- tremely tedious. It is almost transparent and invisible. Therefore its refractive index and its dispersion are very close to those of water. Dissection has proved the meganucleus to be a gel of higher viscosity than the cytoplasm and to be slightly glutinous and elastic. The meganuclear gel has areas, more dense than the surrounding sub- stance, that may be considered granules. A complete study of the micronucleus has not been made. NECTURUS The Striped Muscle Cell. — The living substance of the striped muscle cell of Necturus is the most viscous, elastic and cohesive of the living gels we have so far considered. The muscle substance sticks to a glass needle and can be drawn out into extraordinarily long threads which when released almost regain their previous shape. The absorptive power and turbidity of this substance are compara- tively high. . When the whole or a piece of a muscle cell is stretched the stria- tions become faint or disappear — only to reappear when the tension Physical Properties of Protoplasm 159 is removed. Beautiful but misleading diffraction phenomena are to be observed when a piece of muscle cell is stretched. If the point of a very minute needle be pushed into a muscle cell, it can be moved in one direction about as easily as another. The optical image of striped muscle is very misleading. Dissec- tions have shown that the dark bands seen in living muscle are pro- duced by concentrated areas of muscle substance which absorb enough transmitted light of low intensity to appear as dark bands in the optical image. I have been unable to dissect out definite fibrils. The substance lying between the concentrated regions and appearing as light bands is a highly viscous, elastic gel and has no physical properties that serve to distinguish it from the surrounding sarco- plasmic gel. By cutting the dark band to pieces, small masses of highly concentrated muscle substance, frequently less than one micron in diameter, are partially freed from the dilute enveloping gel and in light of low intensity show well-defined diffraction halos. The appearance of dark bands in the optical image, then, is produced by absorption of light waves by the concentrated muscle substance; the light bands, by the low absorptive power of the diluter inter- mediate gel, and the diffraction of the light waves by the edges of the concentrated substance. Striking changes in the optical image that are well known can be produced by increasing the intensity of illumi- nation. The dark band becomes cloudy and more or less opalescent and the light band may show an intersecting dark line or well-defined diffraction fringes just outside the geometrical shadow of the con- centrated substance. Hence, absorption, diffraction, refraction and dispersion are involved in the formation of the optical image of striped ‘muscle and the former two particularly when the illumination is of a relatively high intensity. The nuclear substance is a gel that is for the most part compara- tively dilute but contains more concentrated areas in the form of granules and an imperfect network. The appearance of a network in the optical image is due not to definite fibrils but to more con- centrated parts of the gel that grade into the diluter nuclear substance. On the outer surface of the muscle cell is found a highly trans- lucent membrane, the sarcolemma, which is extremely elastic and measures about one micron in thickness. it is stuck to the whole outer surface of the muscle cell and is viscous and cohesive enough 160 G. L. Kite to offer an appreciable resistance to a glass needle a micron or less in diameter. The disagreement among investigators concerning the presence of a sarcolemma is due to the fact that it is transparent and that its refractive and dispersive powers are so nearly the same as those of water. Instead of being the delicate structure of the con- ventional descriptions, the sarcolemma of the striped muscle cell of Necturus exhibit physical properties that are very similar to those of the vitelline membrane of an echinoderm egg. If a concentrated solution of isamin blue made by boiling in dis- tilled water or .8 per cent sodium chloride be added to freshly teased muscle cells, blue staining of the sarcolemma occurs in ten to fifteen minutes. An Epidermal Cell. — The epidermal cells are embedded in an intercellular gel of extremely high viscosity and considerable elas- ticity. The substance is tough but softer than many nuclear mem- branes and shows a relatively high absorptive power. It is also quite turbid. A few globules and granules, varying in size from about one to four microns, that can be easily stained with diethyl-safranin- azo-dimethyl-anilin are to be seen scattered through the intercellular gel. The whole cell substance is a gel of even higher rigidity than the muscle substance of the same animal. Small pieces cut out of the nucleus or cytoplasm, in distilled water or .8 per cent sodium chloride, show no appreciable change. The cytoplasm exhibits a high absorptive power and a definite elasticity. Very small granules that seem to be denser cytoplasmic areas are to be seen scattered throughout the turbid cytoplasm. Many cells show radially arranged fibrils, in the outer part of the © cytoplasm, which can be partially freed from the surrounding gel by dissection. Such a fibril is physically and optically more dense than the remainder of the cytoplasm. The nuclear membrane is thin, clear, and quite cohesive and elas- tic, and has a different index of refraction from the cytoplasm and nucleus. The nuclear gel is of a higher viscosity than the cytoplasm. The appearance of a network in the optical image of the nucleus is due to concentrated areas in the form of granules and imperfect threads which are not sharply separated from, but grade into, the surrounding diluter Physical Properties of Protoplasm 161 gel. The whole nuclear substance is quite glutinous. No trace of free liquid could be found in the nucleus. SPIROGYRA The cellulose wall of Spirogyra is enormously cohesive; it is cut or punctured with extremely fine Jena glass needles with considerable difficulty. The outer surface is covered by an almost invisible soft gel, that frequently measures five or more microns in thickness and can be stained red with sodium alizarin sulphonate in a neutral or slightly alkaline solution. A layer of dilute granular gel covers the inner surface of the cellulose wall and is connected by a number of strands of an elastic gel to a central mass of living substance, in which a small nucleus is imbedded. The central mass of gel contains a few granules and is of a higher viscosity and cohesiveness than the surface cytoplasm. ‘This mass also has a higher refractive index and higher absorptive power than the surface cytoplasm. The anchoring strands of gel decrease in viscosity from within outwards. Much of the surface layer of cytoplasm is usually invisible. Hence, it is quite translucent and has refractive and dispersive powers very close to those of water. If the cell wall is cut across the surface cytoplasm shrinks. The chloroplasts either shrink or separate into rounded masses. The chloroplasts have a higher viscosity and elasticity than the gel in which they are imbedded. The pyrenoid is a complex structure. Dissection shows the presence of an optically dense but fragile wall which, when broken, frees a globule that is of considerable interest. This globule shows many of the optical properties of an oil droplet but has too high a viscosity to round up under the influence of surface tension; there- fore it seems to be a true gel. None of the cytoplasm goes into solution very readily even when cut into very minute pieces. The nucleus of Spirogyra is a gel that has higher viscosity and refractive and absorptive powers than the cytoplasm. It is also more cloudy than the cytoplasm. There are denser areas in the nuclear substance in the form of granules and threads that form a sort of network. Small pieces dissected from all parts of the nucleus 162 G. L. Kite into water, not only do not go into the sol state but remain too rigid to show surface tension effects. Pieces of broken glass needles stick firmly to the nuclear gel when imbedded in it. The image of the nucleus is false in important details. The denser areas, when in the focal plane, appear as grayish or slightly opalescent granules and threads and when above or below the focal plane as dark spots and lines. Besides, if the intensity of the illumination be increased the network appears much finer. Very small dense masses of gel could be partly freed from the remaining nuclear substance. It seems proper to term such structures granules. On the other hand, the dense masses that produce the appearance of a network in the image are not actual threads that are sharply separated from the surrounding gel but irregularly shaped dense areas that grade into the immediately contiguous diluter gel. The light spots that change their position at different focal planes seem to be due chiefly to two factors, viz., a relatively low absorptive power of the gel occupying the interstices of the network and diffraction by the edges of the denser areas. : It seems certain that the vacuolar fluid of Spirogyra contains protein and must be considered a hydrosol. Much evidence has been adduced in support of this statement. A number of injections of Millon’s fluid into the vacuole were made with positive results. Extremely small solid particles appeared in the cell sap after the injection of such precipitating agents for proteins, as saturated subli- mate, 40 per cent formaldehyde, saturated picric acid and saturated phosphotungstic acid containing 5 per cent sulphuric acid. The vacuolar fluid is cloudy. This is positive proof of the pres- ence of ultramicroscopic particles which would ordinarily be con- sidered protein even in the absence of a positive color test for protein. The cell sap of Chara seems to be richer in protein than that of Spirogyra. This conclusion is based on the fact that a compara- tively heavy precipitate results from the intravacuolar injection of saturated sublimate or 40 per cent formaldehyde. Hence, it is proba- ble that cell sap containing protein is very common in plants. Mucor, Saprolegnia, Hydrodictyon, Chara and the parenchymatous cells of the leaves of Tradescantia have been dissected for comparison with animal cells. In general, it may be stated that the cellulose walls of plants are extremely cohesive and are cut and punctured Physical Properties of Protoplasm 163 with considerable difficulty. The protoplasm of plant cells is much more dilute or less rigid than that of animal cells. RESTING AND DivipING MALE GERM CELLS OF THE SQUASH Buc (ANASA). GRASSHOPPERS AND CRICKETS A brief note has been published on this subject.” The whole cell substance of resting and dividing spermatogonia and spermatocytes is a moderately viscous gel. Cutting away pieces of the cytoplasm and nucleus in Ringer’s fluid shows that these struc- tures are far too rigid to flow or change shape under such experi- mental treatment. The appearance of a network is due to denser masses of nuclear gel that grade into the diluter surrounding substance. No definite threads or fibrils could be dissected out of resting nuclei. Some of the optical principles involved in a study of the living nuclei of spermatogonia and spermatocytes were discussed in connection with the nucleus of proteus. Very definite statements can be made about the physical proper- ties of chromosomes and spindle fibres. The chromosome has been found to be the most highly concentrated and rigid part of the nuclear gel. Such a mass of gel is less translucent and has a higher refractive index and absorptive power than the diluter homogeneous gel in which it is imbedded. A chromosome when dissected out shows no affinity for water and does not disintegrate readily. Pieces of it stick to the glass dissecting needle but when drawn out show no marked elas- ticity. The spindle fibre is an elastic concentrated thread of nuclear gel and its absorptive power and refractive index are also different from those of the diluter gel in which the spindle fibre is imbedded and from which it cannot be entirely freed. Metaphase spindle fibres that were dissected out with great care seemed continuous with the ends of the chromosomes. The homogeneous gel in which a telophase spindle is imbedded is so rigid, that all the surrounding cytoplasm can be cut away and the spindle and chromosomes show no appreciable change; metaphase, anaphase and telophase spindles can be cut to pieces in Ringer’s fluid and the pieces are so rigid that they undergo no change in shape. 21 KITE and CHAMBERS: 1912, Science, N. S., xxxvi, p. 639. 164 G. Ly Kate Many of the physical and chemical changes of cell-division are reversible. Pressure on the cell plate of spermatocytes in telophase has caused rapid fusion of the daughter cells and extensive swelling and loss in rigidity of the protoplasmic gel in which the spindle fibres are imbedded. If the displaced spindle fibres and chromosomes are dissected out, after such a partial reversal, they are found to have undergone no appreciable change in rigidity. From a preliminary study of mitosis, a few conclusions, that are probably general, can be drawn. It seems that cell-division results primarily from concomitant shrinking and swelling or change in water- holding power of different portions of the cell protoplasm. Many of the structural elements of the mitotic figure separate out of the pro- toplasm and change in rigidity according to their water-content. During the prophase, the nuclear substance becomes so soft that movement of the components of the nucleus is affected by flowing of the nuclear gel. The mechanism at the basis of this flowing seems to be a change in water-holding power of the nuclear components. I wish here to thank Dr. A. P. Mathews for the very helpful interest that he has shown in this investigation. THE American Journal of Physiology VOL. XXXII JULY. a; 1913 NOT ON THE RELATION OF THE BLOOD SALTS TO CARDIAC CONTRACTION By E. G. MARTIN [From the Laboratory of Physiology in the Harvard Medical School] HE importance of the salts of the blood in maintaining the beat of the heart is established beyond question. The pre- cise relation of individual salts in the series of reactions making up a heart-beat is still the subject of study. The problem of the action of salts cannot be attacked directly; too many factors are involved. Conclusions must be drawn, therefore, through comparison of data obtained from numerous more or less indirect experiments. The difficulties of interpretation under these conditions are necessarily very great, and workers have been led to form very diverse opinions from quite similar data. Consider, for example, the question of the ‘‘inner stimulus,” the actual excitant to the cardiac contraction. At least three distinct views as to its nature are at present before physiologists. Lingle,’ correlating heart tissue with skeletal muscle tissue, adopts Loeb’s view that sodium ions constitute the excitant. Langendorff ? looks upon the metabolic products of the heart’s own activity as the means 1 LINGLE: this Journal, 1900, iv, p. 265. 2 LANGENDORFF: Archiv fiir Anatomie und Physiologie, physiologische Abtheilung (Suppl. Band), 1884, p. 1. 166 E. G. Martin of stimulation. Howell* suggests that the origination of the beat is not dependent on any specific stimulus, but results from the spon- taneous break down of the highly unstable substance whose decom- position yields the energy manifested in the contraction. With regard to the rdéle of individual salts there is a like difference of opinion. Thus, in respect to calcium, Lingle * believes that this salt exerts its beneficial effect by antagonizing the poisonous action of sodium. S. R. Benedict * attributes the improved beat which follows its use to the rise in tone which it brings about. Howell ® pictures the réle of calcium in connection with the conversion of energy- liberating substance from a stable into an unstable, easily dissociable compound. From a study of the relation of the tissue to its oxygen supply I was led ‘ formerly to believe that calcium might have something to do with the oxidative activities of the heart sub- stance. Purpose of this Study. — My intention, in presenting the views herein contained, is not to advance any new theory of salt action, but to suggest a means whereby the divergent theories already current can be harmonized in a fashion that will offer satisfactory explanations of the various salt-phenomena thus far described, so, perhaps, simplify- ing the situation. In studies of the action of salts on the heart the chief considera- tion hitherto has been the presence or absence of spontaneous rhyth- micity. Solutions have been classified according as they favor or interfere with contraction. Series of beats have been examined to see whether the media used cause exhaustion or recovery. A some- what different, and perhaps instructive point of view can be gained if attention is directed to the character of the individual contractions. The operation of the “‘all or none” law of cardiac activity makes the height of any contraction the index of the amount of energy-liberating material available for performing it. By observing the effect of various solutions on the height of contraction, therefore, conclusions 3 HoweELL: The Harvey Lecture. Journal of the American Medical Asso- ciation, 1906, xlvi, No. 23. 4 LINGLE: Loc. cit. 5 BENEDICT: this Journal, 1905, xill, p. 199. 6 HOWELL: Loc. cit. 7 Martin: this Journal, 1906, xvi, p. 214. Relation of the Blood Salts to Cardiac Contraction 167 may be drawn as to the influence of the solutions on the elaboration of dissociable energy-yielding material within the tissue.* _ Schultz ® has described another means of gaining information as to the effect of salts on heart tissue, namely by determining their influence on the time required after contraction for the excitability to electric stimuli to return to normal. By the use of this criterion of Schultz and the one I suggest above, in connection with the older criteria, which are concerned primarily with the efficiency of media in causing beats, we should be able to differentiate, if there be any difference, between agencies concerned with the actual production of contractions and those having to do rather with the preliminary process of preparing dissociable material. Is there a Specific, Inner Stimulus ?— That the immediate process of contraction depends upon a definite inner stimulus, in mammalian hearts, at least, seems to be pretty clearly established by some recent observations of Cushny’”’. This author stimulated a spontaneously beating ventricle at a rate faster than its own, forcing it into an abnormally rapid rhythm. At the cessation of the artificial stimula- tion the ventricle showed a period of stand-still before resuming spontaneous activity. The significant observation of Cushny was that during this period of stand-still the heart was as sensitive as at other times to artificial stimulation. The stand-still was not due, therefore, to a loss of irritability, but to a failure of the inner stimulus; and the experiment demonstrates the existence of an inner stimulus quite independent of any particular condition of irritability. Hering " has alsa argued in favor of a specific inner stimulus. He cites various experiments in support of his view, but considers two observations particularly conclusive. The first of these is the reversal of rhythm sometimes seen in perfused mammalian hearts, whereby the auricles, 8 Tf the height of contraction is to be interpreted in this manner care must be used that the tissue under examination is contracting as a whole, and not in part only. The tendency of heart tissue under experimental conditions to show partial contractions is pointed out by Schultz. (This Journal, 1908, xxii, p. 134.) I have observed the same repeatedly. There is, however, little difficulty in dis- tinguishing partial from complete contractions if one is on the lookout for them. ® ScHuttz: this Journal, 1908, xxii, p. 133. 10 CusHNyY: Heart, 1912, ili, p. 257. ; 11 HERING: Archiv fiir die gesammte Physiologie, 1911, cxlili, p. 370, and 1912, cxlviii, p. 608. 168 FE. G. Martin instead of setting the pace, beat in response to stimuli from the spontaneously-beating ventricles. This, according to Hering, is an example of auricular irritability maintained in the absence of spon- taneous activity. Hering’s second significant observation is that in the dying heart the various parts become inactive, not together, but one after the other. This is interpreted as signifying that in this case loss of irritability precedes failure of the inner stimulus. Although the existence of a definite inner stimulus seems, from Cushny’s observations, to be satisfactorily established, the experi- ments of Hering do not appear particularly conclusive in support of it. The failure of the auricles in the perfused heart to beat spon- taneously can be as reasonably explained by assuming a partial loss of irritability, sufficient to prevent spontaneous activity, but not to abolish response to stimuli from the beating ventricle, as by assuming an unimpaired irritability made ineffective through the disappearance of the inner stimulus. In the observation on the dying heart the successive failure of region after region shows, it is true, a progressive loss of irritability, but to stimuli proceeding from the regions still active; there is nothing in the experiment to prove that there is an inner stimulus exerting its influence ineffectively in the inactive regions. Hering '” has pointed out a possible fallacy in such experiments as that of Cushny, in that in them the criterion of cardiac irritability is responsiveness to artificial stimulation, a criterion which may not be valid when applied to the irritability of the heart for its normal stimulus. To settle the question finally, therefore, either responsive- ness to artificial stimulation must be shown to be a valid eriterion of cardiac irritability, or some other undoubted index of irritability must be established. Various considerations incline me to the view that cardiac irri- tability depends upon the amount of available energy-liberating material, per unit of substance, present in the tissue. It is universally “assumed that in the rhythmically beating heart at the end of systole the available source of energy is exhausted, and that during the succeed- ing diastole there is an accumulation of the special material required for yielding the energy of the next systole. That during this accu- mulation there is also a steady increase of irritability until the begin- ning of the next systole is also generally assumed. 12 HERING: Loc. cit.,.1911, cxliii, p. 376. Relation of the Blood Salts to Cardiac Contraction 169 If we grant that in the rhythmically active heart the diastolic accumulation of energy-yielding material is accompanied by increas- ing irritability, must we not admit the probability, at least, that the presence of abundant available material in the quiescent heart signifies high irritability there as well? The index to the amount of energy-liberating material available for any contraction is, as stated earlier, the height of the contraction. If the increase in irritability goes hand in hand with the accumula- tion of this material, we can judge the degree of irritability by the same criterion. As indicating that this suggested relationship between contraction height and irritability actually holds, certain experi- mental results are interesting, even though they are based on the 20 40 60 80 100 120 Moist Chamber NaCl 0.7% Moist Chamber Ficure 1. Curves showing that height of contraction and excitability to induction shocks vary in parallel fashion in freshly excised ventricle strips. The continuous line is the curve of irritability; the broken line is the curve of contraction height. results of artificial stimulation, and cannot, on that account, according to Hering, be accepted as wholly convincing. Schultz reports ™ that freshly-cut heart strips immersed in Ringer’s solution give, for some time after immersion, progressively higher 138 ScHuLTz: Loc. cit., p. 143. 170 E. G. Martin contractions when stimulated, and during this same period show, according to his criterion, mentioned above (p. 167), progressively increasing irritability. By a method recently developed“ I have made accurate determinations of the threshold of response to induc- tion shocks of freshly-cut heart strips, and find that there is a remark- able parallelism between the curve of irritability and of contraction height. This parallelism is shown graphically in Fig. 1, which is the record of the course of the irritability and the height of contrac- tion of a strip during the first two hours after excision. Schultz reports, and I confirm his statement, that in heart tissue several hours after removal from the body, the agreement between excitability to electric stimuli and height of contraction seems not to hold so strictly. Even though there is thus some opposing evidence, the preponderance favors the idea that height of contraction is on the whole a fairly good index to the excitability of the tissue, and we are ju ik Hn tu ae PM a A: ay ae a noe FicurE 2. Tracing showing that a heart strip which is not spontaneously active may give under artificial stimulation, contractions which are much higher than previous spontaneous contractions of the same strip. justified, therefore, in basing tentative judgments as to irritability on observations of contraction height. Let us now apply this criterion of irritability to the problem of the inner stimulus: Can we show that strips which are spontaneously active with a certain degree of irritability may lose their spontaneity and yet develop even greater irritability? Fig. 2 isa tracing obtained from an apex strip of ventricle from Chrysemys marginata. The tracing was taken Nov. 21 and 22, tg11.. The fresh strip was im- mersed in 0.7 per cent sodium chloride solution till beats began. The latent period was one hour and ten minutes. Five minutes after the onset of spontaneous rhythmicity the sodium chloride solu- tion was replaced (2.15). by 50 c.c. Ringer’s solution (NaCl 0.7%, 4 Martin: this Journal, 1908, xxii, p. 116.’ Also the Measurement of Induction Shocks, New York, 1912. Relation of the Blood Salts to Cardiac Contraction LAr CaCl, 0.026%, KCl 0.04%), to which 2 c.c. 0.9 per cent potassium chloride had been added. Spontaneous activity immediately ceased. At intervals, as indicated on the tracing (2.25, 2.35, 2.50, 3.05, 3.30 P.M and 10 A.M), series of three minimal induction shocks were sent through the strip. The liquid was drawn off just long enough in each case for the stimulations to be sent in. The record shows a progress- ive increase in the height of contraction from series to series, con- tinuing for twenty hours after the first activity of the tissue. The first contractions under artificial stimulation were higher than the highest spontaneous contractions, and the last contractions under artificial stimulation were twice as high as the spontaneous contrac- tions. This experiment, which I have performed repeatedly with similar results, proves that a procedure which prevents spontaneous activity completely may have no influence on the development of irritability, and to that extent justifies Cushny’s use of artificial stimuli in judging the irritability of the quiescent mammalian heart In his experiment, cited above. Another experiment, described below, seems to me to give positive indication that spontaneous beats, when they do occur, are the result of the operation of a definite “inner stimulus.’”’ In this experiment I was determining the threshold of irritability of ventricle strips to single induction shocks, measuring the stimuli by the method I have recently described.’ A strip had been immersed in 0.7 per cent sodium chloride solution till spontaneous beats began, and was then removed to moist air. Spontaneous activ- ity ceased in about five minutes, without any indication of the onset of “sodium chloride exhaustion.”’ After fifteen hours in the moist air the threshold stimulus for the strip was 720 Z units.!® The contractions following this stimulation were very vigorous Seven: minutes after determining the threshold the strip was immersed in 0.7 per cent sodium cholride. It contracted spontaneously in less than one minute; the sodium chloride was then withdrawn. One more spontaneous beat occurred, and then the strip remained quiet. Three minutes later the threshold was 660 Z units. Five minutes later still the threshold had fallen to 528. Five minutes after this last stimulus the strip was again immersed in 0.7 per cent sodium chloride solution. Strong spontaneous contractions began within 15 MartTIN: Loc. cit. 16 MartTIN: this Journal, 1910, xxvii, p. 228. 172 E. G. Martin ten seconds. These continued for fifteen minutes, although the sodium chloride solution was withdrawn after one minute. Eight minutes after the strip had ceased spontaneous activity the threshold was still 528. It began to decline soon after, however, and within a half hour threshold values as low as 317 were obtained, without any reappearance of spontaneous beats. During the hour in which the observations just cited were made, a ventricular strip had a declining threshold of irritability, virtually throughout the period. It showed spontaneous activity twice; each time immediately after the applica- tion of asodium chloride solution; and each time upon a different plane of irritability. To one watching a strip behaving as this one did, the impression is overwhelmingly of a definite stimulus thrown into opera- tion with each immersion in the sodium chloride solution. The Characteristic Features of Ventricular Activity in Pure Sodium Chloride Solution. — Careful study of a large number of experiments in which ventricle strips have been placed in pure sodium chloride solutions after various sorts of preliminary treatment, has impressed me with certain features of sodium chloride action which seem to be general, and which I would summarize as follows: (1) For ventricle tissue to be active in sodium chloride solution, it must come from a medium favorable to the production of dissociable substance. (2) The onset of activity is prompter the greater the irritabitity in the preceding medium. (3) The first spontaneous contractions in sodium chloride equal in height the last contractions in the preceding medium. Leaving out of account, for the moment, the effects of sodium chloride on freshly-excised strips, the conditions favorable for the manifestation of the sodium chloride effects outlined above are: long continued immersion in Ringer’s solution; prolonged suspension in moist air following immersion in sodium chloride solution; some- times treatment with sugar solution after sodium chloride exhaustion.” The usual termination of spontaneous activity in these preparatory media is through a period of lessening frequency of beat, without much decline in height of contraction, indicating a diminished spon- taneity, but not a decrease in the production of dissociable substance. The effect of immersion in pure sodium chloride solution is in every one of these cases prompt resumption of rhythmic activity — after 17 For detailed description of the behavior of strips in the media named, see MARTIN: this Journal, 1904, xi, p. 123 ef. seq. Relation of the Blood Salts to Cardiac Contraction 173 Ringer’s solution activity is usually resumed with striking sudden- ness, and in every case the first beats are the highest of the series and are about the same height as the last beats in the preceding medium. Frequently strips in moist air, particularly when placed therein after brief treatment with sodium chloride ® give a rather long series of beats with continuously declining vigor of contraction. Immer- sion in sodium chloride solution at the end of such a period some- times brings about a series in which there is gradual increase of vigor for some time, as long as fifteen minutes in my experiments. This result is significant in that it forms the only exception I have seen to the general observation that the first beats in sodium chloride solu- tion are maximal or nearly so. One of the very familiar phenomena attending the use of sodium chloride on heart strips is the rather long latent period which pre- cedes activity, when the strips are immersed in the solution immedi- ately after removal from the animal. The various explanations of this latent period that have been offered need not be reviewed here. I wish, however, to report certain facts about the condition of heart tissue immediately after separation from the animal, including the latent period in sodium chloride. Schultz reports that heart strips cut while in Ringer’s solution and suspended in moist air are inexcita- ble to induction shocks for a half hour or longer. I have studied the excitability of similar strips, the only difference between my procedure and that of Schultz being that I invariably omitted any rinsing solu- tion; the strips were always cut directly from the heart, through which blood was being pumped. Such strips, placed for a moment on a glass plate, are very irritable to mechanical stimulation and in responding to the stimuli unavoidable in handling them, pump themselves quite free of blood. My observations differ from those reported by Schultz in that they show that the freshly isolated strips, when first placed in moist air, are fairly excitable to induction shocks. I have records _of threshold stimuli ranging from 290 to 850 Z units. In my experi- ence the irritability declines steadily. In an experiment in which the initial threshold was 290 it had increased in twenty minutes to troo. In an experiment with a high initial threshold, 850 Z units, 18 See MARTIN: Loc. cit., p. 124. 19 ScHULTZ: Loc. cit., p. 134. 174 | E. G. Martin no contractions were given after twenty minutes by stimuli of 8000 units. I have demonstrated also, by means of an experiment described in my first paper on this subject,” the steady decline in irritability in moist air of freshly isolated apex strips. In this experiment the entire heart is removed from the body and suspended in moist air and the apex is partially severed from the base. Records of the contractions of the apex are taken. This procedure gives an apex strip, virtually isolated so far as salt relationships are concerned, but subject to rhythmic stimulation from the active venous portion of the heart. Such strips invariably show a steady decline in height of contraction, corresponding, we must believe, with a steady diminution in produc- tion of dissociable material, and with steady decline in irritability. The onset of complete “ exhaustion” usually requires about two hours. However we may interpret these observations they demonstrate the fact that ventricular tissue upon separation from its normal environment loses its excitability more or less rapidly. When a strip is immersed in sodium chloride solution it has first to over- come this tendency to declining irritability before it becomes able to execute spontaneous contractions. Schultz (loc. cit.) states that the return of excitability of excised strips is promoted by immersing them in a saline bath. I have made observations of the threshold stimulus,’at intervals during the latent period, of fresh strips in 0.7 per cent sodium chloride, thé solution being withdrawn only long enough in each case to allow stimulation. I find that the threshold a few minutes after immersion is usually several times higher than at the moment of separation from the body. Thus in one experiment, the threshold of the freshly isolated strip was 400 Z units. Five minutes after immersion in 0.7 per cent sodium chloride the threshold was 1224. In seven minutes more the thresh- old had fallen to 576; eight minutes later it was 343; and three minutes after this reading, and just before spontaneous contractions began, the threshold was 300. Similar variations, although with a . more rapid secondary increase in irritability, I have observed in fresh strips immersed in Ringer’s solution. Such results show that removal from the body to any medium whatever involves in the course of the readjustment of the tissue to its environment a lowering 20 MARTIN: this Journal, 1904, xi, p. 105. » 1904 -) Relation of the Blood Salts to Cardiac Contraction 175 of excitability, which must be overcome before activity is resumed. I believe the necessity for this readjustment explains, at least, in part, the long latent period of fresh strips in sodium chloride solution, as well as the various means of shortening the period that have been described. Further discussion of this point is reserved for a later portion of the paper. The Characteristic Features of Ventricular Activity under Treat- ment with Calcium-Containing Solutions. — Apex strips respond to treatment with solutions containing calcium in a manner perfectly characteristic, and often very striking. The effect of such solutions, when positive, is always to bring about a marked increase in the height of contraction, which is prompt in showing itself. I have seen the effect follow the addition of calcium-containing solutions to sodium chloride solutions surrounding strips in all stages of the typical sodium chloride series. Strips beating in moist air show marked increase of vigor after the application of calctum-containing solutions. Fresh strips immersed in Ringer’s solution show only occasional spontaneous contractions, but such as occur are always very vigorous indeed. That this effect of calcium extends to mammalian heart tissue is shown by Langendorff and Hueck *! and by Gross,” who report that a permanent increase in the calcium content of the circulating blood causes a permanent increase in the force and amplitude of the heart beat. DISCUSSION I have emphasized the characteristic effects of sodium and of cal- cium on ventricular tissue to bring out what I assume, tentatively, to be their respective functions with reference to cardiac activity. The effect of sodium is always such as to suggest the action of a direct stimulus, and this I assume to be its function so far as the heart is con- cerned. In adopting this view I am abandoning my former position ” of support for the Langendorff theory of stimulation by meta- bolic products, in favor of Lingle’s theory that sodium ions consti- 21 LANGENDORFF and Hveck: Pfluger’s Archiv, 1903, xcvi, pp. 473-485. 2 Gross, E.: Ibid., 1903, xcix, pp. 264-322. 23 MarTIN: this Journal, 1906, xvi, p. 201. 176 E. G. Martin tute the “‘inner stimulus”’; but only to the extent of granting to sodium ions a positive stimulating effect. I still believe that the evidence formerly adduced by me” indicates that metabolic products may also stimulate heart tissue. I see no reason why we must confine stimulating properties to single substances, excluding all others. Indeed an experiment reported by Benedict,”? in which galactose solution caused beats in a freshly isolated apex strip, and the commonly observed revival after sodium chloride exhaustion by sugar solutions, can be explained more satisfactorily by granting to these substances certain stimulating powers than in any other way. An objection which may be urged against this theory, so far as it assumes for sodium a direct stimulating function, is that in such an experiment as that cited on p. 171, in which immersion in sodium chloride is followed promptly by beats, the tissue must be saturated with sodium chloride at the time of immersion, and the application of more sodium to a tissue already saturated with it would scarcely be expected to exert a very positive influence. To my mind the best answer to this objection is the experiment itself. The tissue, saturated with sodium chloride, is quiescent; when immersed in more sodium chloride it beats. Obviously there is some difference of condition before and after immersion. The equilibrium existing in the tissue saturated with sodium chloride is instantly upset when it is immersed in more sodium chloride. We have no positive knowledge as to the ionic conditions obtain- ing in the equilibrium of the quiescent tissue. Various hypotheses to account for the equilibrium and its upset by sodium chloride might be offered. A suggestive fact is that equilibrium with quiescence cannot prevail in heart tissue zmmersed in sodium chloride solution, after the initial latent period is over, so long as dissociable material is available, and provided activity is not prevented by the presence of an inhibitory substance, such as potassium. Moreover, after prolonged immersion in sodium chloride solution, strips removed to moist air are nearly always active for many hours. In view of these facts the occasional stand-still shown by strips, after a rather brief immersion in sodium chloride solution, suggests that a true satura- 74 Martin: Loc. cit., pp. 203 and 205. ** BENEDICT: this Journal, 1908, xxii, p. 22. Relation of the Blood Salts to Cardiac Contraction 077 tion may not have occurred under these conditions, and lessens the force of the objection I have mentioned. The effect of calcium seems clearly to be that suggested by Howell,”° namely to promote the production of dissociable energy-liberating material. If the height of contraction is a reliable index to the amount of dissociable substance available, as we must believe it to be, all the reported facts about the effects of calcium point directly to this con- clusion, for the one striking feature of the calcium effect wherever it appears is improvement in the vigor of beat. The dependence of the calcium effect upon the oxygen supply, which, as I have previously shown,”’ is very marked, I formerly interpreted * as indicating a réle for calcium in connection with the oxidative processes in the tissue. That the facts can be interpreted . as satisfactorily in terms of my present view I shall show in a later paragraph. The theory of the action of sodium and calcium on heart tissue presented in this paper may be summarized in a brief statement. Sodium and calcium ions are not antagonistic, but act positively upon different phases of the contractile process. Calcium promotes the conversion of stable material into unstable, energy-liberation material; sodium promotes the dissociation process whereby energy is actually liberated. In other words, calcium makes material avail- able, sodium causes this material to liberate its energy. In neither of these functions are the salts exclusive agents; the production of dissociable material may depend on other factors than calcium; the dissociation process may be affected by other substances than sodium. The notion that sodium and calcium ions are antagonistic in their influence on vital processes has prevailed since the work of Ringer ” and of Loeb * on the interaction of these and other ions. - The greater part of the evidence offered in favor of such antagonism has been derived from experiments on tissues other than heart, chiefly skeletal muscle, Fundulus, and Gonionemus. Loeb has recently presented 26 HOWELL: Journal of the American Medical Association, 1906, xlvi, No. 23. 27 Martin: this Journal, 1906, xv, p. 309. 78 Martin: Jbid., p. 316. 29 RINGER: Journal of physiology, 1886, vii, p. 302; also, xvili, 1895, p. 428. 39 Lorg: this Journal, 1900, iii, p. 337; also, Archiv fiir die gesammte Physiologie, lxxx, 1900, p. 229. 178 | E. G. Martin his ideas on salt antagonism,” taking the position that the antagonism is not a true one in the sense that one salt acts in direct opposition to another, but rather that there is a cooperative action of the salts upon the tissue, of such a sort as to interfere with the manifestation of the peculiar effect of either one. The experimental basis for Loeb’s view is chiefly a series of studies on the effects of salts on developing eggs of Fundulus. Osterhout *® has demonstrated by two distinct methods that in plant cells ‘‘the antagonistic action of salts is largely or entirely due to the fact that they hinder or prevent one another from entering the protoplasm.” Joseph and Meltzer, on the other hand, have described experi- ments on skeletal muscle, which furnish indication that in this par- ticular tissue there may be a direct salt antagonism. All these observations and opinions serve to show clearly that the conclusion reached depends often on the tissue studied To assume, then, an antagonistic action in heart tissue between sodium and calcium ions, on the ground of observations made on very different tissues, is surely unwarranted. ; When I reviewed the literature of the causation of the heart beat, after eliminating from my mind the idea that sodium and calcium are antagonistic, I wondered that the idea could persist so strongly with such meagre evidence in its favor. The observations upon which the belief is based, with reference to heart strips, are the recovery from sodium chloride ‘‘exhaustion”’ which follows the use of calcium, and the better beat in sodium-calcium mixtures as compared with pure sodium chloride solution,** and the observation of Howell *° that sodium tends to relaxation, while calcium tends to tonic shorten- ing. With reference to the improvement in beat caused by calcium, I have shown above that this is apparently a direct calcium effect, and there is abundant evidence that it may be quite independent of a previous injurious influence of sodium. Thus strips which have been vigorously active for hours in moist air often show marked 31 LoEeB: The Carpenter Lecture, Science, N.S., XxXxlV, I9II, p. 653. 32 OSTERHOUT: Science, N.S., XXXiV, 1911, p. 187; and Jbid., N.S., Xxxv, 1912, Pp: 112. 33 JOSEPH and MELTzER: this Journal, 1911, xxix, p. I. “LINGLE: -L06. Cit., Dp: 277. 3° HOWELL: this Journal, 1901, vi, pp. 184 and 199. cue Relation of the Blood Salts to Cardiac Contraction 179 increase in amplitude following the application of calcium-con- taining solutions, a result which can scarcely be due to an antagoniz- ing of sodium by the calcium. Howell’s correlation of the antagonism between sodium and cal- cium in the causation of the beat with their antagonism in relation to tone loses its force if the salts are shown not to be antagonistic in their effects on tone. In a recent paper *® I have analyzed the rela- tions of salts to cardiac tonus and have advanced evidence which seems to me to cast grave doubt on the view that sodium and calcium as present in the blood are definitely antagonistic in their influence on tone. If there is truth in the idea herein advanced that sodium and calcium ions are not antagonistic in their effects on heart tissue, but act positively on different phases of cardiac activity, the various previous observations on the behavior of heart substance when treated by these ions must be explicable in terms of this idea. I purpose, as briefly as possible, to discuss the recorded observations on this basis so as to show the applicability to them of my hypothesis. Ventricular tissue of the turtle in its normal relation to the blood supply we know to be highly responsive to stimuli from the venous end of the heart, but, as the immediate stand-still following excision shows, not capable of spontaneous activity. We have in this situa- tion a high degree of excitability in the absence of an effective inner stimulus. When ventricular tissue is removed from the blood supply, but not otherwise treated, its excitability, both to artificial stimulation and to the normal stimulus from the active venous region, diminishes steadily. This decline in excitability can be explained as due to a disturbance of equilibrium whereby the ions present in the tissue enter other than their normal combinations. A similar change for the ions of shed blood I have already suggested.*’ That the disturb- ance in ionic relationships is a permanent one is shown by the com- plete failure of excised ventricle to recover excitability unless treated with certain suitable solutions. Freshly cut ventricle strips immersed in 0.7 per cent sodium chlo- ride solution show a latent period in which there is at first a decline 36 Martin: this Journal, 1912, xxx, p. 182. 37 Martin: this Journal, 1904, Xi, p. 117. 180 | E. G. Martin in excitability to induction shocks. This is succeeded by a period of increasing excitability, and this in turn by spontaneous activity. Methods of shortening the latent period are by treatment with calcium-containing solutions or carbon dioxide (Martin) or sodium oxalate or galactose (Benedict). On the justifiable assumption that spontaneous beats begin as soon as the intensity of the inner stimulus meets the threshold we may look for shortening of the latent period either by agencies which intensify the inner stimulus or by those that increase the excitability. According to the theory proposed by Howell and supported in this paper the shortening of the latent period by calcium is due to the action of this substance in increasing the excita- bility. Inasmuch as the immediate onset of beats after sodium oxalate is said by Benedict ordinarily to require the use of Ringer’s solution rather than pure saline, we may interpret the effect of the oxalate as due to the precipitation of the calcium of the tissue, thereby preparing the way for immediate influence to be exerted by the calcium of the Ringer’s solution. The effects of carbon dioxide and of galactose are best to be explained, I believe, by attributing to them direct stimulating properties. The ultimate onset of spontaneous activity of strips in pure sodium chloride solution, following a period of increasing excitability, can be explained by supposing that the tendency of the ions contained in the tissue to enter combinations unfavorable to excitability is over- come by the presence of the sodium. This possibility I have previously pointed out.*® The declining series of, beats terminating in ‘‘sodium chloride exhaustion” signifies a continuous decline in the conversion of energy- liberating material from inactive into active form. This decline is probably due, as I have suggested elsewhere,*® primarily to insufficient oxidation, whereby unoxidized waste products accumulate to an extent which interferes with the production of energy-liberating material. The evidence for this view is the excellent recovery which follows treatment with abundant oxygen. Since calcium salts also induce recovery from this exhaustion we must believe that under the stimulation of calcium the production of dissociable material goes on in spite of the clogging effect of the unoxidized waste products, 38 MartTIN: this Journal, 1906, xvi, p. 203. 39 Martin: Ibid., 1906, xv, p. 319. Relation of the Blood Salts to Cardiac Contraction 181 although, as I have shown,” the calcium effect cannot manifest itself in the complete absence of oxygen. Sodium carbonate and sugar are other substances that bring about recovery from sodium chloride exhaustion. That induced by sodium carbonate is probably akin to the improvement which follows treatment with oxygen, at least in that it operates by getting rid of waste products, although in this case the method is neutralization and not oxidation. The beneficial effects of sugar solution may be either through influence on the conversion of material into available form, or, as seems to me equally likely, through a combination of this effect with a powerful reinforcement of the inner stimulus. The Role of Potassium. — That potassium is inhibitory to cardiac activity has long been definitely established. The precise mechanism of its action is, however, unknown. I am inclined to believe that potassium in moderate concentrations acts in opposition to the inner stimulus rather than in antagonism to the elaboration of energy- liberating material, but in higher concentrations opposes both processes. Some observations that bear out this view are reported by Howell * in connection with studies of the relation of the potas- sium content of the circulating medium to vagus inhibition. Howell states that if the concentration of potassium chloride in the medium is gradually increased a point is reached (0.1 per cent) above which there is a definite effect upon the force of the beat, as well as upon its rate. In this diminution of force we have definite evidence of an effect of potassium on the production of dissociable material in the ventricle. The effects of potassium on rate are irrelevant in this connection, since the ventricular rate is established by the venous part of the heart. That concentrations of potassium insufficient to affect unfavorably the production of energy-liberating material may, by opposing the inner stimulus, prevent the manifestation of spontaneous activity in the ventricle is shown by the experiment of which a tracing is given in Fig. 2, p. 170. In this experiment spontaneous rhythmicity developed as the result of immersion in 0.7 per cent sodium chloride solution, and after becoming well established was abolished by trans- ferring the tissue to Ringer’s solution containing a small excess of 40 MartTIN: Loc. cit., p. 316. 41 HowELL: this Journal, 1906, xv, p. 283. 182 E. G. Martin potassium chloride. That the suspension of activity was not due to interference with the production of dissociable material was shown when artificial stimuli were applied. The responses to these stimuli were progressively greater and greater, a result which can be explained only by supposing that the presence of potassium in small concentra- tion is no bar to the conversion of substance into energy-liberating form, although it does suffice to overcome the action of the inner stimulus. According to the view of the action of sodium and calcium set forth above, we may look upon potassium as antagonistic to both sodium and calcium, but to sodium more markedly than to calcium, a much higher concentration of potassium being required to counteract the influence of calcium on the process of preparing energy-liberating material than to counteract the directly stimulating influence of sodium. SUMMARY 1. Various means of studying the influence of salts on the heart- beat are considered. The conclusion is drawn that by virtue of the ‘fall or none”’ law the height of contraction is an index to the amount of available energy-liberating material present at the beginning of the contraction, and as such offers a fruitful means of attacking the problem of salt action. 2. Evidence is presented indicating that to a considerable degree the irritability of heart tissue depends on the amount of dissociable substance per unit of mass present within it, so that the height of contraction may serve to some extent as a criterion of irritability. 3. An experiment is described which may be interpreted as con- firming Cushny’s demonstration of the existence of a specific inner stimulus. 4. The characteristic features of ventricular activity in pure sodium chloride solutions are stated as follows: (1) for ventricle tissue to be active in sodium chloride solution the tissue must come from a medium favorable to the production of dissociable substance; (2) the onset of activity is prompter the greater the irritability in the preceding medium; (3) the first spontaneous contractions in sodium chloride equal in height the last in the preceding medium. Relation of the Blood Salts to Cardiac Contraction 183 5. Freshly excised ventricle tissue is shown to undergo a steady decline in irritability which can be overcome only by treatment with suitable solutions. The decline occurs during the first few minutes of immersion in sodium chloride solution or Ringer’s solution, but is presently succeeded in these solutions by steadily increasing irrita- bility. The initial decline is interpreted as an inevitable effect of cutting the tissue off from its usual environment. Its occurrence explains the latent period in sodium chloride. 6. The characteristic effects of calcium-containing solutions on heart tissue are shown to be always in the direction of increased vigor of beat. 7. To interpret the characteristic effects of sodium and calcium the notion that they are antagonistic in their action on heart tissue is rejected. To each is assigned a positive function on a definite phase of the heart’s activity. For calcium is assumed the function proposed for it by Howell of acting to promote the conversion of stable into © unstable energy-yielding material; and for sodium the function proposed by Lingle of serving as the immediate stimulus to bring about the actual dissociation, and so to initiate the beat. To account for various observations the further assumption is made that neither sodium nor calcium is an exclusive agent; the preparation of dis- sociable substance is hampered to a great degree by accumulating waste products, and is therefore aided by abundant supplies of oxygen or by sodium carbonate; carbon dioxide in moderate con- centration, and perhaps sugar, act to stimulate heart tissue directly, much as sodium does. THE SUGAR CONSUMPTION IN NORMAL AND DIABETIC (DEPANCREATED) DOGS AFTER EVISCERATION ! By J. J. R. MACLEOD ann R. G. PEARCE [From the Physiological Department, Western Reserve University, Cleveland, O.] N connection with the physiology of the carbohydrates no problem more urgently requires solution at the present day than that of the relationship which the pancreas holds to the utilization of sugar in the animal body. The hyperglycaemia which supervenes with such remarkable rapidity after complete pancreatectomy and the subsequent inability of the animal to metabolize any form of carbo- hydrates indicate that in the normal animal this relationship must be of the very greatest importance. When we attempt to explain the manner in which the pancreas exercises this function, however, great difficulties present themselves. We are, for example, not yet certain whether the pancreatic function is a local one or one effected in other parts of the body by an internal secretion (hormone) which the gland discharges into the blood. By a local function is meant one by which some toxic substance, present in the blood and produced in the course of body metabolism, is acted on by the pancreatic cells in such a way as to neutralize it. When this substance is not thus acted on by the pancreas its accumu- lation in the blood paralyzes the power of the tissues to utilize sugar. Although there is no known fact which absolutely disproves this view there is likewise no direct evidence which would encourage us to enter- tain it, so that the tendency at present is to consider the pancreatic influence as being exercised by means of an internal secretion. Even if we accept this view, however, there is no agreement as to the modus operandi of the hormone. Some believe that the hormone is necessary for the utilization of dextrose in the tissues, others, that 1 A preliminary communication of many of the observations included in this article appeared in the Zentralblatt fiir Physiologie, 1913 (March), p. 1311. Sugar Consumption 185 it participates in the control of the mobilization of dextrose from the liver. According to this latter view the disturbance which removal of the pancreas creates is one in which the liver not only loses the power of retaining absorbed dextrose as glycogen but in which it also develops in an exaggerated degree the power of producing sugar out of proteins and certain fats (gluconeogenesis). The sugar which thus passes the liver without having gone through a glycogen stage, and the new sugar which has been produced, accumulate in the blood more quickly than the tissues can utilize them, even although the tissues are not believed to show any less than their normal glycolytic powers. A third view, which is really a compromise between the other two, supposes that the tissues cannot utilize dextrose until after it has undergone a preliminary transformation, one stage of which involves the production of glycogen for which the pancreatic hormone is necessary. It has proved itself to be a most difficult matter to devise experi- ments which offer decisive evidence for or against any one of these hypotheses. Thus, the observations of Forschbach,’” that the removal of the pancreas from one of two animals that had been previously united by sewing skin, muscles and peritoneum together (parabiosis) did not cause the usual degree of glycosuria, is unconvincing. Even if the observations themselves were free of criticism — which they are not——the immunity to diabetes of the depancreated animal might just as well be explained by local action of the pancreas of the anastomosed animal as by the presence of an internal secretion. The same criticism can be made of the observations of Carlson and Drennan * that pancreatectomy in pregnant dogs near full term is not followed by the usual degree of glycosuria. One of these authors (Drennan *) has furnished another type of evidence which if correct would certainly favor the ‘‘hormone”’ hypothesis. He found that the amount of sugar excreted by a depancreated dog in twenty-four hours became less when blood from a normal animal was intravenously injected. Unfortunately, however, the urine alone was examined; 2 FoRSCHBACH: Deutsches medicinische wochenschrift, 1908, xxxiv, p. 910; also Archiv fiir experimentelle Pathologie und Pharmacologie, 1908, ix, p. 131. 3 CARLSON and DRENNAN: this Journal, 1911, xxviii, p. 391. 4 Ibid, p. 306. 186 J.J.R. MacLeod and R. G. Pearce the evidence would be much more convincing if the behavior of the blood-sugar had also been ascertained. Seemingly incontrovertible evidence in favor of the “‘hormone”’ hypothesis has recently been furnished by Knowlton and Starling.® These authors have studied the rate of dextrose consumption in blood perfused through the heart and lungs of normal and depancreated dogs. They found that the normal heart in an hour consumed about 4 mg. of dextrose for every gram of heart muscle, whereas the diabetic heart in four cases did not consume any dextrose in this time. They also found that the rate of disappearance, although low at first, gradually became greater when blood from a normal animal was perfused through a diabetic heart, and conversely, that the per- fusion of blood from a diabetic animal through a normal heart was followed by a practically normal rate of dextrose consumption for the first hour but that this diminished subsequently. The interpre- tation which Knowlton and Starling put on these results is that the normal blood and tissues contain some substance which is necessary for the utilization of sugar in the organism, and they further offer, as evidence that the ‘‘hormone”’ is derived from the pancreas, the observation that the addition of a decoction of the pancreas, made with faintly acid Ringer’s solution and subsequently neutralized with sodium carbonate, caused the diabetic blood to reacquire its normal glycolytic power, when perfused through the diabetic heart. Confirmatory evidence for these remarkable observations has been furnished by Maclean and Smedley ® who found that there was very much less consumption of dextrose from oxygenated ‘Locke’s solution when this was perfused through the heart of a de- pancreated animal than when it was perfused through a normal heart. These authors were however unable entirely to confirm Knowlton and Starling’s statement that the addition of pancreatic extract restores the sugar-consuming power. They point out that the extracts may not have been prepared under exactly the same con- ditions. Although the conclusions which are drawn, at least in so far as 5’ KNOWLTON and STARLING: Zentralblatt fiir Physiologie, 1912, xxvi, p. 169; Proceedings of the Royal Society, 1912, lxxxv, p. 218; The Journal of physiology, 1912, xlv, p. 146. 6 MACLEAN and SMEDLEY: The Journal of physiology, 1913, xlv, p. 470. Sugar Consumption 187 they refer to utilization of sugar by the heart, do seem to be justified by the published results, there are, nevertheless, several technical details in connection with the experiments which demand attention. ‘In the first place, it is unfortunate that an untried method was employed by Knowlton and Starling for estimating the amount of sugar in the blood mixture that was used for perfusion. This method consisted in precipitating the proteins in the blood or serum with copper sulphate and subsequently removing the excess of copper in the protein-free filtrate with sodium hydrate. We have found, by comparison of results obtained on the same blood by this and the well-tried method of Rona and Michaelis, that considerable discrep- ancy is likely to occur unless very great care is taken to add just exactly the amount of sodium hydrate that is necessary to neutralize the cupric sulphate. Even with these precautions, we have been unable to obtain the same close agreement between the duplicates that is observed when the Rona-Michaelis method is employed, nor have we found that the results obtained by the two methods always agree with one another. These facts are shown in the follow- ing table: TABLE I SuGAR IN BLoop OR SERUM AFTER REMOVAL OF THE PROTEIN BY COPPER SULPHATE OR COLLOIDAL IRON Reducing Substance in per cent after precipitation by: Fluid employed CuSO, Colloidal iron 0.098 0.122 I. Dog blood 0.127 0.122 0.153 0.161 Dog serum 0.145 0.161 II. Dog serum and 0.2 ! per cent dextrose ee one Dog serum and 0.4! per cent dextrose ae eee II. Ox blood and 0.41 percent dextrose . ven ee Serum of above 0.967 0.999 1 These percentages are approximate. 188 J.J. R. MacLeod and R. G. Pearce We were never able to obtain accurate agreement between the dupli- cates. by the copper method whereas this is usually the case when the colloidal method is employed. We have probably omitted some pre- caution which Knowlton and Starling adopted, but of which they do not warn us in their published papers. We can only add that we have been most careful to follow their directions. These authors them- selves obtained much more satisfactory results; for example, out of eleven duplicate analyses there was absolute agreement in six, a dif- ference of less than 2 per cent in three. In two cases, however, the error was more than 5 per cent. It is stated by Knowlton and Starling that the amount of gly- colysis in the blood under the conditions of their experiments could not have exceeded o.o1 per cent dextrose per hour. This is without doubt too low a figure. It is at least very much less than that found by one of us (J. J. R. M.) to apply in the case of defibrinated or “hirudin”’ blood of the dog incubated under strictly sterile conditions at body temperature;’ thus, in defibrinated blood the following per- centile glycolysis was observed :— 40 (2 hrs. 15 min.), 55 (2 hrs. somin.), 34 (2 hrs. 30 min.), 27 (19 hrs.) ; and in Hirudin blood these values were: 70 (2 hrs. 30 min.) and 37 (2 hrs. 30 min.). It should be pointed out, however, that in the experiments referred to no dextrose was added to the blood whereas in those of Knowlton and Starling a considerable quantity of dextrose was usually added. This was especially the case in those bloods which exhibited a very slight degree of glycolysis (cf. Table I of Knowlton and Starling). The importance of this observation will be discussed in a subsequent paper; meanwhile it is significant to note that the sugar content of the blood used for perfusing the heart of the diabetic dogs was very high in the three cases recorded in which no sugar disappeared, (Nos. 12, 17, and 18, Table III), possibly because dextrose had been added in excess of the amount usually present, even in diabetic blood. Edelmann,® working with oxalate blood (of the dog) found that after two hours incubation from 11.26 to 24.24 per cent of sugar dis- appeared and Loeb,’ using defibrinated blood, found in ninety minutes 7 Unpublished experiments. The sterility of the blood was tested by bac- teriological examination. 8 EDELMANN: Biochemische Zeitschrift, 1912, xl, p. 314. S LOEB, A.: Lbid., 1913, xix, 1p. .A 53. Sugar Consumption 189 that from 48 to 62 per cent disappeared. The former author also found that a certain amount of glycolysis likewise occurs in diabetic blood and this was also observed to be the case by Knowlton and Starling.!° It is somewhat difficult to harmonize this fact with the statement that in blood perfused through the diabetic heart there should sometimes be no glycolysis whatsoever. Although, as computed from Knowlton and Starling’s tables, it is the case that the general average for sugar consumption by the normal hearts was about 4 mg. per gram heart muscle per hour yet there were very great deviations from this average; thus, leaving out of account cases in which the anaesthetic was left on by mistake, or in which the blood was very venous, the variations ran from 2.84 to 6.29 mg. per gram muscle per hour. In three of the seven hearts of diabetic animals the consumption varied from 1.8 mg. to 4.9 mg. per gram per hour, but this almost normal consumption is explained by the authors as probably due in part, at least, to bacterial growth; in the remaining four observations of this series extra precautions against bacterial growth were taken and no dextrose was used. In five diabetic dogs of another series of observations the consumption was much less than normal. It is stated by Knowlton and Starling that the diabetic heart beat was very slow but that it increased in rate whenever pancreatic extract was added to the perfusion fluid. This and the accompany- ing partial restoration in sugar consuming power may have depended on the fact that the extract was neutralized with sodium carbonate, the presence of which in Locke’s solution as Neukirch and Rona” have shown materially augments the beat and increases the sugar consuming power of the heart. Knowlton and Starling also observed slight quickening of a perfused diabetic heart when some sodium bicarbonate was added to the perfusion fluid. In making these criticisms we do not desire to be understood as denying the possibility that less sugar may be used by the diabetic as compared with the normal heart. We believe however that the observations so far recorded do not unmistakably prove this fact. 10 KNOWLTON and StarLinc: Cf. Proceedings of the Royal Society, 1912, Be EEXxv, (p. 221: 11 NEUKIRCH and Rona: Archiv fiir die gesammte Physiologie, 1912, cxlviii, Dp. 285. 190 J.J. R. MacLeod and R. G. Pearce But even if the diabetic heart should consume less sugar it is not justifiable to conclude that removal of the pancreas brings about the disappearance from the blood of some substance which is necessary for the utilization of carbohydrates in the other tissues of the body. It is necessary before drawing such a conclusion to compare the sugar utilization in the skeletal muscles of normal and diabetic animals. Theoretically, this could most simply be done by ascertaining the rate of sugar consumption in the artificially perfused hind limbs. Since such experiments always involve a certain risk of bacterial con- tamination, and since there are other technical difficulties and sources of inaccuracy connected with them, we have put the question to the test by observing the behavior of the sugar in the blood of dogs from which all the abdominal viscera were removed. Following such evisceration, as Bock and Hofmann, Pavy, and one of ourselves ? have shown, the percentage of sugar in the blood steadily falls because the utilization of sugar in the tissues cannot be compensated by an increased discharge of sugar from the liver. Such preparations may really be considered as perfusions of the muscles with blood pumped by the heart and arterialized by the lungs. In them the conditions are certainly more nearly normal than is the case when an artificial pump and 7m vitro arterialization of the blood are employed. There are however two difficulties which present themselves in the use of such preparations. The first of these is that anaesthetic must con- tinue to be administered, the presence of which in the blood might, as Knowlton and Starling imply, depress the glycolytic power. To control this, in about one half of our experiments, we have tied the innominate and left subclavian arteries just after their origin from the aorta, thus removing the higher nerve centres from the circulation and rendering the administration of anaesthetic unnecessary. As can be seen from the tables of results, however, this did not measur- ably affect the rate with which sugar disappeared. The other dif- ficulty was with regard to the arterial blood pressure. Although this invariably rose considerably, as an immediate result of the liga- tion of the coeliac axis, it subsequently fell, as a rule, until, in some cases, it came to be no higher than about 40 mm. Hg; _ indeed, especi- 2% Bock and HormMann: Experimental Studien iiber Diabetes, Berlin; Pavy and Srau: The Journal of physiology, 1903, xxix, p. 375; MACLEOD: this Journal, 1909, xxiii, p. 278. ' j Sugar Consumption IgI ally in our earlier observations, the blood pressure might steadily decline to zero within a period of about half an hour after ligation of the abdominal vessels. The cause for the fall of pressure in these earlier experiments was probably hemorrhage into some untied small branch of the aorta, for the viscera were not actually removed after ligation of the vessels. This bleeding may have been through vessels coming to the stomach from the oesophagus or on to the rectum from the inferior haemor- rhoidal arteries. In the subsequent experiments, where actual evis- ceration was practiced, these vessels were necessarily tied.” But even when every precaution was taken to avoid any such leakage of blood into untied splanchnic vessels, the blood pressure usually fell, so that within an hour after the evisceration it was no more than 60 mm. Hg. Such a fall was not experienced by Pavy. Since we have found from experience that irregular results in the amount of sugar in the blood are likely to be obtained when the blood pressure is much below 40 mm. Hg., we have, in the later experiments of the present research, kept it at a higher level than this by intravenous injections of adrenalin. The amount of adrenalin injected was always adjusted so as to keep the pressure as constant as possible. The preparation used was 1~1000 adrenalin chloride (Parke Davis); in some cases it was injected undiluted, in others it was diluted five times with Locke’s solution. The amounts required were never large and even in the cases where the dilute solution was employed were never sufficient to bring about any material dilution of the blood. It can be seen, by comparing the results obtained in cases with and without adrenalin, that the injection did not in any way influence the rate of sugar disappearance. In certain of the experi- ments the animals died from cardiac failure in spite of all we could _do to prevent it and we were compelled to take the blood for analysis from the heart chambers after death. We have found, however, that the amount of sugar in such blood is practically always considerably in excess of that present in blood removed even a few minutes pre- viously from the renal vein or carotid artery. The sugar was estimated in the blood by Bertrand’s method after 13 On account of Pavy’s observation that even after ligation of the portal vein there may be some sugar discharged by way of the hepatic veins, we have in all experiments applied mass ligatures to the back portions of the liver lobes. 192 J.J. R. MacLeod and R. G. Pearce removal of the proteins of means of colloidal iron. In as many cases as possible duplicate analyses were made. CONSIDERATION OF RESULTS In order that we might have some standard with which to compare the rate of glycolysis following pancreatectomy, a series of observa- tions were made on samples of blood removed at varying intervals after evisceration in normal dogs. It was hoped that it would be pos- sible so to control the conditions of the experiments, that a constant rate of sugar disappearance for different animals could be determined. The results actually obtained in a series of eleven such observations are given in Table II from which it will be seen that no such con- stancy was attained. The values set down in the fourth column of the table most clearly demonstrate the rate of glycolysis. They represent the milligrams of dextrose which disappeared from too gm. of blood during one minute. TABLE II GLYCOLYSIS IN THE BLooD oF EVISCERATED NON-DIABETIC Docs Amount of dextrose dis- Time after | Dextrose | appearing evisceration | in blood |from 100 gm. Remarks (minutes) |(per cent)| blood per minute (milligrams) Ether anaesthesia. | B. P. fell from 120 to 40 mm. Hg. Morphine and ure- | thane. B. P. fell | ie 70 to 10 mm. | Aceraneal g. vessels * Dead. liga ted (no adrenalin) Cerebral vessels tied. B. P. fell from 50 to 10 mm. Hg. Ether anaesthesia. B. P. 50 mm. Hg. falling to zero. Sugar Consumption 193 TABLE II (Continued) Amount of dextrose dis- Time alter | Dextrose} appearing No. | evisceration | in blood | from 100 gm. Remarks (minutes) |(per cent)| blood per minute (milligrams) Ether and urethane. Morphine and _ ure- thane. B.P. 90-40 mm. Hg. * Heart-blood. {10 min. after opera- | Abdominal vessels tions. ligated and defi- —_________——_ | brinated blood Ether and urethane. [| p/ws dextrose then B. P. 120-20 mm. | injected. Hg. (No adrenalin) 2B Ps practically. zero. 7 15 mm. after opera- tions. Morphine and _ ure- thane. B. P. 80- 20 mm. Hg. Ether anaesthesia. B. P. 160-60 mm. Hg. (viscera not re- moved), 1—5000 adrenalin. Cerebral vessels . i enied eet eats | Abdominal vessels 5000 adrenalin. B. ligated and viscera P, 80-40 mm. He: removed in two ee: : experiments. Starved animal. Adrenalin injected. Hemorrhage during operations, only 1 c.c. 1-1000 adrena- lin used. B. P. 60 mm. Hg. In the first four experiments no special precautions were taken to prevent the fall of blood pressure ensuing upon evisceration; and if we leave out of account those determinations which were made on blood samples removed when the blood pressure was near zero, J.J. R. MacLeod and R. G. Pearce 194 *poAowor JOU L0 6F7'0 06 Aoupryoug § *q “¢ UlvjUreUr OF pozdol : 8970 (pomoyye Fur Ul[eUSIPe QOOS-T YOR so9Iye a0 £17'0 te [se autos) “utd 06 07 dn “SFT “wu QQ dAoqe C0 8870 o£ UT poulveutal “gq “gq “vIsoyJSovuev IY] S £0e'0 0 'T Or (FOTO) ce “Ul[eusIpe 07. cse0 07 (WOOS-T °9°9 SL poyeSIT spassoa yerqar19y a7 : Scr 0 0 87C 6 UOTPEPOSNSaI YA é § LET ainyprey oviIprw~s yuowLodxe sulan(d CC L LEL'O 09 ‘BH WW O8-OZT “d “| = “UlyRuoipe + OST'O CY OOOS-T *9°9 OE +PoreSI] Spassoa [e1qoI9\) ¢ 110 SI Sat 8 *poqieys JUN -lwodxo d10Joq puNnqiiow JsowyR 3soq ‘SH WU OF-08 ‘d “Gq ‘peAotuos (UOI]RAIL}S) JOU BIDISIA "PoPRSI] JOU SOqoy JOATT 09°F S70 CT ee ‘Ul[RUdIpe ON “vIsoyjsovue JOY S Ssoe'0 0 27 } ‘SH “Uru Oz eIT0 SF OF-08 ‘d “@ ‘paqefur ulpeuorpe 98'T SFT 0 o¢ OOOT-T °9°9 OF !Pe7eSI] Sjossoa [e.1qo19;) £ 1e70 0 0'P £ (SuurersTT]IUL) Auto}99 = |aynurur Jod pooyq) (uso 309d) (soqnurUut) SYIVWIDYT -yeoroued |'wWS QQ] Woy Sut} pooyq ur | uoTZeIODSIAD | ONVI‘N ‘d ‘ON Joye sAvqq | -rvoddesip asoi} | asoryxeq{ | J9}e OWI], -xop Jo yunoury sooq SILAaAVIC] GALVAAOSIAY, AO GOOTY AHL NI SISATOOATS) i Tl WIaVeL 195 Sugar Consumption “peep ysnf jewruy , ‘][e} 07 ueZoq uleusipe QOOS-T °9°9 OF ‘peIesl] SAQUPIY YIOG ‘“pozeSIy spassaa [erqaiad i : ST "Yeap Jaqye JRIY WOT] poyda]JOD , “Buryyey Ajyenpeas “ur QZT “d “Aq = “poresiy «1 FEO se sAoupry Jog “Ul[eueIpe QOOT-T *9°9 + aa | 9770 o¢ ¢ UvY} SseT fpazyeSI] sjassaa JeaqeIag P 6970 0 ST al ST OfT 0 Si § 14ST0 o¢ “WU 07-09 ‘dq = ‘paqoel Inks LLSTO “Ul UlTRUsIPe QOOS-]T “9°9 CE ‘poze $970 (vlunjeuary) BI] séoupry YIOg = “visayjsovur J9YIq L L170 0 = eT “poolq BB ics) 8 i i ‘3H WU OF-08 “dA ‘peresyy +PP1'0 9 sAoupry yIOg ‘poazoafut ul[eudIpe LZ 7270 OF (UOI}BAIV}S) OOOS-T “9°92 OOT ‘¥Isaqysovue sJoyIy 9 Te¢e"0 0 se cl “‘pooyq Pa y : £6r'0 “BHT “ww 09 veld Ri ge 6d A “AyMojs poqoafur urpeuorpe est $770 09 QOOT-T “2°9 Z “UM QO Jaye ‘paze €L'0> 0LZ'0 o¢ (UOI}VAIe ys) -s1] sAoupry Pog = “vIsoyysavue 19474 9 7670 0 6¢ Il } 7LV0 990'T O09T'0 IG £170 “SPT WU OFT paeseIoAR MOU "g “{ 01z'0 ‘paqoofur as01}xep S77¢ poojq [CULION 95°0 Z¢7'0 ZT 196 J.J. R. MacLeod and R. G. Pearce it will be seen the dextrose consumption varied between 0.83 to 2.4 | mg. In the next four experiments (Nos. 5 to 8 inclusive) it was at- tempted to maintain the blood pressure in the eviscerated animals by removing usually about 150 c.c. of blood from the animal at the start of the experiment and after defibrination and the addition of an equal volume of Locke’s solution, containing 1 per cent dextrose, reinjecting it into the animal immediately after the evisceration. This was done in order to leave as small a quantity of blood as possible in the tied- off splanchnic vessels. The procedure did not materially prevent the rapid fall of blood pressure. The glycolysis was naturally some- what quicker immediately after the injection (in one case rising to 4.46 mg.) but after some time it fell to between 0.46 and 0.97 mg. dextrose per minute. In the last three experiments of the table, adrenalin (1-1000 or 1—5000 in Locke’s solution) was injected at constant pressure into the renal vein at such a rate as to keep the blood pressure at least above 60 mm.; the dextrose consumption varied between 1.13 and 2.8 mg. per minute, thus indicating, when compared with the previous figures, that the adrenalin had no material influence on the rate of glycolysis. Under the conditions of these experiments there is therefore a considerable variation in the glycolysis of different normal animals and it is not possible to co-relate the variations either with the mean arterial blood pressure or with the tying off of the head arteries which of course also involves opening the thorax and applying artificial respiration. Turning now to the results obtained on diabetic animals, which ~ are given in Table III, it is to be noted that in a much larger pro- portion of the experiments the fall of blood pressure produced by evisceration was compensated by injections of adrenalin. These experiments, being on very valuable material, were naturally not undertaken until after the experience gained by work or normal animals had been obtained, the outcome of which as we have seen was to indicate adrenalin injections as the most satisfactory means of keeping up the blood pressure That such injections did not bear any relationship to the rate of glycolysis in diabetic animals was demonstrated in one or two cases in which the drug did not require to be given until the latter part of the experiment, in one case (No. 10) the rate of glycolysis was the same before and after the injection, Sugar Consumption 197 in another (No. 11) it was less and in a third (No. r5) it was greater. Nor was ligation of the vessels proceeding to the head and fore limbs associated with any demonstrable variation in the glycolysis. Thus, in the cases in which these vessels were untied (Nos. 10, 11, 12, 1 3 and 14), and the anaesthesia consequently maintained the rate of glycolysis varied between 0.5 (No. 10) and 4.6 (No. 4) mg. per minute, whereas in those in which the vessels were tied (Nos. 3, 8, 9, 14 and 15), it varied between 1.5 and 4.3 mg. per minute. When we compare the results obtained on diabetic with those obtained on normal dogs it is plain that no difference can be made out. For some reason which we cannot explain, the results in both cases are extremely-variable and it, therefore, becomes impossible to give any average which would be at all reliable for either series of observa- tions. However, some comparisons may be of interest. The average dextrose consumption for the 11 normal dogs (17 observations) is 1.63 mg. per minute and for the ro diabetic animals (19 observations) it is 1.86 mg. per minute. The maximum and minimum rates for the non-diabetic animals (including those in which dextrose was injected) are 2.4 and 0.83 mg. per minute respectively, and for those that were diabetic, 3.7 and 0.50. In one experiment on a diabetic animal (No. 10) there does appear to be distinctly less glycolysis than in any other case, either normal or diabetic. This was an unusally resistant, although markedly diabetic, animal, the blood pressure remaining above 60 mm. for more than an hour after the evisceration so that no adrenalin had to be injected until after ninety minutes. The dextrose consumption meanwhile ranged between 0.5 and 0.7 mg. per cent per minute. Unfortunately in this experiment the kidney on the right side was not tied off so that the comparatively small decrease in dextrose which did occur might be accounted for, in part at least, by excretion into the urine. In five other experiments, however, the vessels of both kidneys were ligated and in those the glycolysis was very marked. The injection of 250 c.c. defibrinated blood containing 5 per cent dextrose into this dog was followed by more marked glycolysis which might be attrib- uted to the influence of pancreatic hormone. We are not prepared to give any other explanation at present, only we would point out the remarkable rate in the experiments on normal dogs, in which dex- trose was injected, at which it disappeared. Perhaps the dextrose 198 J. J.R. MacLeod and R. G. Pearce introduced in this way finds some depéot other than the liver in which it is converted to glycogen. The possibility of its being retained in the lymph must also be borne in mind. We cannot draw any conclusions from our results regarding the comparative rates of sugar utilization by the normal and the diabetic heart, but we believe that there is no difference in this regard in so far as the skeletal muscles are concerned. It is possible that the difference observed by Knowlton and Starling in sugar consumption between normal and diabetic hearts might have existed under the conditions of our experiments and yet have been insufficient to make an impres- sion on the blood of the eviscerated animal. The conditions which influence the rate of sugar disappearance are so variable in different animals, and even in the same animal at different periods of the same observation, that it has proved impossible to obtain an average figure for sugar utilization by the use of which we could decide whether this was more marked in the one group than in the other. As already pointed out we can offer no explanation for these variations. We are certain, however, that they do not depend on any error in the blood-sugar estimations. | As a criterion of the degree of diabetes in the depancreated animals we have as usual taken the D. N. ratio. In one instance this could not be obtained because of an unaccountable and severe haematuria. Most of the animals received a pint of milk for the day or two follow- ing the pancreatectomy and meat on the succeeding days. In one or two instances they were starved for the day immediately preced- ing the evisceration experiment. It will be seen from the second column that all of the dogs were diabetic to the full degree. Although the operations were performed with every aseptic precaution possible and were kept afterwards in thoroughly clean cages there was always some suppuration of the wound, a condition which we have very rarely experienced in operations on non-diabetic animals. For example, in the experiments numbered three and four in the table the pancreat- ectomy was performed in two stages; in the first one the gland was removed except for the free vertical portion (processus uncinatus) which was grafted, with its blood vessels intact, in the subcutaneous tissues of the abdominal wall. After this operation there was no suppuration. The graft was then removed (without opening the abdomen) and the wound suppurated. Sugar Consumption 199 In conclusion we may point out that even if the isolated heart of the diabetic animal should be unable to utilize dextrose, this need not necessarily imply that it is because of the absence of some hormone which is necessary for utilization of dextrose by the heart muscle. It may be because of the presence in diabetic blood of toxic substances which may interfere with sugar utilization by the heart, under the conditions of the perfusion experiments. The fact that addition of pancreatic extract to the diabetic blood brings about a restoration of the glycolytic power — a fact which is not, we believe, supported by a sufficient amount of evidence (see p. 186) — would certainly offer stronger support to the hormone hypothesis but it would not necessarily render the ‘‘toxic’’ hypothesis untenable, for such an extract might contain antitoxin. ON THE FORMATION OF FAT FROM CARBOHYDRATES By SERGIUS MORGULIS anp JOSEPH H. PRATT [From the Nutrition Laboratory of the Carnegie Institution of Washington, Boston, Mass., and the Laboratory of the Theory and Practice of Physic of Harvard University.] HE experiments described in this paper have all been performed on a dog belonging to one of us (J. H. P.), which showed certain peculiarities in the metabolic activity due to pancreatic achylia. On March 29, nearly eight months before our experiments were begun, the corpus pancreatis of the dog was removed, leaving the processus lienalis and the processus uncinatus in situ. A detailed description of the operation and subsequent history of this dog ‘‘ Flora”’ is given in another paper.' At the time of the operation the dog was very fat and weighed 13.8 kg. There was a steady loss of weight from the time of the exclusion of the pancreatic juice from the intestine to the end of June, when it had fallen to 6.5 kg. From this time to the middle of September the weight was fairly constant. Diabetes did not develop. The administration of pigs’ pancreas, which was begun in September, was followed by a gain in weight. When the feeding of pancreas was discontinued on October 21 the dog weighed 8.35 kg. An absorption experiment was begun on November 12 and con- tinued for four days. During this period the dog ate 3000 gm. of meat. 42.23 per cent of the nitrogen of the food was lost in the feces and 72.73 per cent of the fat. The animal was in nitrogenous equilibrium, losing only .o2 gm. of body nitrogen during the four days. The experiments were performed at the Nutrition Laboratory with the dog apparatus constructed on the principle of the closed circuit, and essentially like that described by Prof. F. G. Benedict 1 BENEDICT, F. G., and Pratt, J. H.: Journal of biological chemistry, July, ror3. Formation of Fat From Carbohydrates 201 in an earlier paper 7 but modified by him for direct determinations of oxygen. It would be superfluous to give here a detailed description of the apparatus; it will suffice to mention that the animal’s muscular activity has been carefully controlled by means of a pneumograph attached to the cage in which the animal is kept, and communicating with a Marey tambour. The cage being suspended by a spring at one end and resting on two knife edges at the opposite end is sensitive to the slightest movement on the part of the animal, and the move- ments are recorded on a kymograph by means of a pointer resting on the tambour. The carbon dioxide, absorbed in soda lime bottles, is ascertained by the difference in weight of the bottles at the beginning and end of each experiment. As there are two sets of soda lime absorbers attached to the apparatus, either one or the other may be brought into operation by a shift of a three-way valve. By this arrangement it was possible to determine the carbon-dioxide produc- tion during successive half-hour periods without interrupting the course of the experiment. The oxygen was admitted into the system from a small cylinder, which was likewise weighed before and after each period. The amount of oxygen thus determined by weight was also checked by readings of a Bohr meter through which the oxygen passed before entering into the system. The relative humidity of the air of the respiration chamber was measured by a psychrometer; samples of the air at the end of the experiment or at the end of each successive period have been analyzed for carbon dioxide with the Sondén apparatus. Knowing the empirical volume of the entire system (including the chamber and accessories) we were able to compute the quantita- tive composition of the residual air at the end of each period by mak- ing use of the data obtained from readings of the psychrometer, barometer, thermometer, and the per cent of carbon dioxide in the air. The difference in the residuals enabled us to make corrections for the consumption of oxygen and the production of carbon dioxide as determined directly by weight. The efficiency and accuracy of the respiration apparatus have been frequently tested, and we did not run regular experiments before complete satisfaction as to its perfection in this regard could be 2 BENEDICT, F. G., and Homans, J.: this Journal, 1911, xxviii, p. 29; Journal of medical research, 1912, xxv, p. 409. 202 Sergius Morgulis and Joseph H. Pratt obtained. The system was tested for tightness and blank experiments showed no changes in the weight of the soda-lime absorbers. Further- more, check experiments were performed from time to time by burn- ing ethyl alcohol in the respiration chamber. Almost invariably the quotients obtained were in complete agreement with the theoretical expectation for the combustion of alcohol, thus giving positive proof of the accuracy of our apparatus. There is nothing essentially novel in the fact that within the organism of an animal one substance may be chemically transformed into another substance. The formation of carbohydrates from fat or protein, and the formation of fat from carbohydrates have been maintained by physiologists on different occasions, and in the ma- jority of cases with sufficient justification. The transformation of fat into carbohydrates, for instance, figured greatly in metabolism studies with hibernating animals where it was thought that this transformation furnished the raison-d’étre for the extremely low respiratory quotients observed by several investigators. This hypoth- esis, however, has serious objections against it, and recently it has been severely criticized by Nagai,’ who showed that with accurate and well-controlled methods one does not find quotients with hibernat- ing animals as low as were hitherto claimed. © The fact of the trans- formation of carbohydrates into fat, on the contrary, has been receiving more and more confirmation ever since Liebig supposed that such a change may occur, and it may be said that today this is fairly beyond questioning. It was corroborated by the experience of breeders and agriculturists that rich carbohydrate food is conducive to the fatten- ing of the stock. Henneberg (1881), Chaniewski (1884),° Munk (1885),° Meissl (1886)? and others® experimenting with various animals, such as sheep, geese, dogs, pigs, found that in striking the balance of the intake and output, and comparing it with the actual acquisi- * Nacal, H.: Zeitschrift fiir allgemeine Physiologie, 1909, ix, pp. 243-367. * HENNEBERG, W.: Zeitschrift fiir Biologie, 1881, xvii, pp. 295-350. ° CHANIEWSKI, T.: Zeitschrift fiir Biologie, 1884, xx, pp. 178-1092. 6 Munk, J.: Virchows Archiv fiir Pathologische Anatomie, 1885, cx, pp. — QI-134. 7 Mersst, E.: Zeitschrift fiir Biologie, 1886, xxii, pp. 63-160. 8’ LEHMANN, K. B., und Vort, E. Zeitschrift fiir Biologie, roo1, xlii, pp- 619-671. ® RuBNER, MAx: Zeitschrift fiir Biologie, 1886, xxii, pp. 272-280. Formation of Fat From Carbohydrates 203 tions of the body they could not evade the conclusion that the carbo- hydrates had contributed to the deposit of fat. These experiments dealt with the assimilation of protein, fat and carbohydrate unassisted by a parallel investigation of the respiratory exchange of the animals under observation. Yet, if the transformation of carbohydrate into fat is an actuality, it should be reasonable to expect some clue as to its occurrence in the gaseous metabolism, since a substance rich in oxygen is thereby changed into one poor in oxygen. In other words this process should become revealed in the respiratory quotient which is the ratio between the carbon dioxide produced and the oxygen consumed during any length of time. When this paper was already written we found an interesting paper in Russian, in which the metabolism of both dogs and rabbits fed only on sugar was discussed. The author of this article apparently did not realize the significance of the respiratory quotient, as through- out his voluminous tables and text he makes no mention of it. He determined, however, the carbon dioxide production and oxygen consumption of his subjects, and we were thus able to work out the respiratory quotients from his data. The experiments were per- TABLE I Original data Computed | | we . CO, | O CO, O bad -| Respiratory aCe as : nae Quotient Average in grams Average in litres per day per day Damka 9 265.1 224.7 134.96 157.29 858 fasting 9 389.2 223.4 || 198.14 156.38 1.267 sugar diet | 29 1094.8 747.6 || 557.36 523.32 1.065 sugar diet Bijka 9 330.7 325.5 || 168.36 227.85 739 fasting | 9 368.5 267.2 187.60 187.04 | 1.003 sugar diet 204 Sergius Morgulis and Joseph H. Pratt formed in the following way: An animal was first subjected to a fast lasting several days, with water, and the urine was analyzed for nitrogen and the various inorganic constituents. Then the animal, after it had recuperated from the fast and attained the initial weight again, was put on a diet of sugar either in the form of solid lumps or in the form of a solution which was given through the stomach tube. The urines were also collected and analyzed as in the preliminary period. In a few instances the gaseous exchange was likewise deter- mined, and we used these data in compiling Table I. We may observe that in either case with an exclusive sugar diet the dog had a respiratory quotient of over one. In the case of the dog ‘“‘Damka”’ the average quotient for the first nine days of the sugar feeding was 1.267, and for the entire experimental period of twenty- nine days, 1.065. The other dog ‘‘Bijka”’ shows only a slight rise of the respiratory quotient over one, but the respiratory quotient of the first dog is likewise a great deal higher when fasting. It is possible that the method for measuring the gaseous exchange employed by Protasov ° was not unimpeachable, as it is very improbable for an animal fasting nine days to have a respiratory quotient of .858, and it may be that the figures for the oxygen are lower than the actual consumption. We believe that Pfliiger first realized the importance of the gaseous exchange and of the respiratory quotient in the transformation of carbohydrate into fat when he instructed his student Bleibtreu ” to carry out a study of the gaseous metabolism. Bleibtreu employed young geese which he fed superabundantly upon rye meal and found large deposits of fat in their bodies when they had been killed at the close of the experiment. He performed also several respiration experi- ments with the result, as was to be expected on theoretical grounds, that in each case the respiratory quotients rose above one, ranging from 1.117 to 1.38, thus furnishing new proof for the origin of fat from carbohydrates. While Bleibtreu’s experiments are extremely interesting, and undoubtedly conclusive, yet owing to deficiencies of ® Prortasovy, I. I.: Metabolism of matter under condition of exclusive feed- ing with sugar, Dissertation, Imperial Military Medical Academy in St. Peters- burg, 1895, p. 67. (In Russian.) 10 BLEIBTREU, M.: Archiv fiir die gesammte Physiologie, 1901, lxxxv, pp. 345-400. Formation of Fat From Carbohydrates 205 his apparatus they leave room for criticism, and it seems to us not unlikely that with methods more properly controlled one would not find quotients as high as 1.38. However this may be, his results are true in the main and the quotients of over one may be taken as a direct proof of the phenomenon which has been recognized and postulated long ago. Although the point may be considered well established, we do not hesitate to contribute to this subject as the conditions. under which the transformation of carbohydrates into fat took place are rather singular. Besides, our experiments present practically the first series of determinations of oxygen and carbon dioxide coincident with this transformation (certainly in the case of the dog) which have been made with a well-controlled and critical method. Our dog was in a very emaciated state and since, as was stated above, she could utilize but a fraction of the protein and fat in the ingested meat, most of it (40 and 70 per cent respectively) being excreted in the form of large ‘bulky stools, she was fed on glucose besides, and on one occasion we observed that the respiratory quotient increased above one. Sus- pecting that our dog was forming fat, we decided to follow up this matter. We proceeded to feed the dog large quantities of glucose, expect- ing by thus over-feeding her with carbohydrates to find a state resembling somewhat that observed by Bleibtreu. The dog was given daily at least 120 gm. of glucose with some meat, and on the days on which respiration experiments were performed she got as much as 200 to 225 gm., with a relatively small admixture of chopped meat. It is interesting to note that in spite of such an abundance of nourishment her body weight remained practically unchanged throughout the experimental period of nearly three weeks. Having given the dog glucose in the food for two preceding days, she was brought into the respiration chamber on November 18, two hours after eating 100 gm. of glucose and 300 gm. of meat. The dog remained in the chamber for two hours and during that time she produced per hour 4.12 litres of carbon dioxide and consumed 3.90 litres of oxygen. A’ week later the dog was fed 300 gm. of meat and 125 gm. of glucose and about three hours later a similar amount of meat and 100 gm. of glucose, after which she was placed in the respira- tion apparatus for two hours. This time she produced per hour 4.96 200 Sergius Morgulis and Joseph H. Pratt litres of carbon dioxide and consumed 4.78 litres of oxygen. The same thing was repeated next day. The animal received 50 gm. of meat and roo gm. of glucose in the morning, then three hours later another portion of 50 gm. of meat and 125 gm. of glucose, and went directly into the respiration chamber. The dog produced per hour 5.40 litres of carbon dioxide and consumed 5.19 litres of oxgyen. On November 26, the dog was again given 50 gm. of meat and 125 gm. of glucose, and a respiration experiment lasting nearly two hours was immediately begun. Then she received another too gm. of meat and 100 gm. of glucose and the interrupted experiment was continued for another two hours. During these two successive experiments the animal produced per hour 4.20 and 4.50 litres of carbon dioxide and consumed 3.92 and 4.22 litres of oxygen respec- tively, but the respiratory quotients in both experiments were prac- tically the same. The last experiment of the series was performed a week later during which time the dog was fed on meat and glucose. On the day of the experiment she received 50 gm. of meat and 125 gm. of glucose, but could not be induced to eat any more, and vomited at the mere sight of food. Under these circumstances we were obliged to resort to injecting the glucose subcutaneously, and we thus intro- duced into the body 240 c.c. of a 20 per cent aqtieous solution of glucose, making a total of 48 gm. of glucose. The dog was put directly into the respiration chamber and this time the highest respira- tory quotient of practically 1.1 was obtained. The respiratory quo- tients obtained in this series of experiments range from 1.038 to 1.099 and the data are presented in tabular form in Table IT. Since the respiratory quotient is the ratio between the carbon diox- ide production and the oxygen consumption, the high quotient con- tingent upon the formation of fat from carbohydrate may be either due to an increase of the numerator, or to a decrease of the denomina- tor; the numerical outcome in both events remains the same. The latter course is in agreement with Liebig’s conception of this trans- formation process. Pfliiger’s idea of an intramolecular migration of the oxygen atoms resulting in a formation of carbon chains of which the fat molecule is afterwards reconstructed, is*probably the more correct one. The postulate that the transformation is accompanied by an increased output of carbon dioxide which is to be expected on this assumption, finds corroboration at least in some of our experi- Formation of Fat From Carbohydrates 207 ments. Thus we observed a gradual rise in the carbon dioxide pro- duction in successive periods of thirty minutes each while the oxygen consumption remained practically constant. In one of the experi- ments the dog having been placed in the respiration chamber two Body weight in kilo- grams: TABLE II Carbon | Oxygen dioxide | consump- production tion Respiratory _| quotient per hour Nov. 18 Nov. 25 9.30 A.M. 300 gm. Meat + 100 gm. Glucose 11.30 A.M. 300 gm. Meat + 125 gm. Glucose 3.00 P.M. 300 gm. Meat + 100 gm. Glucose 11.00 a.m. 50 gm. Meat + 100 gm. Glucose 2.00 P.M. 50 gm. Meat + 125 gm. Glucose 9.00 A.M. 50 gm. Meat + 125 gm. Glucose 11.00 a.m. 50 gm. Meat + 100 gm. Glucose 9.45 AM. 50 gm. Meat + 125 gm. Glucose 3.00 P.M. Injected subcutaneously 240 c.c. Glucose Average of a 20 per cent solution of| hours after a meal of meat and glucose the carbon dioxide output was continually increasing from 1.88 to 2.21 litres for thirty minutes. In another experiment tabulated below, the carbon dioxide output was gradually increasing from 2.25 to 2.65 litres in successive thirty- minute periods, but the oxygen consumption, if we take into considera- tion that the amounts for the second and third periods probably 208 Sergius Morgulis and Joseph H. Pratt compensate for each other, remains practically 2.39 in each period. Although in the early part of the fourth period there was some mus- cular activity, the dog has been very quiet in the second and third periods, as can be seen on the kymograph records, while she was less ‘quiet in the first period. This must be taken as good evidence that the increasing output of carbon dioxide has not been caused by an increased muscular activity of the subject. TABLE III Carbon Oxygen | dioxide | i Date BETES Diet | quotient per hour | Nov. 25, 1912 | 3.30 — 4.00 p.m. | 2.251. | 2.391. 941 11.30 a.m., 300 gm. Meat + 4.00 — 4.30 p.m. | 2.411. | 2.19 / 1.100 125 gm. Glucose 2.391. 4.30 — 5.00 p.m. | 2.611. | 2.58 \ 1.012 3.00 p.m., 300 gm. Meat + 100 5.00 — 5.30 p.m. | 2.651. | 2.401. ° 1.104 gm. Glucose Total for 2 hours} 9.921]. | 9.56.1. 1.038 It would be an extremely difficult task to figure out with some degree of accuracy the amount of fat which is formed from carbo- hydrates during the experimental period. Furthermore, as we did not analyze the urine excreted during a sufficient number of the respiration experiments, we lack the most important data for this purpose. Without pretending to estimate with any degree of precision the quantity thus formed, we may attempt to compute that amount approximately by making the justifiable assumption that the animal has been burning carbohydrates for its maintenance during the ex- periment. Following this line of reasoning, we should expect the carbon dioxide output to be equal to the oxygen intake during the same experiment. The excess of carbon dioxide set free in the chemi- cal process of transformation of carbohydrates into fat is not a measure of the metabolic activity of the organism, nor is it connected with the production of heat. It is an extra quantity superimposed upon Formation of Fat From Carbohydrates 209 the carbon dioxide that results from the maintenance combustion of the metabolism. Theoretically, we know that 2.7 gm. of glucose may give 1 gm. of fat with the liberation of .55 gm. of water, and 1.16 gm. of carbon dioxide. Hence it follows that for every gram of fat originating from carbohydrates there should be set free 1.16 gm. or .59 litre of carbon dioxide above the amount resulting from the com- bustion of the body materials. If we avail ourselves of Hanriot’s formula for the transformation of carbohydrates into fat, where a hypothetic substance (stearo-oleo-palmitin) is imagined to be formed by satisfying the three valencies of a glycerid with the fatty acid radicles of the three chief representatives of animal fatty acids (stearic, palmitic, oleic acids), we would expect instead .60 litre of carbon dioxide. On the line of reasoning suggested above this extra amount of carbon dioxide resulting from the formation of fat can be computed by subtracting from the ascertained quantity of carbon dioxide an amount equal to that of consumed oxygen, which on the above sup- position must have been directly produced in the process of combustion of carbohydrates. For the sake of convenience, we may make use of the average figures given in Table II which may serve as representative for the entire set of experiments recorded therein. We will observe that there were 4.78 litres of carbon dioxide produced and 4.50 litres of oxygen consumed per hour, and that the average respiratory quotient was 1.062. If carbohydrates alone had been burned for maintenance during that time 4.50 litres of carbon dioxide would have been derived from that source. The excess of .28 litre (4.78-4.50) of carbon dioxide is what on this view has been set free in the transformatory process which was going on simultaneously with the other. The theoretical expectation of the liberation of about .60 litre of carbon dioxide when one gram of fat is newly formed from glucose, is equivalent to a rise in the respiratory quotient by .131 when the animal’s consumption of oxygen per hour is 4.50 litres, as may be gotten by dividing .60 by 4.50. The average respiratory quotient observed in our experiments is .062 above one, or roughly one-half of the theoretically expected rise of the quotient, when one gram of fat is newly formed. In other words, we may assume that in the course of one hour.5 gm. of fat are being formed from carbo- hydrates on the average in such an extreme case as when carbohydrates alone are being burned for maintenance. As a matter of fact, how- 210 Sergius Morgulis and Joseph H. Pratt ever, the experiments with our dog have shown that on a mixed diet, though the respiratory quotient was very high, it never reached unity, but was usually about 0.940. We may, therefore, assume further that under the experimental conditions and the condition of the dog described here, the animal deposited on an average a gram of fat per hour. In concluding we wish once more to emphasize the fact that a weak and emaciated dog with severe disturbance in the absorption of fat and protein was still able to form fat from carbohydrates. Apart from the additional proof which our investigation brings to the theory of the transformation of carbohydrates into fat, it also shows that the transformation may and actually does take place even in the carnivorous dog. THE ACTION OF THROMBOPLASTIC SUBSTANCE IN THE CLOTTING OF BLOOD By F. W. MacRAE, Jr. anp A. G. SCHNACK [From the Physiological Laboratory of the Johns Hopkins University] N the theory of the coagulation of the blood which we owe to Morawitz it is assumed that the prothrombin is converted to active thrombin by the combined influence of calcium and thrombo- kinase, the latter element being furnished by the tissue cells, including the blood corpuscles. Howell in several papers has contended that thrombokinase, or the thromboplastic substance of the tissues, is not concerned in the activation of the prothrombin, but exerts its favor- ing influence upon coagulation by neutralizing the antithrombin present in blood. This latter author’ has shown, moreover, that the active constituent of thromboplastic substance is one of the lecithans or phosphatids; not the one designated as lecithin, but a related substance which in its solubilities coincides rather with the fraction known as kephalin. A somewhat similar conclusion in regard to the nature of the thromboplastic substance has been reached independently by Zak.” In order to determine whether or not the thromboplastic sub- stance (kephalin) acts as a kinase, it would be desirable to isolate prothrombin and determine directly whether calcium alone suffices to convert it to thrombin, or whether the action of thromboplastic substance is needed in addition. Unfortunately no method of isolating prothrombin has been devised, so that this direct mode of approach- ing the problem is not feasible at present. An indirect method of attack is suggested by the fact, emphasized by Morawitz,’ that the favoring influence of thromboplastic extracts upon the coagulation of blood is not exhibited when the blood is deprived of its calcium 1 HoweELt: This Journal, 1912, xxxi, p. I. 2 Zak: Archiv fiir experimentelle Pathologie und Pharmakologie, 1912, lxx, Dp. 27. 3 Morawitz: Handbuch der Biochemie, 1909, ii, pt. 2, p. 51. 212 F. W. MacRae, Jr. and A. G. Schnack by the addition of oxalate solutions. Thromboplastic substances (tissue extracts) have a very remarkable influence in hastening the coagulation of peptone-bloods. It is upon such bloods in which, owing to an excess of antithrombin, spontaneous clotting is greatly delayed or entirely prevented, that the effect of thromboplastic extracts is shown most clearly, and it is a matter of interest to know whether in peptone-blood the presence of calcium is absolutely neces- sary for this action of thromboplastic substance. At Dr. Howell’s request we undertook to study this point. We began our experi- ments with the idea that a peptonized blood which was oxalated as soon as it was drawn from the animal might behave differently from one which, after removal from the animal, was allowed to stand for a certain time, thirty minutes to an hour, before being oxalated, since in the latter there would be an opportunity for the conversion of some of the prothrombin to thrombin. To test this idea a dog was pep- tonized by the injection of peptone in amounts equal to 0.4 gm. per kilogram of animal. The blood, after twenty minutes, was withdrawn in two lots. The first lot was drawn at once into a solution of sodium oxalate in the proportion of nine parts of blood to one part of oxalate (one per cent sodium oxalate made up in o.g per cent solution of sodium chloride). The second lot was allowed to stand for thirty minutes to one hour and was then mixed with sodium oxalate in the same proportions as in the first lot. Both lots were then centri- fugalized to obtain a clear plasma. It was believed that the second lot, because of standing for a time before its decalcification, would have enough thrombin formed so that kephalin solutions added to it might be able to cause clotting in the absence of calcium. The experiments carried out in accordance with this plan failed to dem- onstrate the point. Neither the plasma of lot one nor that of lot two would clot upon the addition of solutions of kephalin alone. Addition of calcium chloride-alone caused prompt clotting, when the plasma was first diluted with an equal volume of water, and calcium solutions together with kephalin solutions were even more effective. A single example (see opposite page) will suffice to illustrate this point. Similar results were obtained from other experiments in which the calcium was removed by the addition of sodium metaphosphate, sodium citrate, or sodium fluoride. An interesting although inexplicable result which came out of Thrombo plastic Substance in the Clotting of Blood ane these experiments may be referred to briefly although it has no direct bearing upon the main problem which we were investigating. Ina successful peptone plasma which does not clot spontaneously, even upon the addition of an equal volume of water, the addition of solu- tions of calcium chloride alone does not produce clotting while solu- tions of kephalin alone cause clotting in a few minutes. If this same Lot I. Prptonizep Doc BLED AT ONCE INTO OXALATE SOLUTION Peptone Water CaChk (1% Kephalin plasma solution 1. 10 drops 10 drops 6 drops 10 drops Clot in 2.5 min. 2. 10 drops 10 drops 0 10 drops No clot in 24 hr. 3. 10 drops 20 drops 6 drops 0 Clot in 11 min. 4. 10 drops 20 drops 0 0 No clot in 24 hr. Lor II. Prpronizep BLoop KEpt THIRTY MINUTES BEFORE OXALATING Peptone Kephalin plasma Water CaCh (1%) solution 1. 10 drops 10 drops 6 drops 10 drops Clot in 2 min. 2. 10 drops 10 drops 0 10 drops No decisive clot in 24 hr. Slight membrane formation 3. 10 drops 20 drops 6 drops 0 Clot in 8 min. 4. 10 drops 20 drops 0 0 No clot in 24 hr. plasma is decalcified by the use of oxalate solutions addition of cal- cium chloride in quantity sufficient to overcome the excess of oxalate now causes clotting, while solutions of kephalin alone are without effect. It should be added that the promptness with which calcium causes clotting under these circumstances varied with the condition of the plasma, for example, with the amount of contained antithrombin, or with the dilution. If calcium is added to the undiluted plasma clotting may occur very slowly, whereas if the plasma is diluted once or twice with water clotting takes place promptly. As is well known, dilution weakens the effect of antithrombin. Several hypotheses may be suggested to explain this result, but as they are entirely specu- lative it is scarcely worth while to enumerate them. This result, however, together with other known facts regarding the action of solutions of kephalin, suggested that possibly the assumed effect of kephalin upon the antithrombin in the peptone plasmas was inter- fered with by the presence of an excess of the oxalate. The experi- 214 F. W. MacRae, Jr. and A. G. Schnack ments were modified, therefore, by providing for the removal of this excess of oxalate. For this purpose the oxalated peptone plasma was dialyzed in collodion tubes against large volumes of solutions of sodium chloride, 0.9 per cent, the outside solution being renewed once or twice. The results obtained from this series of experiments differed some- what in details, for reasons which were not clear, but which depended probably upon certain variations in conditions that could not be con- trolled, such, for example, as the varying amounts of fibrin factors in the several bloods used, the character of the dialyzing membranes, etc. The important fact is that by this means we have been able to show that solutions of kephalin alone can cause clotting in peptone plasmas free from calcium. The following experimental results may be quoted in proof of this point. Experiment.— Fasting dog. Eight per cent solution of Witte’s peptone injected under pressure into the femoral artery in amount to give 0.4 gm. per kilogram of animal. Blood withdrawn after 20 minutes. One lot oxalated at once (g pts. blood to 1 pt. solution of sodium oxalate made up in solution of sodium chloride 0.9 per cent) and one lot oxalated after standing 30 minutes. Each lot was centrifu- galized to get a clear oxalated peptone plasma. Each lot was then dialyzed over night (15 hrs.) against a solution of sodium chloride, 0.9 per cent. The plasmas, free from oxalate and calcium, were then tested as follows: Lor i Peptone Kephalin plasma Water solution CaCl, (1%) 10 drops 20 drops 0 0 No clot in 24 hr. 10 drops 10 drops 10 drops 0 Clot in 45 to 50 min. 10 drops 0 20 drops 10) Clot in 1 hr. 10 drops 20 drops 0 1 drop Clot in 15 min. — feeble Lor II Peptone Kephalin CaCh (1%) plasma Water solution 10 drops 20 drops 0 0 No clot in 24 hr. 10 drops 10 drops 10 drops 0 Clot in 5 min. 10 drops 0 20 drops 0 Clot in 15 to 20 min. 10 drops 20 drops 0 1 drop Clot in 15 min. Thromboplastic Substance in the Clotting of Blood 215 The kephalin solution used in these experiments was calcium free as was demonstrated by incinerating a large amount of the kephalin used and dissolving the ash in dilute hydrochloric acid. This solu- tion gave no precipitate on the addition of ammonia and ammonium oxalate. It is not possible, therefore, to explain the clotting obtained with the solutions of kephalin on the hypothesis that the kephalin acted as a thrombokinase in conjunction with calcium as demanded by Morawitz’s theory. Nor is it possible to assume that the kephalin acted as a kinase to activate alone the prothrombin present in the plasma. Other experiments, made with oxalated normal (non-pep- tonized) plasma, subsequently dialyzed for twenty-four hours to remove excess of oxalate, showed that the kephalin has no such action. Such plasmas clot readily on the addition of thrombin or of calcium solutions, but are entirely unaffected by solutions of kephalin. The only hypothesis that explains satisfactorily the result obtained is that the kephalin by neutralizing the antithrombin contained in the peptone plasma allowed the thrombin present to react with the fibrinogen. The specimen — lot II1—allowed to stand before oxalat- ing clotted more readily than the other specimen — lot I — because more thrombin had formed. Some subsequent experiments carried out in a manner similar to the one described above gave a different result in that the solutions of kephalin did not cause clotting in the dialyzed plasma. Investigation showed that one difficulty lay in the length of time that the oxalated plasma was submitted to dialysis. As is well known thrombin is easily adsorbed and it is, therefore, probable that in plasma in which little thrombin is present this sub- stance may be removed partially or completely either as a result of dialyzing off or because of adsorption by the substance of the dialyz- ing tube. If such an action takes place kephalin of course can no longer induce coagulation, if its action is limited to neutralizing the antithrombin. In later experiments, therefore, the plasma was examined from time to time during the dialysis to determine whether oxalate was still present and whether kephalin solutions caused coagu- lation. While each specimen of peptoneplasma used gave somewhat different results when treated by this method, it was possible in all cases to demonstrate that for a certain time after dialysis had pro- ceeded the plasma when treated with kephalin alone gave a clot. Before the dialysis the kephalin had been ineffective and usually 216 F. W. MacRae, Jr. and A. G. Schnack when the dialysis had gone over a certain period the kephalin was again without effect, probably, as suggested above, because the ready-formed thrombin in the plasma had been destroyed or removed. Two examples may suffice to indicate the variations exhibited by different plasmas. In each experiment the dog was peptonized as described above and the withdrawn blood after standing was oxalated and centrifugalized to obtain a clear plasma. It was shown first that this oxalated plasma was not clotted by solutions of kephalin and it was then submitted to dialysis and tested from time to time. I Period of Peptone Kephalin dialysis plasma Water solution 15 min. 10 drops 10 drops 10 drops Clot in 26 min. 30 min. 10 drops 10 drops 10 drops Clot in 41 min. 60 min. 10 drops 10 drops 10 drops Clot in 37 min. 120 min. 10 drops 10 drops 10 drops No clot At each interval a control, consisting of 10 drops of plasma and 20 drops of water, was prepared. Each remained unclotted for twenty-four hours. II Period of Peptone Kephalin dialysis plasma Water solution 15 min. 10 drops 10 drops 10 drops No clot 30 min. 10 drops 10 drops 10 drops No clot 45 min. 10 drops 10 drops 10 drops Feeble clot over night 60 min. 10 drops 10 drops 10 drops Feeble clot over night 90 min. 10 drops 10 drops 10 drops Firm clot over night. 120 min. 10 drops 10 drops 10 drops No clot but precipitate Controls with water alone (peptone plasma to drops, water 20 drops) tried throughout the experiment gave a negative result in each case. These dialyzed plasmas after dilution were tested also by the addi- tion of solutions of calcium chloride. As would be expected this reagent caused clotting in most cases within a few minutes owing to its effect in activating the prothrombin present in the plasma. Since in most cases this reaction was obtained even after prolonged dialysis Thromboplastic Substance in the Clotting of Blood 277 lasting over several days it is evident that prothrombin unlike the thrombin is relatively stable. In one case, however, for reasons which were not apparent, the reaction with calcium was slower and slower as the dialysis proceeded and disappeared entirely after two hours. In the above-described experiments it is assumed that the kephalin alone causes clotting of a calcium free plasma because by antago- nizing or neutralizing the antithrombin it permits whatever ready- formed thrombin may be present to exert its action On this view it is evident that if thrombin is added to the peptone plasma, in amounts insufficient to neutralize the antithrombin, the effect of the kephalin in causing clotting should be shown more clearly. Experiments demonstrated that this inference is correct. For example: Experiment.— Dog weighing 73 kg. Injected into the femoral artery 3 gm. of Witte’s peptone. After twenty minutes the blood was drawn from the carotid into an oxalate solution (g parts of blood to r part of oxalate 1 per cent). The oxalated blood was centrifugalized at 3000 for thirty minutes. The clear plasma was drawn off and was dialyzed in a collodion tube against a solution of sodium chloride 0.9 per cent until the oxalate was completely removed. With this plasma the following experiments were performed in duplicate. Peptone Solution of Solution of plasma Water thrombin kephalin Time of clotting 10 drops 15 drops 15 drops 5 drops 43 min. 10 drops 10 drops 15 drops 10 drops @ 53 min. 10 drops 20 drops 15. drops 0 Clot between 8 and 16 hr. 10 drops 25 drops 0 10 drops No clot in 24 hr. 10 drops 35 drops 0 0 No clot in 24 hr. It will be seen from this experiment that while the amount of thrombin added, 15 drops, caused clotting only after eight hours, the same amount of thrombin together with 5 or 1o drops of the kephalin caused prompt clotting in four to five minutes. In this plasma, owing to the promptness with which it was oxalated and centrifugalized, none of the prothrombin apparently was converted to thrombin or if so the latter was removed in the dialysis, since kephalin alone caused no clotting. A word may be added in regard to the action of the oxalate in retarding the normal reaction of kephalin. This retarding influence 218 F. W. MacRae, Jr. and A. G. Schnack is indicated clearly enough by the experiments described above, but other observations have shown that even in the presence of an excess of oxalate the effect of kephalin in neutralizing antithrombin may be demonstrated, provided sufficient active thrombin is present in the mixture. In one series of experiments, for example, a peptone plasma was oxalated and was then divided into two portions. To one of them was added an equal volume of water, to the other an equal volume of a solution of kephalin and to these two portions a thrombin solution was added in increasing amounts to determine for each the minimal amount of thrombin requisite to cause clotting. In all cases the solu- tion containing the kephalin clotted with the fewer drops of thrombin indicating that in spite of the oxalate the kephalin continued to exert some favoring influence. Under the conditions of the experiment the nature of this favoring influence can hardly be interpreted other- wise than on the hypothesis that it exerted a neutralizing effect of some kind upon the antithrombin. SUMMARY Calcium-free (oxalated) peptone plasma may be made to clot by the addition of calcium-free solutions of thromboplastic substance (kephalin), provided the excess of oxalate is removed by dialysis, properly controlled. This action of the kephalin is demonstrated more easily if some thrombin is added previously to the dialyzed oxalated plasma in an amount insufficient in itself to overcome the effect of the antithrombin. This result is opposed to the theory (Morawitz) that the throm- boplastic substance acts as a kinase in conjunction with calcium, but is in accord with the view (Howell) that thromboplastic substance (kephalin) facilitates clotting by neutralizing the action of anti- thrombin. ERRATA in June number of the American Journal of Physiology (Vol. XXXII., No. IL). Substitute ‘apparatus”’ for “ apparati”’ in the following places: page 110, lines 7, 11, 23. page 120, line r. page 137, line 28. page 138, lines 1, 3. page 144, line 6. Substitute “7.7 c.c.” for “‘ 1.1 c.c.”? on page 144, line 20. In figure 1, page 120, correct as indicated the following drawing: THE American Journal of Physiology VOL. XXXII AUGUST 1,°1913 NO. IV STUDIES IN FATIGUE I. FATIGUE AS AFFECTED BY CHANGES OF ARTERIAL PRESSURE By CHARLES M. GRUBER [From the Laboratory of Physiology in the Harvard Medical School.] HAT increased muscular effort causes an increased arterial pressure has long been known. The reverse of this relation —the effect of arterial pressure on muscular efficiency — seems not to have attracted much attention. After carefully searching through the literature I have been unable to find any references to observa- tions on the effects of changes of arterial pressure, either increase or decrease, on neuro-muscular fatigue. In a recent paper! Cannon and Nice have called attention to the need of more exact study of the relation between the circulation and muscular ability, and the work here reported was undertaken with the purpose of making clearer these relations. THE MeEtTHOD Cats, anaesthetized with urethane (2 gm. per kilo, by stomach) were used in the experiments. By making a small slit through the skin on the outer side of the left thigh, the sciatic nerve was isolated, cut, and its distal end fastened in a Sherrington shielded electrode. The electrode was then held in place by fastening around it, with paper clips, the two flaps of skin. 1 CANNON and NIcE: this Journal, 1913, xxxii, p. 80. 222 Charles M. Gruber Through another small slit in the skin the tendon of the left tibialis anticus muscle was isolated from its insertion. The tendon was then fastened to a muscle lever by a string passing about a series of pulleys. These pulleys were arranged so that the muscle pulled in its normal direction. One leather loop about the hock and another around the foot just below the fastening of the tendon bound the leg to the board and made a very satisfactory nerve muscle preparation. This preparation had its normal blood supply, unaltered except by the cutting of the sciatic nerve. The stimulating current in every case was a break induction shock, obtained from a Martin vulcanite knife-blade key * operated by an electro-magnet as follows. A soft iron bar was pivoted near its centre to an upright board. Below one end of the bar was an electro- magnet, working in opposition to a flat steel spring attached to the other end of the bar. An upright iron rod, fastened near the centre of the bar, was connected at the other end (by a wire link) to the inverted brass triangle of the Martin key. Thus as the upright rod was moved to and fro it moved the knife blade making and breaking the circuit through the mercury in the key. A motor running at a uniform rate was used to revolve a metallic cylinder provided with projecting points, which made and broke the current in the electro- magnet circuit, usually 160 times per minute. In a few experiments lower rates were used. This rate was slow enough to produce not vasoconstriction but vasodilation * in the vessels of the stimulated muscle. The secondary of the inductorium was connected with the shielded electrode on the sciatic nerve. The muscle lever consisted of a piece of light straw 20 cm. in length from the axis to the writing point. The tendon was attached 4.5 cm. from the axis and at the moment of contraction began to pull against the tension developed in a spring which was attached at the same position on the lever. This spring, in the majority of cases, had a tension of 120 gm. the moment the muscle began to contract, but in a few cases it had an initial tension of as much as 250 gm. For each 2.5 cm. excursion of the muscle lever on the drum the spring increased 15 gm. above the original 120. * MARTIN: this Journal, 1910, xxvi, p. 181. 3 BowpitcH and WARREN: Journal of physiology, 1886, vii, p. 416; BRAD- FORD: Jbid., 1889, x, p. 390. } } } | } | | { Studies in Fatigue i) i) WwW The blood pressure was registered from the right carotid or femoral artery by means of a mercury manometer. A time marker which indicated intervals of thirty seconds was placed at the atmospheric pressure line of the manometer. Thus, at any given muscular contrac- tion, the height of blood pressure was simultaneously recorded. The blood-pressure style, muscle lever and time marker were all placed in a vertical line on the kymograph surface. The rate of the drum was always slow and the muscle contractions were recorded close together. Several methods were used to vary the blood pressure. Those employed to raise it were: (1) stimulation of the spinal cord in the cervical region with platinum elect- rodes, and (2) stimulation of the left splanchnic nerves with the adrenal glands tied off. The electrode used on the splanchnic nerves was similar to that used by Cannon and Nice.t. The methods employed to lower the pressure were: (1) simple compression of the FicurE 1. Inthis and all following records, the upper curve indicates the blood pressure the middle line muscular contraction, and the lower line the time in 30 seconds (also zero blood pressure). Between the arrows the exposed cervical spinal cord was stimulated. thorax; (2) pulling on a loop placed around the aorta just above its iliac branches, and (3) injection of very small doses of adrenalin 7) through a cannula in the left external jugular vein. 4 See CANNON and Nice: Loc. cit., p. 71. 5 See CANNON and Lyman: this Journal, 1913, xxxi, p. 376. Charles M. Gruber ho to — THE EFFECTS OF INCREASED ARTERIAL PRESSURE In taking up the results of variations of arterial pressure on fatigue, it is convenient to consider first, the effect of rise of pressure. This rise was brought about in the experiment represented in Fig. 1, by stimulation of the cervical spinal cord, and in Figs. 2 and 3 by stimu- A B c FicurE 2. Stimulation of the left splanchnic nerves (left adrenal gland I 8 tied off) during the period indicated by the arrows. lation of the left splanchnic nerves after the left adrenal gland was tied off. The original blood pressure in Fig. 1 was 120 mm. of mercury. This was increased 62 mm. with an increase of only 8.4 per cent in the height of muscle contraction. In Fig. 2 the original pressure was 100 mm. of mercury. By increasing this pressure 32 mm. there resulted a synchronous betterment of 9.8 per cent in the height of muscular contraction. In Fig. 2B the arterial pressure was raised 26 mm. and the height of contraction increased correspondingly 7 per cent. In Fig. 2C no appreciable betterment can be seen although the blood Studies in Fatigue 225 pressure rose 18 mm. In Fig. 3 the original blood pressure was very low — 68 mm. of mercury. This was increased in Fig. 3A 18 mm. with an increase in the height of contraction of 20 per cent; in Fig. 3B 24 mm. with a corresponding increase of 90 per cent and in Fig. 3C 30 mm. with a betterment of 125 per cent. That this increase in the height of contraction is due to the increase in blood pressure seems almost beyond dispute. It is evident from A B Cc FicureE 3. During the period indicated in the time line the left splanchnic nerves were stimulated. The vessels of the left adrenal gland were tied off. these observations that when the blood pressure is low a small rise has many times the effect that it has when the pressure is high. There is abundant evidence that fatigue products accumulate in a muscle doing work.® As the pressure rises, thus bettering the circulation through the active muscle, these products are carried away more rapidly. Moreover, since the stimulation of the sciatic nerve used § DuBots, ReyMonp: Archiv fiir Anatomie, 1859, p. 849; RANKE: Archiv fiir Anatomie, 1863, pp. 422-450; Morescuorr and BArristini: Archives ital- iennes de biologie, 1887, viii, pp. 90-124; Geiss: Archiv fiir die gesammte Phy- siologie, 1887, xl, pp. 69-75; LANDSBERGER: Archiv fiir die gesammte Physiologie, 1891, 1, pp. 339-363; FLETCHER and Hopkins: Journal of physiology, 1906-07, XXXV, p. 247; LEE: this Journal, 1907, xvill, p. 267. 226 Charles M. Gruber in these experiments was too slow to cause vasoconstriction, but instead caused vasodilation the opportunity for the blood to pass readily in large volume through the vessels and thus to carry away a large per cent of the accumulated waste products of fatigue, is obvious. Ranke‘ found that if a muscle is deprived of its circulation and fatigued to a standstill, and then the circulation restored, it again contracts for a short time due to the neutralization of the waste- products by the blood. THE EFFECT OF MECHANICAL DECREASE OF ARTERIAL PRESSURE If an increase in blood pressure produces an increase in the height of muscle contraction it is natural to suppose that a decrease in A B Cc D FicurE 4. The arrows indicate the point at which the thorax began to be compressed. blood pressure would have the opposite effect. Such is the case only when the blood pressure falls below the region of go to 100 mm. of mercury. Thus, if the arterial pressure is 150 mm. of mercury it 7 RANKE: Archiv fiir Anatomie, 1863, p. 446. Studies in Fatigue 227 has to fall approximately 55 to 65 mm. before it produces a decreased effect in the height of contraction. Fig. 4 is a record in which the blood pressure was decreased by compressing the thorax. The record shows that, when the pressure dropped from 120 to 100 mm. of mercury there was no appreciable decrease in the height of contraction; when to 90 mm. of mercury there resulted a decrease of 2.4 per cent; when to 80 mm. of mercury a 7 per cent decrease and when j£0° yO mm. a 17:3 per cent decrease. Thus about go to roo mm. of mercury may be called the critical region at which the decrease in blood pressure is accom- panied by a concurrent decrease in the height of muscular contraction. It is near that point that the blood flow is in danger of being insufficient. Results similar to those represented in Fig. 4 were obtained by pulling ON @ FycuRE 5. During the period in- string looped about the aorta just above __ dicated in the time line 0.3 c.c. of a 1:100,000 solution of adre- the iliac branches. nalin was injected into the left external jugular vein. THE EFFECT OF DECREASING THE ARTERIAL PRESSURE BY ADRENALIN In the third series of experiments adrenalin was used to lower the blood pressure. Cannon and Lyman found that adrenalin injected in small doses —o.1 to 0.2 c.c. of 1: 100,000 solution — produces a fall in blood pressure until a critical region was reached. Below this region the same amount injected produces a rise.* In Fig. 5, 0.3 c.c. of 1: 100,000 solution of adrenalin was injected slowly into the right external jugular vein. The blood pressure dropped from 120 mm. to 96 mm. of mercury. With this decrease in arterial pressure there was a resultant betterment of 14.3 per cent SSEOG. Git. D300: 228 Charles M. Gruber in the height of muscular contraction. Since this fall of pressure is within the critical zone a uniform contraction or a decrease would be expected. A similar drop in arterial pressure is shown in Fig. 4B. This was brought about mechani- cally and there resulted a decrease of 2.4 per cent in the height of contraction. When at 100 mm. no change resulted. This betterment may be accounted for by the effect of adrenalin.® A quite different effect is shown in Fig. 6A. The same amount of adrenalin — 0.3 €:€: 1: ‘100,000 solution — was injected as in the case represented in Fig. 5. In this case, however, the blood pressure fell from a low region to a region below the critical region — 108 to rt yp ae go mm. of mercury. Instead of a betterment in the height of contrac- A B Ficure 6. In A, atthe pointindicated tion, as in the preceding experi- by an arrow, 0.3 c.c. of a 1:100,000 solu- ment, there was a decrease of 18.7 tion of adrenalin was injected intrave- nously. In B, the arrows indicate the Per cent. The same result followed period during which the thorax was when the pressure was lowered by compressed. compressing the thorax. In Fig. 6B an almost equal fall of blood pressure was thus produced and a fall of 17.7 per cent in the height of contraction resulted. Fig. 7 confirms Fig. 6 very well. In Fig. 7A, as in Fig. 6A, 0.3 c.c. of 1: 100,000 solution of adrenalin was injected. There was a fall in blood pressure from 102 to 80 mm. of mercury and a corresponding fall of 17.7 per cent in the height of contraction. In Fig. 7B the same quantity of adrenalin was injected but here the blood pressure was maintained above the critical point by stimulating the left splanchnic nerves (with the left adrenalin vessels tied) at the points indicated by arrows. No fall in the height of muscular contraction resulted. This seems to indicate that, in these cases, the muscular contraction is ® See CANNON and NIcE: Loc. cit., p. 74. Studies in Fatigue 229 Ficure 7. In A, the arrow indicates the point at which 0.3 c.c. of a 1:100,000 solution of adrenalin was injected. In B, the lower arrow indicates the point at which the same quantity of adrenalin was injected. The upper three arrows indicate the points at which the left splanchnic nerves were stimulated. The left adrenal gland was tied off, dependent upon the blood flow rather than upon the action of adrenalin. SUMMARY 1. Increasing the arterial pressure, thus bettering the circulation, increases the height of muscular contraction too to 125 per cent when the blood pressure is below go to 100 mm. of mercury but only 5 to 25 per cent when the pressure is above this region. 2. As the blood pressure or the circulation is decreased, the height of muscular contraction is lowered, but this takes place only when the arterial pressure falls below go to 100 mm. of mercury. 3. Small doses of adrenalin — 0.1 to 0.3 c.c. of a 1: 100,000 solu- tion — slowly injected intravenously, cause a fall of arterial pressure. When this fall is not below the critical region — about 90 to 100 mm. of mercury —a betterment in the height of contraction results; when below this zone the result is the opposite. I wish to express my thanks to Dr. W. B. Cannon for valuable suggestions offered me during these experiments. CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE, No. 238 ON CERTAIN DISTINCTIONS BETWEEN TASTE AND SMELL By GEORGE HOWARD PARKER anp ELEANOR MERRITT STABLER INTRODUCTION ERHAPS the most widely accepted distinction between taste and smell is that taste is excited by materials in the state of solution and smell is called forth by substances in a vaporous or gaseous condition. This distinction is based upon an experiment on sensory stimulation published by E. H. Weber in 1847 in which he showed that a dilute solution of eau de cologne, though highly odorous when sniffed, produced no sensation of smell when poured into the nose. Nagel, many years later (1894), was so fully persuaded of the correctness of this view that he went so far as to deny a sense of smell to water-inhabiting vertebrates, such as the fishes, etc., maintaining that solutions were inodorous and that the so-called olfactory organs of fishes and other water-inhabiting animals were more in the nature of organs of taste than organs of smell. Meanwhile, Aronsohn (1884), who had repeated Weber’s experi- ment, declared that substances in solution when poured into the nose, could be smelled, provided they were dissolved in a physiological salt solution that was kept warmed to about the temperature of the human body. This conclusion was confirmed by Vaschide (1901), and by Veress (1903), though the latter showed that the solutions tested were rather in the nature of heterologous than homologous stimuli. Thus, notwithstanding the opinion of Zwaardemaker (1895), Haycraft (1900), and others, it seems to us that solutions must be admitted to Certain Distinctions Between Taste and Smell 231 be stimuli for the olfactory surfaces, a conclusion in agreement with the belief expressed many years ago by Miiller (1837, p. 484) and reiterated recently by von Frey (1904, p. 334) and even by Nagel (1904, p. 600), that in normal olfaction in man the odorous particles are caught on the moist olfactory surfaces and dissolved there before they can act on the nerve terminals. The belief that solutions are stimuli for the olfactory surface is also favorable to the view that fishes and water-inhabiting amphibians may have as true an olfactory organ as air-inhabiting vertebrates do, a conclusion suggested by the observations of Aronsohn (1884, p. 164) on goldfish, and of Baglioni (1909a, p. 719) on Balistes, and proven by the experiments of Parker (1910, 1911) on Ameiurus and Fundulus, of Sheldon (1911) on Mustelus, and of Copeland (1912) on Spheroides. It must, there- fore, be admitted that in both air-inhabiting and water-inhabiting vertebrates the stimulus for the olfactory organs is in reality a solution and, since this is also true for the sense of taste, the problem naturally presents itself of the real difference between smell and taste. In attacking this question it seemed well to select as a stimulus a substance that was relatively simple chemically, that was easily obtainable in pure form, and that was at once a stimulus for both smell and taste. Such a substance was found in ethyl alcohol. This well-known reagent has a characteristic odor and a well-marked sweetish taste. Our preliminary tests were carried out with a high- grade laboratory product, but our final tests were made with Kahl- baum’s alcohol of the highest obtainable purity and guaranteed to contain not over o.2 per cent water. The alcohol was received in sealed tin containers and was kept by us in glass-stoppered bottles. In carrying out our tests we endeavored to determine the lowest dilutions at which the sweet taste and the characteristic smell could be excited as well as that which would call forth the first signs of sting from those surfaces of the mouth that are unprovided with gustatory organs. In order that the various concentrations of alcohol used as stimuli for the several sense organs might be compared, we have made all dilutions on the basis of the relative number of molecules and expressed them in terms of molecular solutions or corresponding gas dilutions and not in per cents as has been done heretofore by many investigators. 232 George Howard Parker and Eleanor Merritt Stabler TASTE FROM ETHYL ALCOHOL So far as the taste of ethyl alcohol was concerned, it was our object to ascertain the weakest dilution at which the sweet taste could be distinguished. We therefore prepared a 1o mol. solution of the purest alcohol and this we used as a stock from which weaker solutions were made as they were needed. In each test the subject was required to close the eyes, close the nose by pinching it between thumb and finger, and receive on the extended tongue near the tip two drops of the fluid to be tested. The glass dropping apparatus that we used delivered 297 drops of dilute alcohol per 5 c.c. The two drops generally em- ployed in these experiments represent, therefore, about 0.017 C.c. The fluids used in these tests were dilute alcohol and, as a check, dis- tilled water. After the fluid had been on the tongue a short time and before the nose was released, the subject was required to state whether the fluid was distilled water or dilute alcohol. Preliminary tests showed that a fold of filter paper wet with dilute alcohol and held in the cavity of the mouth, but not in contact with its walls, could not be distinguished from a similar fold wet with distilled water. Hence there was no reason to suppose that the determinations were influ- enced by any diffusion of alcoholic vapor from behind into the nasal chambers. It is believed that the determinations depended abso- lutely on the action of the materials on the surface of the tongue. The subjects of the tests were the authors of the paper, one experi- menting on the other. After each test the subject washed out the mouth with tepid tap water and rested for a period of about five minutes before another test was made. To a solution of alcohol of 5 mol. strength both subjects responded with perfect accuracy and, on repeated trials, they always distin- guished the dilute alcohol from the distilled water. At a dilution of 3 mol. the sweet taste of the alcohol was very faint. In ten tests nine were correct and one a failure. On applying the two drops of dilute alcohol to the tongue, both subjects regularly experienced a slight indescribable sensation before the characteristic sweet taste of the alcohol appeared. ‘This preliminary sensation was quickly and completely obliterated by the sweet taste that followed. At a dilu- tion of 2 mol. the subjects failed five times in ten trials and we there- Certain Distinctions Between Taste and Smell 233 fore conclude that the weakest solution that can call forth the sweet sensation with certainty is of about 3 mol. strength. A comparison of the sweet taste of cane sugar with that of ethyl alcohol showed the former to be much the more effective stimulus. While it was possible to distinguish with certainty a solution of ethyl alcohol of not less than 3 mol. concentration, a solution of cane sugar of only 3'5 mol. concentration was easily recognized and, judging from the results of Lemberger (1908, p. 303), still more dilute solu- tions can be easily distinguished. Ethyl alcohol is to be regarded, therefore, as a not very efficient stimulus for the sweet taste. IRRITATION FROM EtHyL ALCOHOL Distilled water and solutions of alcohol were applied to certain non-gustatory surfaces of the mouth in the same manner as to the tongue, with the intention of determining the weakest dilution that would call forth the slight stinging and warming effect of alcohol on such sufaces. Three regions were selected for these tests: first, the region on the floor of the mouth between the lower incisors and the root of the tongue; secondly, the space between the lower lip and the lower incisors; and thirdly, the inner face of the cheek. In the region between the lower incisors and the root of the tongue, the results were most uniform. At a concentration of 10 mol. ten correct determinations were made in ten trials. At 5 mol., however, there were six failures in ten trials. In the region between the lower lip and the incisors the results were less uniform than on the floor of the mouth proper. One sub- ject (E. M. S.) distinguished with certainty the 5 mol. solution of alcohol but failed generally on the 3 mol. solution; and the other subject (G. H. P.) distinguished the 10 mol. solution but failed on the 5 mol. solution. : The records of the tests on the inner surfaces of the cheeks show a somewhat similar difference. A 10 mol. solution of alcohol was always distinguished with certainty from distilled water by one subject (E. M. S.) and poorly distinguished by the other (G. H. P.). At a dilution of 5 mol. both subjects failed to distinguish between the water and the distilled alcohol. In this, as in the preceding trials, one subject (E. M. S.) proved to be somewhat more sensitive than the 234 George Howard Parker and Eleanor Merritt Stabler other (G. H. P.), a condition probably dependent upon the greater youthfulness of the former. The sensations produced in these tests were described in a variety of ways, such as faintly warming, warmish sting, slightly stinging, prickling, etc., but they all partook in general of the nature of slight irritations. They probably result from the stimulation of sense organs which have recently-been designated as those of the common chemical sense (Parker, 1912), and which probably pervade many of the peripheral mucous surfaces of the body. THE SMELL FROM ETHYL ALCOHOL In determining the weakest dilution at which the characteristic smell of alcohol could be detected, a method of procedure different from that used for taste had to be employed. ‘This was in essentials the method that had been used by Valentin (1850), Fischer und Penzoldt (1886), and others. Our own procedure was as follows: Two glass battery jars of equal capacity, 1340 c.c., were cleaned till they gave the least possible odor. It was found impossible to free such jars entirely from smell. A careful examination always dis- closed a faint clay-like odor, which, from its constancy, we were led to believe was due to the glass itself. When the two jars were indis- tinguishable in this respect, a drop of distilled water was put in one and a drop of dilute alcohol in the other. Both jars were covered and the fluids allowed to evaporate completely, a process which was facilitated by the introduction of a small electric fan, through the cover of the jar. After the complete evaporation of the fluids, the jars were tested in sequence by the subject, who, with eyes closed, was allowed a full breath through the nose from first one and then the other jar. The subject was then required to state in which jar the alcohol had been evaporated. The jars were then carefully washed and, after having been dried, were tested for their own smell and prepared for another trial. The results obtained from these tests were remarkably uniform and constant for the two subjects. Both subjects distinguished with invariable correctness the jar in which a drop of a to mol. solution of alcohol had been evaporated from the one in which an equal amount of distilled water had been vaporized. When a drop of dilute alcohol Certain Distinctions Between Taste and Smell 235 of 5 mol. concentration was evaporated in the jar, six failures in ten were made. ‘Taking into account the dilution due to the evaporation into the known volume of air in the jar, the results may be stated as follows: both subjects detected the alcohol in an aerial dilution of y960 mol., but failed to detect it at zs,459 mol. In other words the most considerable dilution at which the alcohol could be detected by the olfactory apparatus was about s,)) mol. This determination was obtained by the use of Kahlbaum’s purest grade of alcohol. The preliminary trials made with the high- grade laboratory alcohol gave a very different result. The jar in which the laboratory alcohol had been evaporated could be distin- guished when the contents were at a dilution of s9o'p99 or even 400-000 mol. The odor noted at these dilutions, however, had a sharp and penetrating quality quite unlike that of alcohol and was without question due to some impurity. Jn the test with the Kahl- baum alcohol, the odor remained constantly alcoholic to the weakest dilution that could be smelled. We therefore believe that the limit of dilution, 3999 mol., found by us for the odor of ethyl alcohol is a reliable determination uninfluenced by impurities. The only previous record of such a determination that we have been able to find is that given by Passy (1892, p. 1140), who states that 0.250 mg. of ethyl alcohol in a litre of air is the least concentration at which alcohol can be detected by its odor. This is equivalent to a dilution of 434'p99 mol., which is so near that of the least per- ceptible dilution of our laboratory stock that we suspect that this determination, like that for our laboratory stock, was influenced by an impurity and does not represent the real limit for pure alcohol. DISCUSSION It appears to us that the evidence is sufficient to justify the con- clusion that in vertebrates the stimulus for smell is a substance dis- solved in the fluids that bathe the olfactory surface and that in this respect smell and taste are similar. The difficulty in imitating a normal stimulation of the olfactory organs by solutions experimentally 1 Passy (1892,? p. 307) elsewhere states that x}o gm. of ethyl alcohol in a litre of air is only slightly perceptible. This concentration is twenty times that referred to by Passy as the lowest concentration that can be smelled. 230 George Howard Parker and Eleanor Merritt Stabler introduced into the nose of air-inhabiting animals is due in our opinion to the inability of the investigator to reproduce the olfactory solvent. This material, the slimy covering of the olfactory surface, is very different from water or even warmed physiological salt solution. Hence, it is not surprising that odorous substances dissolved in these media should not act as normal stimuli for the olfactory surfaces of air-inhabiting vertebrates. Were it possible to imitate closely the olfactory solvent, we believe that there would be no difficulty in stimulating the olfactory organs with solutions made up in this solvent. The fact, known even to Weber (1847, p. 351), that water introduced into the human nose will cause a temporary loss of the power of smell, is enough to show the extreme sensitiveness of the olfactory organ and to suggest that any fluid, except that which is normal to it, may be physiologically disturbing to its surface. Hence, we put at naught those experiments that have thus far yielded negative results on introducing odorous solutions into the nose and believe that these results are dependent upon the disturbing effect of the solvent rather than on the inability of the olfactory organs to be stimulated by dissolved materials. In fishes the olfactory surfaces are apparently undisturbed by the water that bathes them, but in air-inhabiting vertebrates these surfaces seem to have become adapted to a well- developed slimy covering and thus to have lost their ability to respond normally to simple aqueous solutions. It is this loss of responsive- ness that has led, in our opinion, to the degeneration of the olfactory apparatus in such mammals as the whales, whose aquatic habits, in comparison with those of fishes, have been secondarily acquired. We therefore definitely abandon the idea that taste and smell differ on the basis of the physical condition of the stimulus, a state of solution for taste, a gaseous or vaporous condition for smell, and maintain that both senses are stimulated by solutions, though in smell, at least for air-inhabiting vertebrates, the solvent is of a very special kind. If the senses of taste and smell are stimulated by solutions, the probability that they are both chemical senses, as maintained long ago by Nagel (1894), is thereby greatly increased, and from this standpoint a partial distinction between them may be drawn on the basis of the solutes. Most substances that we smell have no taste, and most substances that we taste are without smell. Thus the olfactory sense is attuned to one set of substances and the gustatory Certain Distinctions Between Taste and Smell 237 to another. But this distinction has numerous exceptions, for not a few organic substances, like alcohol for example, have both taste and smell, and, judging from the work of Veress (1903), there are inorganic salts with smells as well as tastes. Nevertheless the dis- tinction pointed out seems to us to have some value. But the difference between taste and smell that we believe to be of a still more general character is a quantitative one; we smell very dilute solutions, we taste only relatively strong ones. This difference appears most clearly when we deal with the dilution of the stimulus as expressed in terms of molecular solutions rather than in per cent, for this method allows us to compare mixtures of gaseous materials with solutions. If, from this standpoint, we compare the stimuli for some of the most penetrating odors with those of the strongest tastes, the contrast becomes very striking.. One of the strongest tastes is the bitter taste of quinine hydrochloride and this can be excited by a solution as dilute as =s,)99 mol. One of the strongest smells known is that of mercaptan of which, according to Fischer und Penzoldt (1886, p. 8), o.or mg. evaporated in 230 c.c. of air gives a perceptible odor. Assuming the substance used to have been methyl mercaptan and stating the matter in terms of a molecular solution, thissubstance can be smelled at a dilution of ;.494,000,000,000 mol. Thus it appears that when we compare the most powerful tastes with the most powerful smells, we find that the olfactory organ of man is responsive to a dilution over 44,000,000 times greater than that to which the sense of taste responds. But this comparison is obviously inexact since the stimuli for the two senses are different substances. Hence, the importance of making this comparison with a substance that is a stimulus for both smell and taste. Ethyl alcohol, as recorded in this paper, can barely be recognized by taste at a con- centration of 3 mol., but it is discernible by smell at a dilution of about go000 mol. This comparison, then, like that between mercaptan and quinine, shows that the olfactory organs of man are much more sensitive to ethyl alcohol than are his gustatory organs, the ratio of the stimuli being about 1 to 24,000. Although we believe that the distinction between taste and smell as presented in the preceding paragraph is a valid one, we are per- fectly aware that the measurements upon which this opinion is based are not measurements of the real stimuli. They show the relative 238 George Howard Parker and Eleanor Merritt Stabler concentration of molecules in the materials supplied to the tongue and to the moist olfactory surfaces; they do not show the molecular con- centrations in contact with the actual end-organs. It is probable, however, that in both smell and taste the concentration of stimula- ting material at the end-organ is not far from that in the adjacent source, the air in the olfactory organ or the solution on the surface of the tongue. We therefore believe that we are correct in concluding that we smell enormously attenuated solutions and taste only relatively strong ones. In this respect the two senses may be said to differ from each other more or less as ordinary scales do from a chemical balance; taste is used in determining the presence of relatively large amounts of substance, smell for only the most minute quantities. Hence, taste is inoperative except when the source is very near at hand, usually in the mouth, whereas smell may be active when the source is far distant, the dilution suffered by the stimulating sub- stance in its spread not having been sufficient to have brought it below the concentration necessary for stimulation. SUMMARY 1. The weakest aqueous solution of ethyl alcohol that could be tasted was of about 3 mol. concentration. 2. The weakest aqueous solutions of ethyl alcohol that just stim- ulated the non-gustatory surfaces of the mouth were as follows: for the region between the lower incisors and the root of the tongue, 1o mol.; for the region between the lower lip and the incisors, from 5 to 1o mol.; and for the inner surface of the cheek, 10 mol. 3. The weakest aerial dilution of ethyl alcohol that could be smelled was about ga/o> mol. 4. Ordinary grades of ethyl alcohol may excite smell at dilutions as low as 00 00 mol., but this is probably due to impurities. 5. Both smell and taste are stimulated by solutions. In air- inhabiting vertebrates the olfactory solvent is a slimy fluid of organic origin and not easily imitated. Hence the olfactory organs of these animals are not appropriately stimulated by ordinary aqueous solutions. 6. Beside the different chemical nature of the stimuli, that for the sense of taste is a relatively strong solution, that for smell a rela- Certain Distinctions Between Taste and Smell 230 tively weak solution. The dilutions of ethyl alcohol as minimum stimuli for smell and taste are as 1 to 24,000. BIBLIOGRAPHY ARONSOHN, E.: 1884. Beitrige zur Physiologie des Geruchs. Arch. fiir Anat. u. Physiol., physiol. Abt., Jahrg., 1884, pp. 163-167. BAGLIONI, S.: t909a. Zur Physiologie des Geruchsinnes und des Tastsinnes der Seetiere. Zentralbl. fiir Physiol., Bd. 22, pp. 719-723. BAGLIONI, S.: tgogb. Contributions expérimentales a la physiologie du sens olfactif et du sens tactile des animaux marins (‘Octopus” et quelques poisson). Arch. Ital. Biol., tome 52, pp. 225-230. CopELAND, M.: 1912. The Olfactory Reactions of the Puffer or Swell- fish, Spheroides maculatus (Bloch and Schneider). Journ. Exper. Zool., xii, pp. 363-368. FIscHER, E. unD F. PENzotpT: 1886. Ueber die Empfindlichkeit des Geruchsinnes. Sitzungsber. phys.-med. Soc., Erlangen, Heft 18, Pp. 7—1O: Frey, M. von: 1904. Vorlesungen iiber Physiologie. Berlin, 8vo, x + 392 pages. Haycrart, J. B.: 1900. The Sense of Smell. In E. A. Schafer, Text- book of Physiology, ii, pp. 1247-1258. LARGUIER DES BANCELS, L.: 1912. Le Gout et l’Odorat. Paris, 8vo, x + 94 pages. LEMBERGER, F.: 1908. Psychophysische Untersuchungen tiber den Geschmack von Zucker und Saccharin (Saccharose und Krystallose). Arch. fiir ges. Physiol., Bd. 123, pp. 293-311. Miter, J.: 1837. Handbuch der Physiologie der Menschen. Band 2. Coblenz, 8vo, 780 pages. 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THE American Journal of Physiology VOL: XXXII SEPTEMBER 2, 1913 NOW: IS THE PRESSOR EFFECT OF PITUITRIN DUE TO ADRENAL STIMULATION By R. G. HOSKINS anp CLAYTON McPEEK [From the Laboratory of Physiology of the Starling-Ohio Medical College] UR first experiments were made upon dogs under ether-urethane anaesthesia. In most cases the experiments were begun under ether, then urethane (two grams per kilo) was immediately introduced into the stomach and the ether gradually discontinued as the urethane became effective. In some instances, however, a slight amount of ether had to be continued throughout the experiment in order to maintain satisfactory anaesthesia. Cannulas were introduced into the carotid artery for the registration of blood pressure and into the femoral vein for the administration of the drug under investigation. The belly was opened in the median line and, while the viscera were protected by warm towels, needles were thrust through the body wall at each side of the hilus of each adrenal gland. By means of these needles double ligatures were drawn through so that they could be pulled tight from outside the body and occlude adrenal circulation. The ligatures were carefully placed to avoid including any considerable number of splanchnic nerve fibres, the blocking of which would intro- duce an extraneous factor. The ligatures were used double to permit removal with a minimal laceration of tissues; they were looped together and so adjusted that the junction came at the hilus of the gland. Then when either member of either ioop was cut, a slight traction on its fellow-member released both loops. The ligatures 242 R. G. Hoskins and Clayton McPeek having been adjusted, the abdominal cavity was closed and the experi- ment begun. This technique has previously been used by several investigators.! Blood pressure was recorded by means of an ordinary mercury manometer and float. Having established the normal pressure for a given animal, a standard dose of pituitrin (Parke, Davis and Co.) was introduced into the femoral vein. In order to avoid a diminu- tion of effect when the dose was repeated, small quantities only were used, — from 0.6 to 1oc.c. of a r:1o solution. It was found, as a matter of fact, that such dosage could be repeated at intervals of 1o to 15 minutes without significant decrease in pressor effects. Having . recorded the results following a given standard dose, the adrenals were ligated by traction upon the previously arranged ligatures. After a brief fluctation blood pressure immediately returned to its previous level. Then the same dose was repeated and the effects again observed. When this effect had worn off, the ligatures were released. Again there was a brief fluctation of pressure, which, how- ever, soon returned to normal, permitting another repetition of the standard dose. Allowing for a slight progressive diminution in the sensitiveness to the drug, there was no appreciable difference between the effects when the adrenals were intact and when their circulation was occluded. Our results confirm the observations of Young and Lehmann and of Hoskins and McClure? that adrenal ligation in the dog has no immediate influence upon blood pressure. An obvious source of error in such results is a possible exhaustion of the adrenal glands as a result of the anaesthetic and of the sensory stimulation necessarily involved in the preparation of the animal. In carrying out another research we obtained evidence which indicates that in the dog such exhaustion actually does occur.’ Cannon and Nice? have shown, however, that there is still dischargable epinephrin in the adrenals of cats even after evisceration. We decided, therefore, to repeat the observations on this animal. Three such experiments 1 YounGc and LEHMANN: The Journal of physiology, 1908, xxxvii, p. liv. Hoskins and McCivre: Archives of internal medicine, 1912, x, p. 343. 2506. Cth. 3 Hoskins and McPreEek: The Journal of the American Medical Asso- lation, Word, lv, p: 1777. 4 CANNON and RicE: This Journal, 1913, xxxii, p. 49. Pressor Effect of Pituitrin due to Adrenal Stimulation 243 were made, using correspondingly smaller doses,— about 0.5 c.c. Ether alone was used for anaesthesia. The results in each case, how- ever, confirmed our previous findings. One experiment was par- ticularly convincing. The cat was in a late stage of pregnancy and the adrenals therefore supposedly hypertrophied. That they were actually secreting during the experiment was shown by the fact that the blood pressure was lowered during the time they were ligated off and, after a characteristic epinephrin wave, was re-established at the original level after their release. Pituitrin was injected before, during, and after adrenal ligation; the rise in blood pressure was closely similar in each case. The presence of accessory chromaffin tissue can scarcely be con- sidered a source of error. If the adrenal glands contain by far the greater portion of such tissue and if the removal of these is without effect, the intact supply can safely be ignored. The foregoing observations are not without interest in their bear- ing upon the general problem of the interrelations of the endosecretory organs. Our findings have been offered for publication because in the present state of this subject definite negative observations are nearly as much to be welcomed as further positive results. So far as a relationship between the adrenals and the pituitary is concerned, there is to be found in the literature little evidence. The fact that both adrenal and pituitary extracts raise blood pressure and cause hyperglycemia *® suggests that one organ might stimulate the other. There are on record observations by Hallion and Alquier ® and by Rénon and Delille’? that the prolonged use of extracts of the posterior lobe of the pituitary causes a hyperplasia of the adrenal cortex. In our present ignorance of the physiology of the adrenal cortex, how- ever, the significance of such observations is obscure. On the whole, it now seems probable that there is no direct dependence of the ad- renals upon pituitary functioning. 5 WEED, CUSHING, and Jacopson: Johns Hopkins Hospital bulletin, 1913, XXIV, Pp. 40. 6 HALLION and ALQuIER: Comptes rendus de la Société de biologie, 1908, iv, Dp. 5. , 7 RENON and DELILLE: ibid., p. 499. 244 R. G. Hoskins and Clayton McPeek SUMMARY 1. Intravenous injections of pituitrin in small dosage can be repeated at intervals of ten or fifteen minutes without significant failure of their pressor effect. 2. The adrenal glands of the dog can be ligated off without affect- ing general blood pressure; in the pregnant cat, however, such ligation has been observed to cause fall of blood pressure with subsequent rise when the ligatures were released. 3. In either animal occlusion of the adrenal circulation does not diminish the pressor effect of a standard dose of pituitrin. 4. There is probably, therefore, no direct dependence of adrenal functioning upon pituitary secretion. CONTRIBUTIONS TO THE PHYSIOLOGY OF THE STOMACH V. THE INFLUENCE OF STIMULATION OF THE GASTRIC MUCOSA ON THE CONTRACTIONS OF THE Empty SToMACH (HUNGER CONTRACTIONS) IN MAN By A. J. CARLSON [From the Hull Physiological Laboratory of the University of Chicago ANALYSIS OF THE PROBLEM HE character of the periodic and continuous motor activity of the empty stomach in man has been reported. It has also been shown that the contractions of the empty stomach give rise to the sensation of hunger, or the “ hunger pangs”’ by stimulation of _ afferent nerves endings in the walls of the stomach, and not by stimu- lation of nerve endings in the gastric mucosa. The hunger contrac- tions of the stomach are inhibited, reflexly, by all stimuli acting on the end organs of taste and general sensation in the mouth cavity, so that in the case of chewing palatable food when in hunger we have the so-called psychic secretion of gastric juice preceded and paralleled by a psychic inhibition of gastric motility and tonus. The fact that the hunger contractions of the stomach lead to increased excitability of the central nervous system and to vase-motor changes has also been placed on record.!' In the present paper an attempt is made to determine more specifically the cause of the hunger contractions themselves, so far as this is possible in man. ‘The contractions of the empty stomach may be due to: (1) The Condition or the Stimulation of the Gastric Mucosa. — The absence of food means absence of mechanical stimuli, and cessa- tion or diminution of the secretion of gastric juice, and hence a dimin- ished acidity. Carbon dioxide may be secreted into the empty stomach and may act as the primary stimulus. Carbon dioxide and 1 CARLSON: This journal, 1912-13, xxx], Pp. 15I, 175, 212, 318. 246 A. J. Carlson other gases may enter the stomach from the intestines, and act as stimuli. Succus entericus, pancreatic juice, and bile may enter the stomach and act as the primary stimulus through alkalinity or by means of specific substances such as the bile acids. The reader will recall that a number of workers maintain that bile facilitates the intestinal movements. (2) The Condition of the Blood, such as the relative concen- tration of nutrient substances, tissue metabolites, and hormones. It is possible that the neuro-muscular apparatus of the stomach is specially sensitized to slight variations in these substances. While we recognize the condition of the blood as a possible factor, it does not seem a probable one; in the first place, because the composition of the blood is on the whole more constant than the composition of the tissues, and because in young and vigorous individuals the hunger contractions of the stomach begin as soon as the stomach is empty, and while digestion and absorption is still in progress. in the intestines, so that there can be no lack of nutrient substances in the blood. In view of the relative constancy of the composition of the blood as shown by all past work on the serum, the existence of a periodic fluctuation in the concentration of any one substance in the blood parallel with the periodicity of the hunger contractions seems Improbable. (3) The Nervous Impulses Through the Vagi.—JIt is well known that the tonus of the stomach depends, in part, on impulses from the vagi, and that the stimulation of the peripheral end of the vagi induces strong contractions in the stomach whether empty or filled with food. It is also known that the stomach is capable of carrying out the movements of digestion to a fair degree of efficiency after section of both the vagi and the splanchnic nerves. In other words, the neuro-muscular apparatus of the stomach seems to be primarily automatic, as regards the genesis of the movements of digestion. The experiment of sectioning the vagi does not prove this point, however. The experiment does prove the plasticity of the gastric motor mechanism. One would expect that the extrinsic gastric nerves bear the same relation to the movements of the filled and of the empty stomach. This phase of the problem cannot be studied on man. If it should develop that the periodic hunger contractions Contributions to the Physiology of the Stomach 247 of the empty stomach are caused by periodic discharges through the vagi, the ultimate question of the cause of hunger would again become a problem of physiology of the central nervous system. (4) A Primary Automaticity of the Local Neuro-Muscular Mech- anism of the Stomach. — This can be established only by exclusion of the three other possibilities outlined above. A primarily auto- matic mechanism might still be influenced by the blood, by the extrinsic nerves, and by local reflexes from the gastric mucosa. The periodicity of the automatic activity might be due, not to a parallel periodicity in any essential stimulus, but to some peculiarity in the metabolism of the stomach developed as a_ special adaptation, similar to periodicities in other organs. The absence of the hunger contractions during digestion, or possibly the modifications of the hunger contractions into the movements of digestion, must, in this case, be due to specific inhibitory or regulatory impulses from the gastric mucosa. 3 Mr. V. is admirably adapted for determining the relation of stimulation of the gastric mucosa to the hunger movements, as the fistula is large enough to permit the balloon and connecting tube, and a tube for the introduction of liquids and gases, to be placed in the stomach simultaneously. The liquids and gases can be intro- duced with or without the man’s knowledge. Furthermore, the contents of the stomach (fluid and gas) can be withdrawn for analysis at any stage of the hunger movements and without any material disturbance. RESULTS (1) The Action of Water.— Water, at body temperature or nearly ice cold, inhibits the tonus and the hunger contractions of the stomach. The inhibition following the introduction of a glass of water (100~200 cc.) directly into the stomach lasts on the whole only three to five minutes, and is never followed by any augmenta- tion of the tonus or the hunger contractions. The cold water causes greater inhibition than the water at body temperature. If the water is introduced into the stomach during very intense hunger contrac- tions (“hunger tetanus’) there may be no perceptible inhibition. In other words, the degree of inhibition by water in the stomach is inversely proportional to the intensity of the hunger contractions 248 A. J. Carlson present at the time the water is introduced. Water, warm or cold, introduced directly into the stomach during a period of relative relaxation and quiescence does not increase tonus or initiate a con- traction period. A typical tracing showing this temporary inhibi- tion by water is reproduced in Fig. 1. The statement that cold water causes on the whole greater in- hibition than water at body temperature requires the following qualification. The record of the stomach movements were taken by means of an air-inflated balloon in the stomach cavity. Now, when cold water is introduced the water surrounds the balloon, at least partly, and cools the air in the balloon. This itself will lower the tension somewhat, until the temperature of the air is restored to that of the body by the warming of the water or by the passing of the water into the intestine. I do not think that this is a serious source of error, for this reason. A few experiments were made with water at 50°C. This causes greater inhibition than the water at 38°C. Water at 50°C. will, of course, increase the air tension in the balloon, yet the inhibition of the stomach tonus and movements is sufficiently marked to mask the effect of shght warming of the air. How does water in the stomach produce this temporary inhi- bition? It goes without saying that in these experiments the water was not introduced fast enough to cause contractions by distension of the stomach walls, although this occurred unavoidably in a few instances. The only possible ways that water at body temperature can stimulate the nerve endings in the mucosa seem to be (1) by mechanical pressure, or (2) by osmosis. Cessation of the inhibition probably marks the passing of the water out of the stomach into the intestine, or the addition of sufficient salts to prevent stimula- tion by hypotonicity. The greater inhibitory action by cold water and by water above the body temperature is evidently due to stimu- lation of the protopathic temperature nerve endings in addition to those acted on by pressure and osmosis. It is clear that the action of water on the stomach mucosa is in the direction of inhibition of the hunger contraction. How can this be reconciled with the view that a glass of cold water induces or augments hunger? It is to be remembered that in these experiments the water had no chance to act on the nerve endings in the mouth and the csophagus. The alleged action of a cold drink on hunger Contributions to the Physiology of the Stomach 249 and appetite is probably reflex effects (cold) from the mouth and cesophagus. In the writer’s own case a glass of ice water causes increased mucular tonus, sometimes even to the point of shivering and formication. ‘This increased kinestetic sense probably acts in the way of “ bahnung ” for the hunger sensation, if it is not actually a part of the hunger complex. Cannon and Washburn? suggest that the effect of a cold drink on the hunger sensation is due to “ the power of cold to induce contraction in smooth muscle.” Although their meaning is not clear I take it that they have in mind primarily the contraction of the stomach musculature. This could not come about by the cold acting on the stomach musculature directly. The reflex effects of cold water from the mouth and cesophagus are very complicated as regards the stomach, while cold water acting on the gastric mucosa directly causes inhibition. (2) The Action of Acids.— All acids, or liquids containing acids, including normal human gastric juice, cause inhibition of the movements and the tonus of the empty stomach when introduced directly into the stomach cavity. No acid has been tested in stronger concentration than o.5 per cent. The duration of the inhibition is on the whole directly proportional to the concentration and the total quantity of acid introduced. 200 c.c. of 0.5 percent HC1 may cause complete inhibition of the contractions and a relaxation of the tonus for 40-60 minutes, while 200 c.c. of 0.25 per cent HCr will usually inhibit for a period of 25-30 minutes only. This inhibition by acids can be made evident during all stages of activity of the empty stomach. If the acid is introduced during relative quiescence of the stomach the appearance of the next period of hunger contractions is delayed; if introduced during the active contractions these are abolished or depressed. The duration of the acid inhibition is probably determined by three factors, namely, (1) passing of the acid into the duodenum, (2) fixation and neutralization of the acid of the mucous gastric secre- tion, (3) neutralization by bile and intestinal juice which at times pass into the stomach through the dilated pylorus. While it is a striking fact that gastric juice of full normal acidity (0.48-0.53 per cent) and other acid solutions inhibit the hunger con- tractions, it does not follow that a neutral or alkaline reaction in the 2 CANNON AND WASHBURN: This journal, 1912, xxix, p. 452. 250 A. F. Carlson gastric cavity is a prerequisite for these contractions. During the strong contractions the stomach secretes a juice rich in mucin and combined HC1, but usually containing some free HC1. After the introduction of acids the contractions reappear before all the acid has passed out of the stomach or has been completely neutralized. And in case Mr. V. chews palatable food during a strong hunger period, the hunger contractions reappear before there is complete cessation of the psychic secretion of gastric juice. In other words, the hunger contractions are not inhibited by weak concentrations of acids in the stomach. A neutral or alkaline reaction of the mucosa is not necessary for these contractions. A typical tracing showing the inhibition of the hunger contrac- tions by V.’s own gastric juiceis reproduced in Fig. 2. When V. chews palatable food during a hunger period two inhibitory factors come into play, namely the reflex inhibition from the mouth, and the acid inhibition from the stomach. If the food is sufficiently palatable and the mastication is continued long enough the inhibition pro- duced reflexly from the mouth fuses with the acid inhibition from the stomach. If the food is not especially palatable or the mastication period brief, the contractions may resume on cessation of the chew- ing and then again be inhibited for a time during the period of most rapid secretion of the gastric juice. The degree of inhibition produced by normal gastric juice is the same as that caused by an equal quantity of hydrochloric acid of a concentration equal to the free acidity of the gastric juice. It would thus seem that the hydrochloric acid in the gastric juice constitutes the stimulus that leads to the inhibition. This acid inhibition of the hunger contractions is of peculiar interest in connection with the neuro-muscular mechanisms of these hunger movements and the gastric movements in normal digestion. The movements of the stomach in digestion are not inhibited by acids in the stomach, that is, at least not by acids in concentrations equal to that of the gastric juice. The fact that the intensity of movements of the antrum increases as the gastric digestion advances may even indicate that a certain degree of free acidity facilitates the movements of digestion. At first it occurred to me that since acid in the stomach inhibits the hunger contractions, but not the digestion contractions, the mechanisms involved in these two types of gastric Contributions to the Physiology of the Stomach 251 activity are different, at least as regards the character of the afferent impulses from the gastric mucosa. But on further reflection it became apparent that this is not necessarily the case. For the digestive movements involve primarily the pyloric end, while the hunger movements (as studied by our method) involve the fundus of the stomach. It is possible that acid stimulation of the nerve endings in the gastric mucosa leads, reflexly, to inhibition of the fundus, and peristalsis of the pyloric region of the stomach. This hypothesis is, of course, capable of experimental verification or refu- tation. (3) The Action of Alkalies.— The tests were made with sodium carbonate in concentrations varying from 0.2-1.0 per cent, and in varying quantities. In concentrations of 0.2 per cent or less the sodium carbonate solution appears to have the same influence on the hunger contractions as equal quantities of water, that is a slight temporary inhibition. This inhibition is evidently due, not to the alkalinity but to the bulk of the solution. In concentrations from 0:2 per cent to 1.0 per cent the degree of inhibition produced is on the whole directly proportional to the concentration and the quantity of the solution put into the stomach. 200 c.c. of one per cent sodium carbonate causes about the same degree of inhibition as 200 c.c. one-half per cent hydrochloric acid. It is thus clear that alkalinity has the same effect as acidity, only to a less degree; both acids and alkalies causing inhibition without any after effect of the nature of augmentation. The fact that 0.2 per cent sodium carbonate has no more effect on the hunger movements than equal quantities of water seems to show that a slight alkalinity of the gastric mucosa is compatible with the hunger contractions of the empty stomach. It makes it also evident that the entrance of bile or intestinal juice into the stomach will have little or no effect on these movements, while any concen- tration that influences these movements produce inhibition. (4) The Action of Local Anaesthetics. — Solutions of some local anaesthetics were tested with the view of determining whether the sensory nerves in the gastric mucosa plays only an inhibitory role in the processes of gastric hunger contractions. Phenol, chlore- ton, orthoform, quinine-urea-hydrochloride, and adrenalin chloride were used in quantities and concentrations compatible with absolute 252 A. J. Carlson safety to Mr. V. It was not considered advisable to use cocaine. The solutions of the drugs were introduced in quantities of too and 200) CIC: In the concentrations employed no specific action of any of the above substances could be demonstrated. For example, too c.c. of phenol (dilution 1-10,000) has the same effect as 100 c.c. of water, that is, a slight temporary inhibition. The same applies to the other drugs. No appreciable anaesthesia of the gastric mucosa was produced by any of the drugs. It seems probable that the solutions of these drugs pass out of the stomach just as rapidly as equal quanti- ties of water, and hence do not remain long enough in the stomach to produce local anaesthesia. Because of the danger attending the use of local anaesthetics In strong concentrations, further work on V. was deferred until complete orientation was at hand from work on dogs. It seemed, however, that adrenalin chloride introduced into the stomach even in considerable quantities could not be par- ticularly-injurious. But even in large quantities (100 c.c. of a dilu- tion of 1-10,000) the adrenalin acting in the gastric cavity has no other effect on the hunger movements than equal quantities of water. (5) The Action of Alcoholic Beverages. — Tests were made with sour and sweet wines, beer, brandy, and pure alcohol. The taking of alcoholic beverages with the meals is a habit with many people. It is claimed by many people that a glass of wine, beer, or some mixture of alcohol taken before meals increases the appetite (and possibly the hunger). The writer is neither a total abstainer nor a habitual user of alcoholic beverages. But it is his experience that a glass of beer or brandy taken at meal time seems to awaken or increase appetite. This effect is rather immediate and therefore not due to the absorption of the alcohol. Powlow has recorded an instance from his own experience where a drink of wine seemed to initiate the sensation of hunger (?) the very minute the wine reached the stomach. From enquiries as extensive as opportunities have permitted, I am inclined to believe that this apparent augmentation of the appetite by alcoholic beverages is rather a common experience. In view of this fact I expected to find that these alcoholic beverages increased the tonus and the contractions of the empty stomach, since it is the tonus and the contractions of the empty stomach that give rise to the hunger sensation. To my surprise the results proved to Contributions to the Physiology of the Stomach 253 be the very opposite. Wane, beer, brandy, and pure alcohol introduced directly into the stomach inhibit the hunger contractions and the tonus of the empty stomach instead of increasing them. This is true whether these fluids are cold or at body temperature. If these alcoholic bever- ages are greatly diluted with water, a degree of dilution can be reached which has the same action on the stomach as equal quantities of water, although the specific beverage is readily detected when the mixture is placed in the mouth. In no instance have I been able to make out any undoubted augmentation of the stomach tonus and hunger contractions after the inhibition period. In other words, alcoholic beverages when introduced directly into the empty stomach in quantities and concentrations that directly affect the tonus and the contractions of the stomach cause inhibition, and inhibition only. The pure alcohol was never used in stronger concentrations than to per cent. The brandy was usually diluted one half with water, while the beer and wines were put into the stomach undiluted. We have seen that acids in the stomach cause inhibition of the hunger contractions. Pure alcohol also causes inhibition. It is therefore evident that the alcohol and acids are primarily responsible for the inhibition following the introduction of alcoholic beverages into the empty stomach. But for the sake of brevity we may desig- nate it “ the alcohol inhibition.” The duration of the alcohol inhibition varies directly with the quantity and concentration of the beverage introduced in the stomach. Thus 50-100 c.c. of 10 per cent alcohol may inhibit the hunger con- tractions for one to two hours; or if introduced during a period of relative quiescence it delays correspondingly the onset of the next hunger period. 200 c.c. of beer causes inhibition for 30-60 minutes. The sour wines on the whole cause greater inhibition than the sweet wines, probably through their acids. A typical record of the alcohol brandy inhibition of the hunger contractions is reproduced in Fig. 3. This tracing is from a series of experiments on the author himself. It must be stated that these alcoholic beverages were put into the stomach of Mr. V. with his consent and without any protest, resentment, fear, or disgust on his part, which might account for the stomach inhibition. Mr. V. takes wine and beer occasionally. At times I had him bring his own choice of wine and beer, and occa- sionally I had him, himself, introduce into the stomach the desired “suorovzyuoo Jesuny oy) JO UOTTIQIYUT [OYooye oy} Surmoyg “yeuro}s ay} oyUL ApPOVIIp Joy WARM *9°9 ¢Z Ul ApuRIq *9°9 SI jo uononpoayyut & Wy OO *f[ “Vv Jo yoruiojs Ajduia ay} Woy sploday «= “g AMNOI mec ‘a ni ve Ad | | | | | ' i in| “UONIGIYUL plov sy} SULMOYS “YORUTOYS oY} OJUT poonpogzUT (AJsNorAdid sanoYy OM} paaNoas ‘UOTJaIOas oIYOASd ‘aoInf dysv5 UMO Ss, A) aoInf o1ajses ueumny jox2 67K IV A CAIN JO Yorwmoys Ajdura oy} JO SUOI}IvIZUOD Jo PIOOY —-“aZIS [LUISIIO ay} FLY 9uU0 ynoqy “7 AXA i ait ) Ni ‘ty wm i alenmeaaine i wre a ip after wf i A. J. Carlson euolqryur Arerodura} oy} SuIMoYyS “YyORUIoO}s ay} OJUT AT}OaIIP paonpodut JOJeM PfOI ‘9°9 COT & IW “A “AIX Jo yorutojs Aydt oy} Jo SuOIIVAJUOD JO PIOIDY *9ZIS [VULSIIO dy} Spsly} OM] YNOGY *] AYNOT] x r poy py aN roan i. v ‘J angi Thon aoe x ene vv re f\ = pt Af \ } st N | 5 255 ‘saqnurm o¢ ‘paonpoider Suloes} oy} Jo yzed oy} jo ou} pejoy, *AIAODOI enpeis YUM “UoOrIqIyUr plore pesuojoid ay} SuIMOYS —- “YO VU} 0FUT Ay}oaIIp peonposrzut (WARM) TFT 7Ued jad c’Q JO 99 CZ X IV ‘RINIsY I14jSeB YIM Sop Jo YyovUo}s Ayduia oy} WoIy pO ~—s “9 ZIS jeulsiio0 94} yey ou yNoqy “¢ ANNO WT ayer anysrenyer to i *suOT]OvI]UOD yosuny oy} Jo woNTqryUL Suryse] Suc] pur oyeIpouruUy Surmoys ‘yoeuroys oy} OUT ApOeAIp paonpomUl Jaoq JO “9° 00z | 3V ‘squowaAout 1a8uny Suoms Jo powed ev jo zed Surmp “A “IJ JO YoRUUO}s Aydwa oy} wor proxy =—«*9ZIS [eUISIIO ay} JPY OUD “f AMAL] Wynd wnat x Contributions to the Physiology of the Stomach 256 A. J. Carlson quantities. The effect on the hunger contractions was always the same. I am therefore confident that we are here dealing with a characteristic alcohol and acid inhibition, and not with a masked “‘ psychic ”’ inhibition. How are these results to be harmonized with the seeming stimu- lation of the appetite by alcoholic beverages taken by the mouth? In the first place the local inhibitory action of alcoholic beverages in the gastric cavity is so marked and so invariable, that I feel confident that this is always the gastric effect of these beverages, whether taken normally by the mouth, or introduced into the empty stomach with- out coming in contact with the mouth and cesophagus. Alcoholic beverages can therefore not initiate or increase hunger, since hunger is caused by the stomach contractions, and these are inhibited by the alcohol. Since most alcoholic beverages stimulate the end organs of taste and smell as well as those of general sensibility in the mouth cavity and in the cesophagus it is possible that this stimulation in some way augments or initiates appetite for food. If this is the case we have the singular condition of alcoholic beverages augmenting appetite and inhibiting hunger at the same time. There can be little doubt that cerebral states as modified by training and habit are also a factor in this apparent action of alcoholic beverages on appetite. It is certain that the individual’s first taste of alcohol, beer, or sour wines does not focus his attention on food and eating. If alcoholic beverages in the stomach caused as marked inhibition of the stomach movements in digestion as they do in the case of the stomach movements in hunger even moderate drinking with meals would lead at once to acute indigestion. As this is not the case, it is evident that alcoholic beverages affect the mechanisms of these two types of movements differently. (6) The Action of Carbon Dioxide and Air.— The action of carbon dioxide in the cavity of the empty stomach was studied in two ways: (1) by introduction of water charged with COs2, (2) by intro- duction of CO. gas. It is well known that an excess of carbon dioxide in the blood of the abdominal vessels initiates and augments the tonus and movements of the digestive tract. An excess of CO, is sometimes found in the gaseous contents of the empty or partly filled stomach. It is known, furthermore, that carbon dioxide in sufficient concentration acts as a powerful stimulus to the nerve endings in ee ae Contributions to the Physiology of the Stomach | such membranes as those of the mouth and nose, and of the cornea and conjunctiva. Carbon dioxide in the cavity of the empty stomach was at first considered a possible stimulus to the gastric hunger contractions, but this hypothesis proved entirely erroneous. In so far as the carbon dioxide in the stomach cavity affects the hunger movements the influence is in the direction of inhibition. Water saturated with CO, under pressure has practically no more effect than similar quantities of pure water. It produces the same degree of temporary inhibition without any after effect in the way of augmentation. As such carbonated water stimulates the nerve endings in the mouth in the characteristic way, it follows that the nerve endings in the stomach are less affected by CO, than are the nerve endings in the mouth. When the CO, is forced into the stomach in the form of gas and under pressure, the results are complicated by the mechanical action of the gas in forcibly distending the walls of the stomach and raising the intragastric pressure, and hence increasing the pressure in the balloon in the fundus. ey Beginning one Per cent Weight after Per cent ; gain 48 hrs. gain I 0.62 25 100 0.68 ie) 2 0.74 1.40 IOI 0.84 ified 3 1.07 2.40 124 Tae 24 4 1.09 2.45 125 Ty, 7 The actual swelling of the skin is thus seen to have about the same order of magnitude as that of other tissues immersed in dis- tilled water, while the great proportion of the gain in weight is due to free liquid lying within the sac. The free liquid was always found to be more or less tinged with blood when the sac was opened at the end of twenty-four or forty-eight hours. This was true even when the inside surface of the skin had been carefully washed with water or 3 NaCl at the beginning of the experiment. A not negligible amount of blood had been contained in the cutaneous vessels and affected the result. On this account a few experiments were made of the kind illustrated in Table III. The inner surfaces were washed with water and 2 c.c. of distilled water were placed in each sac before it was tied. The skins were then weighed and placed in salt solu- tions of varying concentration. So long as these concentrations were low their effect upon the total quantity of liquid absorbed was little different from that of distilled water, but even a small amount of salt tended to inhibit the injurious action on the skin. to go S. S. Maxwell TABLE III SKINS RINSED INSIDE AND 2 C.C. DISTILLED WATER PLACED IN EACH Empty Skin Solution bath- ing Skin Experiment | Beginning | 48 | MW ee peat Distilled H.O | M/xoo NaCl M/so0 NaCl N/25 NaCl * Note that the calculations of per cent gain assume that the 2 c.c. of water placed inside weighed 2 grams The error in this assumption does not exceed the necessary experimental error and may properly be neglected. In the experiments thus far described the skin was immersed in distilled water, and the presence of any of the tissue fluids in the inner layer of the skin would tend by simple osmotic processes to carry water through. Some experiments were then arranged by means of which the liquids in the sac of skin would be diluted while the liquid outside would have a considerable osmotic pressure. The direction and amount of transfer of liquid was noted and this was compared with the osmotic pressure as inferred from the electrical resistance of the liquid without and within the skin at the end of the experiment. The order of magnitudes is illustrated in the Table IV. TABLE IV Specific Conductivity in reciprocal ohms mies |\Liquid bath-|Liquid placed Baxpe EES ing the skin| inside sac Per cent! Of bathing Of liquid gain solution in sac | | a —_ \Tap water |None Baie 1.45 X 10—3 2.14 X 10—3 M/25 NaCl | 5 c.c. H20 x 5-19 X 10—3 5.90 X tO—* | 'M 15 NaCl} 5 .c.c. H20 : | y. 10.26 X 10—3 9.19 X 10—3 | | 'M ro NaCl | 5 c.c. H20 68 as x 13.33 X 10—8 | 12.90 K 10o— 8 Note: In experiments 2, 3'and 4 the per cent gain is calculated after deducting 5 gm. as the weight o the water added. On the Absorption of Water by the Skin of the Frog 291 A comparison of the results in Table IV shows as would be ex- pected that the movement of water is in the direction of the lower ' electrical resistance, that is towards the higher osmotic pressure. It would have been better to have made determinations of the freez- ing point of the liquids, but the apparatus at hand was not suited to the small amounts of liquids obtainable in some of the experiments. It is perfectly evident, however, that any error due to the presence of non-electrolytes, suspended colloids and the like, would be in the sense of increased resistance, and hence would give appearances favoring the idea of vital activity as opposed to osmotic transfer. A few experiments were arranged with skins filled with the same liquid as that in which they were immersed. The liquid used was Loeb’s solution composed of § NaC! too parts, ¢ CaCl 2 parts, 3 KClL2 parts. Ten cubic centimetres of the solution were placed in each skin and the skin was immersed in fifty cubic centimetres of the same solution. It should be pointed out that these experiments are far from being so crucial as they would seem. Notwithstanding the fact that the skins were rinsed inside and out in the solution, before they were filled and tied, they nevertheless carried with them a con- siderable quantity of water in the mucous layer on the one side, and of blood and lymph on the other. The attempt to meet this objection by turning some of the skins wrong side out was not wholly satis- factory and it is planned to continue this part of the work in a new form. The sample experiments shown in Table V_ will, however, give some idea of the conditions observed: TABLE V SKINS IN LOEB’s SOLUTION; I0 C.C. OF THE SAME SOLUTION INSIDE EACH SKIN Weight Specific Conductivity Experi- Side a= ment out : aA : Begin- | After | Per cent Of bathing | ning | 24 hrs. | gain solution | | Of liquid in sac | | 14.01 | 13-45 13.09 II.10 14.93 12.06 292 S. S. Maxwell It is probable that a careful removal of excess of liquid from the skins used in experiments 2 and 3 of Table V would have changed the result of the conductivity experiments considerably, but it is a question how much of this can be done without injury to the skin. Experiment 3 would seem to indicate that transfer of liquid can take place without difference of osmotic pressure; but it is possible that such a difference had existed and that in this case equilibrium was established within twenty-four hours. III. Discussion oF RESULTS It will be seen that while the above experiments show an enor- mous tendency of the frog skin to transport water by osmosis, the skin appears to be highly impermeable to inorganic salts. Some of the experiments, however, seem to show an ability to transfer water from one side to the other when the same osmotic pressure exists on both sides. The amount of such transfer is strikingly small as compared with that obviously due to osmotic pressure. and also as compared to the work done by secreting organs like the kid- neys or the salivary glands. This difference raises the question whether the inference is justified that any transfer of water taking place through the skin is due to vital activity of the living cells. The experiments of Reid ® commonly cited in support of such a view are not very convincing. He found, for example, that the osmotic effect of a 5 per cent solution of glucose in normal saline in one side of the osmometer, against a normal saline solution on the other, was more effective when the inner side of the skin was turned towards the glucose than when in the reverse position. In the clearest set of experiments reported by him‘ the amount of liquid passed through in twenty-four hours in the one direction was 174.093 cubic milli- metres in the other 79.035 cubic millimetres. The difference of the two is 95 cubic millimetres. According to the secretion theory this difference is due to an acceleration in the one direction and a retardation in the other and is brought about by the vital activity of the cells. The actual secretion would then be approximately equal to one half the difference or 47.5 cubic millimetres. The area 6 Locs ci. Cos Cie pa 208 On the Absorption of Water by the Skin of the Frog 293 of skin exposed in the osmometer used by Reid was 95 square milli- metres and the total effect was equal to the transfer of a layer of liquid one half a millimetre deep over the entire surface of the skin, an amount which could be very well due to the physical differences of the two sides of the skin. On the one side is a layer of tissues infil- trated with blood and lymph; on the other an adherent layer of watery mucous secretion. This arrangement would favor movement of water by osmosis from the outer to the inner side of the skin. When liquids of equal osmotic pressure were placed on the two sides of the skin, movement of water from outside to inside would proceed until the salt concentration of the mucous layer became equal to that of the surrounding liquid. The irreciprocity of permeability to sodium ions described by Bayliss* is harder to understand but may be accounted for on analogous grounds. Moreover, Starling * has given theoretical considerations to prove that under certain conditions substances may actually pass through a membrane against osmotic pressure without the interference of vital activity. It is highly desirable that the conditions which he assumes should be worked out experimentally. It has also been shown by Reid that the rate of transfer of water through the frog skin is affected by the addition of chloroform and of alcohol to the liquids in the osmometer, and the inference has been drawn that this is due to an effect of these agents upon the proto- plasmic activities of the epithelial cells. Im view of the slender evi- dence for the apparently sight amount of secretion by the skin, it would seem much more reasonable to suppose that these agents bring about some change in the physical state of the epithelium itself or in the adherent layers of material. It has been pointed out already in the introductory part of this article that a difference in rate according to direction of transfer of liquid through the skin does not of itself prove a vital activity as the cause of the difference, for such a difference exists also in dead skin. In the latter the direction of easier transfer is from within outwards. The differences dealt with are differences in rate of trans- fer. It is noteworthy that the emphasis in most of the work on osmosis has been laid upon equilibrium rather than on rate, while 8 Bayliss, Zeitschrift fiir Biochemie, 1908, xi, p. 226. 9 STARLING: The fluids of the body, London, 1909. 204 S. S. Maxwell diffusion has been studied from the standpoint of movement. When we have an adequate knowledge of the dynamics as well as of the statics of osmosis it will be easier to understand the apparent anom- alies of the movement of water through the frog’s skin without invoking vital activity as a means of explanation. SUMMARY t. It has been shown that an empty frog skin immersed in water takes up a relatively enormous quantity of water. 2. The taking up of large quantities of water depends upon the permeability of the frog skin to water and its relative impermeability to inorganic salts. 3. A skin exposed to liquids of equal osmotic pressure on both sides may still transfer water through itself from without inwards, but the amount is relatively small and is probably due to physical differences of the layers of liquid carried by the skin on n and within its opposite surfaces. 4. The assumption of a vital activity on the part of the frog skin in transferring water through itself is shown to be unnecessary. THE American Journal of Physiology VOL. XXXII OCTOBER 1, 1913 NO. VI PHYSIOLOGICAL OBSERVATIONS FOLLOWING DESCENT FROM PIKE’S PEAK TO COLORADO SPRINGS By EDWARD C. SCHNEIDER [From the Department of Biology of Colorado College, Colorado Springs, Colorado] MONG the numerous physiological observations on the influence of high altitudes upon man, only a few have dealt with the changes following the’descent to the lower level, and these have been on men who have resided at the high alti- tude from a few days to five weeks. It has been interesting, therefore, to follow the changes in the blood, circulation, and respiration of a man who has lived long on the summit of Pike’s Peak, altitude 14,109 feet. Mr. Howard H. Robison,’ the resident manager of the Sum- mit House on Pike’s Peak, very kindly consented to serve as the subject for this study. He has resided on the summit six months—from early in May to November — for seventeen consecutive years. He first went up when a young man in his early twenties. He is a man of athletic physique, excellent habits, and leads a very active life. He holds the record for the most rapid ascent ever made of the Peak, walking from Manitou to the summit, up the “Cog” railway track, a distance of 8.9 miles and a rise of 7485 feet, in 2 hours 31 minutes. 1 The writer wishes here to express his sincere thanks to Mr. Robison for so kindly serving as subject for this study, and to Mr. Leon C. Havens for help with blood-counts and air analyses. 296 Edward C. Schneider In 1912 he went to the summit the morning of May 8 and came down the evening of November 12. During this period of over six months he had been down only once and then for only one night. The first observations on him were made on the sum- mit of the Peak October 12, 13, and 14. The first study in Colo- rado Springs, altitude 6000 feet, was on the morning following his descent. An attempt was made to have the examination made at the same hour each day so that daily rhythms need not be considered. Observations were made at frequent intervals throughout a period of ten weeks, and to these have been added a few isolated exami- nations made at other times. Changes were followed carefully for the first six days after the descent. Mr. Robison then went on a hunting trip for two weeks to Lamar, altitude 5765 feet, and was available again for the investigation several times during the next ten days. December 12 to January 25 he spent at San Antonio, Texas, near sea-level, after which he subjected himself to further observations. THE CHANGES IN THE BLOOD The changes in the percentage of haemoglobin, number of red corpuscles, total oxygen capacity, total volume of the blood, and specific gravity of the blood have been followed. The results ap- pear in Table I. The total oxygen capacity and blood volume were determined by the carbon monoxide method of Haldane and Lorrain Smith.?, Care was taken to allow at least twenty minutes to elapse, while the subject still continued to breathe into the con- fined space of the apparatus, after having received the carbon monoxide, so that the gas would distribute itself evenly through- out the body. The blood samples then taken were titrated in duplicate, and sometimes in triplicate, with a standardised carmine solution against a north light. The percentage of haemoglobin was determined by the Haldane-Gower’s haemoglobinometer and the blood-counts were made with a Thoma-Zeiss haemocytometer. For the specific gravity a series of wide-mouthed bottles containing mixtures of glycerine and water of different densities was used. 2 HALDANE and LoRRAIN SMITH: Journal of physiology, 1900, xxv, p. 33I- Physiological Observations from Pike's Peak 207 The changes that followed Robison’s descent from the summit of Pike’s Peak to Colorado Springs agree in general character with TABLE I OBSERVATIONS ON THE BLOOD OF Mr. ROBISON | ; rs | oe = Sauls Sina of Ed| es |oq 2 Sela] &O/ So] & | 3 Date 338 SIE cea lbs ncaa ass one) se). | wees tel eure 2 Seen su || ig | a | & | Oe | Scene ooo | a Se) meena He | slea|5e/58) 8g as BER S| d/eS| a |9s 5 eae | fice . 12,1912} 3 p.m| 148 | — | 274 | — | — |(7.7)| Summit of Pike’s | Peak 13 8.45 a.m.| 150 = 27.8 — — |(7.5) | | | 14 Ssh 148 | 1101 | 27.4 | 4018 | — | — | | 13 | 7.35 “ | 144 | 1062 | 26.6 | 3992 |1.073) — | Colorado Springs 14 10.40 “ 142 | 1085 | 26.3 | 4125 | — | — | | | | | 15 7.45 “ | 145 | — 26.8 — |1.073 — 16 fe 144 1088 266 4090 1.073 7.6 | | | 17 e2O e 147 — 27.2 |. — —|— 18 lato = 143 | 1125 | 26.5 | 4245 |1.072) 7.7 3 8.15 “ | 134 | 1050 | 24.8 | 4234 |1.071) 7.4 | Colo. Springs after | return from Lamar 5 8. 134 — 24.8 — /|1.071) 7.6 We (6: Sie 132 | 1054 | 24.4 | 4320 |1.070| 7.5 5 26;,1913)10: - 122 — | 22.6 — /|1.068 7.2 | After return from sea-level 28 | elle Ye 121 965 | 22.4 | 4308 1.068) 7.2 30 8.20 122 973 | 22.6 | 4305 | — | — May 1 Meo. 122 — 22.6 — 1.067 7.0 In Manitou three months, altitude 6620 feet 1 Counts made in July and August, 1911. 298 Edward C. Schneider those observed by Douglas, Haldane, Henderson, and Schneider ® in the English-American Pike’s Peak Expedition of 1911. They observed on themselves following their return to Colorado Springs that there was an immediate reduction in the percentage of haemo- globin, which fell in a day or two to about 110 per cent, which is near the normal for the altitude of 6000 feet. Simultaneously in them there was a distinct decrease in the total oxygen capacity of the blood, but this was not so marked as the change in the haemo- globin percentage. In each man, except one, the blood volume increased for thirteen days following descent, after which it re- turned to the normal for the lower altitude. In Robison the blood changes delayed in appearing and then took place at a very slow rate. The percentage of haemoglobin did not clearly alter during the first six days. At the end of three weeks it had fallen from 148 to 134 or about 9.4 per cent. Nine days later it had only reached 132,so in one month it had not nearly approached the average for the altitude. Sometime during the next six weeks while Robison was near sea-level the haemo- globin fell to 122, the level to be maintained throughout the re- mainder of the stay at the foot of Pike’s Peak. The previous winter Robison’s percentage of haemoglobin fell from 145 on Pike’s Peak to 116 at Manitou. This also is high, the average being 110 in men at 6000 feet. In 1912-13 the entire fall in the haemoglobin percentage was 17.6 per cent. The destruction of haemoglobin and alteration in blood volume followed a somewhat different course than that observed in the English-American Pike’s Peak Expedition. Two determinations of Robison’s total blood volume and total oxygen capacity were made on Pike’s Peak; unfortunately the figures, which were on a loose sheet of paper, for the titration in the experiment made October 13 were lost. An approximate estimate made at the time showed the results to agree well with the data of the 14th. Unlike the imme- diate change observed in Douglas, Haldane, and Schneider there was in Robison no destruction of haemoglobin during the first six days after his descent. However, while the total oxygen capacity remained stationary there was some diluting of the blood on the 3 DouGLAs, HALDANE, HENDERSON, and SCHNEIDER: Philosophical Transac- tions of the Royal Society of London, 1913, Series B, CIII, pp. 271-208. Physiological Observations from Pike’s Peak 299 sixth day which gave an increase of 5.4 per cent in the total blood volume. During the next three weeks the total oxygen capacity diminished 4.3 per cent; while on the other hand, the blood reached its maximum volume. This was 7.5 per cent greater than the volume on Pike’s Peak. While Robison was at sea-level the blood volume does not appear to have increased. There was, neverthe- less, during this period a marked destruction of haemoglobin; the total oxygen capacity of the blood the last of January was 11.9 per cent less than it had been on the summit of the mountain. Apply- ing Hiifner’s value that the amount of oxygen which combines with one gram of haemoglobin is 1.34 c.c. there were 822 grams of hae- moglobin in the subject’s blood on Pike’s Peak. Sometime within the ten weeks he destroyed a surplus of 98 grams of haemoglobin. The reductions in the number of red corpuscles and in the specific gravity of the blood are roughly parallel with the other blood changes. OBSERVATIONS ON THE ARTERIAL PRESSURE AND PULSE-RATE _ For six years at widely separated periods arterial pressure and pulse observations have been made on Robison, and these indicate that long residence at an extremely high altitude has in no way altered the efficiency of his heart action and circulation. The arterial pressure determinations were always made on the subject while resting in the sitting posture. They have been found to range as follows: — Systolic pressure Diastolic pressure Pulse pressure On Pike’s Peak 106 to 122 mm. 75 to 86 mm. 26 to 38 mm. In Colorado Springs 114to126 “ 80 to 90 * 28 to 39“ A uniform difference in the pressures at the two altitudes has not been found. On the whole, however, the data agree with the observations of Schneider and Hedblom.* They found in a series of eighteen observations on Robison, in 1907, that his systolic and diastolic pressures averaged somewhat less on the summit of Pike’s Peak. 4 SCHNEIDER and HEpBLom: This journal, 1908, xxiii, p. 101. 300 Edward C. Schneider Robison’s resting heart-rate on Pike’s Peak during the three days October 12, 13, and 14 varied between 80 and 92 beats per minute. The normal tempo on Pike’s Peak per minute as shown by frequent observations was about 82. The slowest rate yet noted in him at this high altitude was 68, recorded by Schneider and Hedblom in 1907. A very marked slowing of the pulse-rate, such as was observed by Durig and Kolmer,’ occurred following Robison’s descent to Colorado Springs. During the first five days the rate remained constantly at 60 but on the sixth morning it had increased to 64. After the trip to Lamar the resting pulse had accelerated to 72 and throughout the remaining period of study it never returned to the slow tempo of the early days, but varied between 68 and 78. This increase in the pulse-rate does not appear to be definitely associated with the blood changes although the haemoglobin percentage had fallen ten points at the time the rate increased and the total oxygen capacity of the blood was slightly lowered. Very likely the expla- nation is to be found in the fact that the alveolar oxygen pressure in the lungs had fallen almost to the normal for the lower altitude. Benedict and Higgins® have shown that the pulse-rate at sea- level in normal individuals breathing oxygen-rich mixtures is less than when breathing ordinary air. Parkinson” confirmed their ob- servations and suggests in that the blood is capable of taking up more oxygen, when an excess is present, the heart muscle is better supplied with oxygen and thus works to better advantage, supply- ing the tissues by fewer beats. It is evident that the reaction of the organism to high altitudes is in large measure due to deficiency of oxygen and, therefore, we may expect the heart to benefit when oxygen is administered. This was found to be the case. Robison was set to breathing oxygen through the apparatus employed for administering carbon monoxide and oxygen in the blood volume studies. In each of two experiments on the summit of Pike’s Peak there was almost an immediate slowing of the heart. Thus in the first experiment in two minutes after beginning to breathe the pure 5 Duric: Physiologische Ergebnisse der im Jahre 1906 durchgefihrten Monte Rosa Expedition, p. 48. 6 BENEDICT and HiGGINs: This journal, 1911, xxviii, p. 25. 7 PARKINSON: Journal of physiology, 1912, xliv, p. 54. Physiological Observations from Pikes Peak 301 oxygen his pulse-rate was reduced from 80 to 72 and at the end of seven minutes had fallen to 64. The second experiment a day later was briefer but again the pulse-rate after having remained at 82 for some minutes was reduced in two minutes to 72 and a minute later was down to 7o. During the first month after the descent to Colorado Springs repeated attempts were made to reduce the cardiac-rate with oxy- gen but without success. It should here be remarked that the normal individual at an altitude of 6000 feet will respond to the breathing of oxygen-rich mixtures by a slowing of the pulse-rate. In our laboratories we have often confirmed the earlier observa- tions on healthy young men. For example, one subject after sit- ting quietly for ten minutes had a pulse-rate of 71; this was lowered to 62 during ten minutes breathing of pure oxygen and four min- utes after return to air it had again accelerated to 70 per minute. In a majority of the men studied, the character of the pulse while breathing the oxygen changes, becoming fainter and softer. With Robison this change could not at first be noted with certainty. However, on January 28 and 30 after he returned from sea-level the character of the pulse while he breathed the oxygen, although the rate was still unaltered, was found by several observers to be softer. A definite slowing of this subject’s cardiac-rate was obtained in an experiment on May 1, five and one half months after the descent. For ten minutes his pulse remained constant at 68 per minute; he was then given oxygen for ten minutes and during this interval the rate varied between 64 and 62. After the return to air the rate slowly increased and within nine minutes it had re- turned to 68. The observations indicate that the accelerated heart-rate ob- served in the majority of persons during residence at very high altitudes is one of the several adaptive responses to the influence of the shortage of oxygen. They furthermore may possibly offer a confirmation of Parkinson’s explanation that an excessive supply of oxygen in the blood favors the heart muscle. That there was less oxygen available for oxidative processes in the blood at 14,000 feet was indicated by the decidedly dark color of the blood when it was drawn for examination; while in Colorado Springs the color 302 Edward C. Schneider was always a good arterial red. In addition, the partial pressure of oxygen in the arterial blood was less at the high altitude. Thus Douglas, Haldane, Henderson, and Schneider® found the mean partial pressure of oxygen in the arterial blood on Pike’s Peak to be 88.3 mm. while Douglas and Haldane ° have shown the mean at Oxford to be 99.1 mm. Miss FitzGerald has pointed out that the symptoms of oxygen deficiency at high altitudes are due not to the amount of oxygen in the arterial blood but to the partial pressure of this gas in the blood. When Robison came down to Colorado Springs there must have been, because of his deep breath- ing (this is discussed later) and of the high content of haemoglobin in the blood, much more oxygen rendered available by the rise in the partial pressure of the arterial oxygen. This excess of ‘oxygen may have acted by destroying easily oxidizable substances which are very abundant in the blood at very high altitudes and even to some extent at sea-level. The withdrawal of the stimulating action of these metabolites which may act through their hydrogen ion-concentration,” or the excess of oxygen alone, ‘reduced the heart-rate of Robison below his normal for the lower altitude; and inhalation of pure oxygen, therefore, failed to further slow the heart. Later the breathing was shallower and the total oxygen capacity of the blood less, hence the supply of oxygen in the blood was not sufficient to completely destroy these oxidizable meta- bolites, or to permit the heart to work as economically as during the early days after the descent. It was, therefore, then possible to show the heart-rate when oxygen was administered. THE RESPIRATION Lung Ventilation.— The ventilation of the lungs for those dwelling at high altitudes is greater than that of mankind living 8 DouGLAs, HALDANE, HENDERSON, and SCHNEIDER: Joc. cit., pp. 197-08. ® Douctas and HALDANE: Journal of physiology, 1912, xliv, p. 331. 10 FitzGERALD: Philosophical Transactions of the Royal Society of Lon- don, 1913, Series B, CIII, p. 361. 1 Doucitas, HALDANE, HENDERSON, and SCHNEIDER: loc. cit., p. 300. 12 See FELDMAN and HiLi: Journal of physiology, 1911, xlii, p. 439. 13 Mathison — Heart, 1911, II, p. 60 — finds in his study of the heart-block that the cardiac tissues are sensitive to want of oxygen. Physiological Observations from Pike's Peak 303 at sea-level. It has been known since the researches by Haldane and pupils '* that the volume of fresh air taken into the lungs per minute during rest is so regulated as to keep the partial pressure of carbon dioxide in the alveolar air practically constant for the individual. The carbon dioxide content of the alveolar air is, therefore, taken as an index of lung ventilation. A diminution of the alveolar carbon dioxide pressure indicates an increase in the lung ventilation, while an increase in carbon dioxide means a reduction in the alveolar oxygen pressure. A number of workers ?° have demonstrated that the alveolar carbon dioxide pressure falls, and as a consequence the volume of air breathed increases, with a diminution of atmospheric pressure. According to Douglas, Hal- dane, Henderson, and Schneider the alveolar carbon dioxide pres- sure required to excite the respiratory centre of man on Pike’s Peak falls to about two-thirds that of the normal value at sea-level. © This causes the breathing of 30 per cent more air per minute and an increase of 50 per cent in the alveolar ventilation. The partial pressure of carbon dioxide in the alveolar air on Pike’s Peak is about 27 mm. as compared with 40 mm. at sea-level. A series of observations on Robison’s alveolar air under resting conditions were made while he was on the Peak and at intervals for a period of five and a half months after his descent. Hal- dane’s '® gas apparatus was used for the analyses and the samples of alveolar air were obtained by the direct method of Haldane and Priestley.’ Table II contains the results of this study. The figures as given are with two exceptions the average of the analyses of two samples. The content of alveolar carbon dioxide and oxygen on Pike’s Peak agreed closely with that obtained on the members of the 14 HALDANE and PRIESTLEY: Journal of physiology, 1905, xxxii, p. 225, and Douctas and HALDANE: ibid., 1909, XXXvlii, p. 420. 15 See Boycott and HALDANE: Journal of physiology, 1908, xxxvii, p. 25} Warp: ibid., p. 378; Dovuctas: ibid., 1910, xl, p. 472; Zuntz, Loewy, MULier, and Caspari: Héhenklima und Bergwanderungen, 1905, p. 428; Duric: Uber das Verhalten der Atemmechanik und der Alveolartension, 1910, p. 61; and Doucras, HALDANE, HENDERSON, and SCHNEIDER: Joc. cit., pp. 206-220. 16 HALDANE: Methods of Air Analysis, 1912, p. 47. 17 HALDANE and PRIESTLEY: loc. cit., p. 228. 304 Oct. Nov. Jan. Date 12, 1912 12 13 14 13 or Barometer in mm. Hg. Edward C. Schneider TABLE II gases in dry alveolar air Partial pressure | of gases in mm. Percentage of | Hg. in alveolar air at 37° satu- | rated with mois- | ture | | C054) 205. | GOs) ats 458 |° 6.96 | 12.42 | 28.6 | 51.0 458 | 6.72 | 12.91 | 27.6 | 53.1 457 | 7.16 | 12.34 | 29.4 | 50.6 457 | -6.27 | 12.59 | 25.7 | 51%6 6164), 4:77) | 15:59) | 27a) Weeer 612 | 5.19 | 15.62 | 29.3 | 88.3 613 | 5.44 | 14.90 | 308 | 843 623 | 5.52 | 15.51 | 31.8 | 89.3 620) |e S46al. 15:37 J) 61-3 ugh Pee 616 | 5.95 | 14.62 | 33.9 | 83.2 608 | 6.28 | TE GSO |) 7 609 | 6.42 | 13.27 | 36.1 | 74.6 615 | 6.93 | 13.26 | 39.4 | 75.3 613 | 6.98 | 12.39 | 39.5 | 70.1 612 | 6.67 | 12.29 | 377%) 604 614 | 689 | 12.65 | 39.1 | 71.7 607 | 6.64 | 13.70 | 37.2 | 76.7 lPike’s Peale \Colorado Springs /After return from Lamar After return from near sea- level In Manitou three months Physiological Observations from Pike’s Peak 305 English-American Pike’s Peak Expedition after they had been two weeks on the summit. The first sample of alveolar air which was taken from Robison fourteen hours, or the next morning, after his descent showed no change whatever in the carbon dioxide partial pressure. Hence he still continued to ventilate his lungs as much as on the summit of the Peak, which resulted in an alveolar oxy- gen pressure at least 35 mm. greater than he had on the summit and at least to mm. above that found in men acclimatised to the altitude of Colorado Springs. During the first six days following the descent the alveolar carbon dioxide pressure very gradually increased and as it did the ventilation decreased. However, on the sixth day the alveolar oxygen pressure was still more than 5 mm. above the normal for the altituce of 6000 feet. The next two weeks while the subject was hunting near Lamar the decrease in lung ventilation must have greatly retarded, for on December 3, three weeks after the descent, the alveolar carbon dioxide content was 35.2 mm., which was still below normal; his normal for the altitude of Colorado Springs being about 37 mm. Sometime between December 3 and 12 the carbon dioxide pressure reached normal and may have passed above if the read- ing 39.4 mm. may be regarded as correct and it is the result of several analyses. It was impossible to study this condition further because the subject left that day for the journey to near sea-level. The observations made in January, immediately after the re- turn from this journey, indicate that at sea-level he adapted his breathing so that the ventilation of the lungs was similar to that of the normal man at that level. It appears that Robison readjusted his breathing on returning to the altitude of Colorado Springs after a residence of six months at 14,109 feet far more slowly than men who have sojourned only a few weeks at a high altitude. Thus Ward‘ after a residence of six days at Capanna Regina Margherita on Monte Rosa, alti- tude 14,965 feet, and Douglas, Haldane, Henderson, and Schneider after their sojourn of five weeks on Pike’s Peak, on descending found an immediate response, by an increase in carbon dioxide pressure and lessened lung ventilation, to the rise in the barometric pressure. The time required for complete adjustment in the mem- 18 WarD: Journal of physiology, 1908, xxxvii, p. 383. 306 Edward C. Schneider bers of the English-American Expedition at 6000 feet was not determined because they later went down to sea-level. Here, however, they observed that the change became complete within two weeks of the day of leaving the summit of Pike’s Peak. This slow change in Robison’s respiration suggests that some condition affecting the respiratory centre and due to the altitude stimulus, want of oxygen, becomes more permanently fixed by longer residence at the high altitude. This acquired condition or habit is then very slowly readjusted on return to a low level. It has been suggested by Douglas, Haldane, Henderson, and Schneider !* that the fall in alveolar carbon dioxide pressure at high altitude is due to diminished alkalinity of the blood. They deem it probable that the diminished alkalinity is not due merely to an excessive production of lactic acid, as is the case after muscular activity, but to some adaptive alteration in the regu- lation of blood alkalinity; this regulative function they attribute to the kidneys. ‘‘ A slight and gradual adaptive alteration in what one may call the exciting threshold of alkalinity for the kidneys would explain the reduced fixed alkalinity of the blood in acclimatised persons.” Power to hold the Breath. — Mosso * found on Monte Rosa that the power to hold the breath voluntarily was less than in Turin. The subject of this report was able to hold his breath on Pike’s Peak for not longer than 25 to 28 seconds but in Colorado Springs he was able to hold it 46 to 56 seconds. No change in the power to hold the breath occurred during the winter. Vital Capacity. —It is a popular belief, also held by numerous medical men, that the chest is greatly enlarged by residence at high altitudes. Humboldt”! claimed to find an increase in the capacity of the thorax among the inhabitants of the Andes, and Williams * reports an increase in the size of the chest as a result of a residence in high mountain resorts. With these exceptions 19 DouGLas, HALDANE, HENDERSON, and SCHNEIDER: loc. cit., p. 301. 20 Mosso: Life of Man on the High Alps, 1899, p. 201. *1 HUMBOLDT: Voyage aux régions équinoctiales du nouveau continent, fait en 1799-1804, Paris, 1814. * See STRAUCH: American Journal of the Medical Sciences, 1911, cxlii, p. IIS. Physiological Observations from Pike's Peak 307 all observers agree that for the majority of persons the vital capacity actually diminishes at high altitudes. Mosso* showed the members of his expedition had on Monte Rosa a vital capacity that was less than in Turin. Zuntz and his co-workers,”* Durig,”® and Fuchs *° have confirmed Mosso’s report. The morning after Robison came down to Colorado Springs and frequently throughout the period of study his vital capacity was determined. The records of the first day taken at intervals of five minutes are 4000, 3975, 4120, and 4070 c.c. The second day shows 4225 c.c. The difference undoubtedly should be at- tributed to lack of experience with the spirometer and not to a change in the thorax. After his return from sea-level there was no change in the capacity. Robison’s endurance and strength as a mountain climber are certainly not to be explained by chest development as the follow- ing comparison with Born’s”’ statistics of Yale men well shows: Track Average Athlete Student Robison Height 68.3 in. 68.7 in. 67.8 in. Weight 145.0 lbs. 143.5 lbs. 137.0 lbs. Girth of Chest (normal) 34.2 in. 36.3 in. 34.4 in. Girth of Chest (inflated) 35.8 in. 38.1 in. 36.0 in. Vital capacity 42255 “C:Cs ATS53> (GC. 3934. c.c. Even though Robison is an active man and has lived at an altitude of 14,109 feet for six months during each of the last seventeen years his chest measurements, considering his height, compare not with the athlete but with the average student at sea- level. The two keepers*® of the Regina Margherita hut on Monte Rosa who remained from the beginning of July until the end of 23 Mosso: loc. cit., p. 342. 74 ZuNTZ, LOEwy, MULLER, and CASPERI: loc. cit., p. 335. 75 Duric: loc. cit., pp. 54-60. 26 Fucus: Sitzungsberichten der Physikalisch-Medizinischen Sozietét in Erlangen, 1908, xl, p. 240. 27 BorN: Yale Alumni Weekly, April 1, 1908, pp. 1-s. % Mosso: loc. cit., p. 154. 308 - Edward C. Schneider September at an altitude of 14,965 feet and continually ascended and descended for provisions showed a similar chest development. Francioli with a height of 68.5 inches and weight 169.8 lbs. had a vital capacity of 4017 c.c.; while Quaretta, height 64.6 in.., weight 154.4 lbs., had a vital capacity of only 3790 c.c. SUMMARY 1. The percentage of haemoglobin in the blood decreased very slowly after the descent from Pike’s Peak, falling from 148 to 132 in 30 days and to 122 during the following six weeks. 2. The number of red corpuscles decreased from 7.7 to 7.0 millions; the specific gravity of the blood from 1.073 to 1.067. 3. The total volume of the blood showed an increase of 5.4 per cent on the sixth day and a maximum, 7.5 per cent, on the 30th day. 4. The total oxygen capacity of the blood did not alter the first six days. At the end of the third week it had decreased 4.3 per cent and at the end of 10 weeks had diminished 11.9 per cent. 5. During a period of six years the arterial pressure has re- mained normal. 6. The pulse-rate on Pike’s Peak was about 82. The first days after descent it remained at 60 and later accelerated to 7o. 7. The breathing of an oxygen-rich mixture slowed the heart- rate from 82 to 64 per minute on the Peak, but after the descent did not alter the rate the first ten weeks. Later at the lower altitude a slight reduction in the pulse-rate was obtained with oxygen. 8. The alveolar carbon dioxide pressure required to excite the respiratory centre did not alter immediately. After 24 hours it began to rise and increased slowly for 30 days, at which time it was above the normal for Colorado Springs. It later returned to the normal. For more than three weeks the amount of lung ventilation was excessive for the altitude of 6000 feet. g. The power to hold the breath on Pike’s Peak was one-half of that in Colorado Springs. 10. The vital capacity and chest measurements are not greater than those of men of similar physique at sea-level. THE EFFECT OF WATER INGESTION ON THE FATTY CHANGES OF THE LIVER IN FASTING RABBITS ! By M. R. SMIRNOW Unstructor in Pathology and Bacteriology, Med. Dept. Yale Univ., New Haven, Conn.] N a paper entitled “‘ Hydropic Changes in the Liver Cells of Rabbits,” soon to be published by Dr. C. J. Bartlett and my- self, mention is made that the peculiar picture described is not seen in fasting rabbits. It was to establish this point and to emphasize the fact, that the ‘“ hydropic”’ changes were probably due to hyperfunctional activity, that the work here outlined was undertaken. Five rabbits were taken for experimentation, and emphatic instructions were given to the attendant not to feed them; but no instructions were given concerning water. Our rabbits are at all times given plenty of green stuffs,so the attendant as a rule finds no need of watering them, and it was this fact that caused him to neglect to water the rabbits placed under observation. The first and second rabbits were killed on the fourth and fifth day respectively, but showed nothing abnormal in gross. The third was killed on the seventh day, and showed a moderate degree of fatty change in liver. The liver was enlarged, quite yellow, softer than normal and tore easily. It was this unexpected condition coupled with the lack of water ingestion that resulted in the work here to be reported. Twenty-seven normal rabbits were used. Of these, eleven were fasted from four to ten days, no water being given, and are here designated as “ Group A.” A second group of nine rabbits were fasted from four to fourteen days, but were given ordinary tap water, and are here called: ‘“‘ Group B.” The last, a group of seven rabbits, were fasted five to eleven days, were given distilled water, and are here designated: “‘ Group C.” 1 Read before the American Ass’n of Pathologists and Bacteriologists, in Washington, D.C., May, 1913. Z 310 M. R. Smirnow The rabbits were separated into groups of three to five, and placed in clean cages, which allowed ample room for them to move about; but not room for excessive exercise. Water was placed in dishes for the animals that were to receive it. Most of the rabbits were weighed at the beginning and at the end of the experiment, and showed an average loss of two fifths their original weight, varying from between 250 to 600 grams. This average was the same for both the watered and unwatered animals. The livers were examined in the fresh state with Sudan III in all cases. Frozen sections were also made and stained with Sudan III, haematoxylin and eosin, and tissue was fixed in Zenker’s fluid and formalin for inbedding. Eight of the first group of eleven rabbits showed a moderate or advanced fatty infiltration. These animals were fasted, with no water given, from five to ten days, were then killed, and autopsied immediately. The livers of these animals were quite yellow, flabby, almost semi-fluid in consistency, tore readily, and left considerable fat on the knife in cutting. Scrapings stained with Sudan III showed numerous small and large fat globules. Osmic acid gave positive test for fat where tried. Microscopically, with haematoxylin-eosin stain, the typical picture of fatty infiltra- tion was seen, the more numerous fat vacuoles nearest the central veins. One animal of this group was killed at the end of four days. The liver showed nothing in gross, but gave a few fat globules when stained with Sudan III. Microscopically, the cells were somewhat smaller, coarsely granular, and showed here and there a few small vacuoles, which might have been interpreted as fat. The remaining two were animals that fasted seven and nine days respectively. These animals died. The livers showed a marked degree of coccidiosis and were quite congested. Scrap- ings stained with Sudan III and microscopical examination were negative. Of the second group of nine normal rabbits deprived of food from four to nine days, water being allowed, only one showed a fair degree of fatty infiltration of the liver, and gave the Sudan III test. The livers of five others showed very slight vacuola- tions in the cells, microscopically with the use of the oil: immer- sion. The cells were smaller and more granular and the nuclei Effect of Water Ingestion in Fasting Rabbits 311 stained well. The livers did not give the Sudan III test in the fresh state, but microscopically the vacuoles mentioned might have been interpreted as fat. The remaining three animals of this group showed only a slight increase in size of the cells and a more granular cytoplasm than normal. Of the seven that makeup the third group of fasting animals, and which received distilled water, only one, an eleven-day rabbit, gave a positive Sudan III test, and was found to have a fair amount of fat microscopically. The others showed a slight vacuolation of the cells with the oil immersion, but did not give the Sudan III test. Of these rabbits, the one that showed the distinct fatty change, and one of those that showed vacuolations, were killed, all the others died. Most of the animals of this group showed a marked coccidiosis, which might have contributed to their deaths; but apparently did not favor fatty change. Summing up the results obtained as shown in the accompany- ing charts, we find that nine of the eleven hungered and un- watered rabbits gave both the Sudan III test and the microscopic picture of fatty infiltration. This is in striking contrast to the findings in the rabbits fasting under the same conditions, but receiving water, wherein only two of the sixteen animals showed a fair amount of fat, evidenced both with Sudan III and the microscope; seven showed slight microscopic -vacuolations, but gave no Sudan III test; and seven were entirely negative. Whether or not a greater percentage of watered fasting rabbits would show the fatty change, if more time were given them, is questionable. Offhand it would appear that it is not only a matter of time, in-as-much as six of the seven animals that were negative both to Sudan III and microscopical examination were kept under observation until death. It is worthy of mention in this connection that all of the last mentioned animals suffered from extreme coccidiosis, which must have at least hastened their deaths before fatty changes could develop. The literature on this subject is very scant and conflicting. Statkewitch 2 and Nikolaides * and others have shown fatty changes in the livers of fasting dogs, cats, rabbits and guinea pigs; but 2 MotrraM: Journal of physiology, 1909, vol. 38, page 281. 3 GILBERT AND JANNIER: Quoted by Mottram. 312 M. R. Smirnow regard a decided fatty change taking place only after prolonged hunger, and consider these changes to be degenerative in charac- ter. Water was given the animals during their fast. On the other hand, Gilbert’s and Jannier’s* experiments show that only a very mild degree of fatty change is seen in rabbits fasting for one to eight and one half days. These investigaors do not regard the change as degenerative. GROUP A Killed Ss : Macroscopic ; : Microscopic or Died P P Killed . +icells swollen, granular, few | show vacuolation + definite fatty vacuoles in cells Soft, friable; + definite, fatty infiltration iyellow color | | : | ‘Soft, friable, | +)|Moderate fatty infiltration flabby, yellow color, coccidia | | | } | Soft, friable, flabby _ + /Good amount of fatty infiltra- | _ tion | Soft, friable, mushy + |Good amount of fatty infiltra-| tion Soft, friable, flabby | -+/Good amount of fatty infiltra- | tion | mushy | +|Moderate amount of fatty in- filtration Congestion and coccidiosis Natural cell structure “ “ce Moderately fatty yellow mottling Congestion and coccidiosis Normal cell structure Rh Watty, V = Vacuoles — = Negative 4 Mrxatarpes: Archiv fiir Physiologie, 1890, page 518. Effect of Water Ingestion in Fasting Rabbits 213 Mottram ® claims that a marked degree of fatty infiltration is evident in fasting and watered rabbits, and guinea pigs in from twenty-four to forty-eight hours. He states that this change is GROUP B Killed pete lor Died Macroscopic Sud. ; Microscopic 4 | Killed |Pale, otherwise normal Cells slightly swollen, more granular Normal Slight vacuolation, with 7,” objective Fair vacuolation Cells quite vacuolated for fat globules Congested, otherwise Cells granular, moderately vac- normal uolated Very pale Cells granular, moderately vac- uolated Normal Cells granular, slightly vacuolated i\Congested, coccidiosis Congestion coccidiosis, cells normal Congestion, coccidiosis, cells normal microscopically visible, and uses the oil immersion for its demon- stration. In conclusion the following suggestions may be offered: 1. Fasting, unwatered rabbits, from four days and upwards, show a decided fatty infiltration of the liver, apparent in gross and microscopically. 2. Fasting, watered rabbits, from ten days and upwards, may 6 STATKEWITCH: Archiv fiir experimentelle Pathologie, 1894, page xxxiil. 314 M. R. Smirnow show similar changes in the liver, but the percentage of incidence is very low, as compared with that of the unwatered animals. 3. In half the number of the fasting, watered rabbits under observation, microscopic vacuolation was observed. This vacuo- lation may be interpreted as a fatty change, but the picture is by no means comparable to that seen in the non-watered animals. GROUP C | Killed | \ gies a NMaseescans or Died Tacroscopic Sud Microscopic Died |Congested, extreme Cells normal, congestion coccidiosis |Congested, extreme coccidiosis Congested, extreme coccidiosis Congested, extreme coccidiosis Congested, extreme Cells smaller, more granular,) coccidiosis occasional vacuole | Pale color, otherwise . +|Moderate fatty infiltration ‘normal ‘Normal \Cells swollen and_ granular, slight vacuolation ON THE INFLUENCE OF MUSCULAR EXERCISE ON THE ACTIVITY OF BULBAR CENTRES By. E. G. MARTIN anp C. M. GRUBER [From the Laboratory of Physiology in the Harvard Medical School] USCULAR exercise is accompanied by certain very definite adaptive changes in the circulation and in respiration. That there is an increased heart rate is a matter of common experience. The increase has been studied in detail by Hering, Bowen,” Cook and Pembrey,’ and others.’ An increase in arterial pressure has been demonstrated by Zuntz and Tangl’° on dogs working in a tread-mill, and by MacCurdy, Bowen, Cook and Pembrey, and others on men.® An increase in the rate and depth of respiration is a familiar accompaniment of muscular exertion. This has been studied in great detail by Geppert and Zuntz.’ The mechanism of these adaptations remains obscure. Vari- ous factors may be involved in bringing them about, and the task of determining which of the several possible factors are actually responsible is by no means easy. In each of the adaptations we have to do with a bodily function governed by bulbar centres. These centres have been shown to be susceptible to influences reaching them either by way of the blood stream or over afferent nerves. Our first task is to decide, if possible, for each of the 1 HERING: Archiv fiir die gesammte Physiologie, 1895, lx, p. 483. 2? BowEN: Contributions to Medical Research, Ann Harbor, 1903, p. 462. § Cook and PEMBREY: Journal of physiology, xlv, p. 1. 4Lows.Ley: This journal, 1911, xxvii, p. 446. 5Zuntz and TANGL: Archiv fiir die gesammte Physiologie, 1898, lxx, Pp. 544. 6 For references see Lowsley: Joc. cit., p. 446. 7 GEPPERT and Zuntz: Archiv fiir die gesammte Physiologie, 1888, xlii, p. 189. 316 | E. G. Martin and C. M. Gruber adaptive changes, the relative importance of the two great channels of influence. Hering,® Hunt,’ and Bowen! have concluded that so far as cardiac acceleration is concerned the adaptation is medi- ated chiefly through the nervous system. Johannson" agrees with their view as to the mechanism of immediate acceleration, but offers evidence that the persistent increase in heart rate fol- lowing exercise depends on stimuli conveyed to the bulb by the blood. Johannson’s view has recently been supported by Mans- feld,’ to the extent of making the blood an important agent in the persistent acceleration of exercise, although the latter author differs from Johannson as to the details of the mechanism. When arterial pressure is considered we deal with a function depending only in part upon the activity of special bulbar centres, changes in heart rate and mechanical effects of muscular move- ment tending likewise to modify it. We cannot, therefore, draw direct conclusions as to the influence of exercise upon these centres from simple observations of blood-pressure changes. Bowen,” in fact, interprets the rise of blood pressure accompanying exer- cise on a basis wholly exclusive of vasomotor influences. Evidence that the vasoconstrictor centre is affected positively by exercise seems to be lacking, although Hooker '* postulates a compensatory splanchnic vasoconstriction in accounting for the rise of venous pressure observed by him. There is, moreover, direct evidence of vasodilation within the active muscles,!® perhaps dependent on activity of the vasodilator centre; and of cutaneous vasodilation in the later stages. of prolonged exercise,!® indicating a depres- sion of the constrictor centre. Whether the mechanism for bring- ing about vasodilation in active muscles operates through nervous influences upon the vasodilator centre, or in some other way, has 8 HERING: loc. cit., p. 483. * Hunt: This journal, 1899, ii, p. 464. 10 BOWEN: loc. cit., p. 462. 1 JOHANNSON: Skandinavische Archiv fiir Physiologie, 1895, v, p. 20. 2 MANSFIELD: Archiv fiir die gesammte Physiologie, 1910, cxxxiv, p. 508. 8 BOWEN: This journal, 1904, xi, p. 60. 14 HOOKER: This journal, ro11, xxviii, p. 235. 1 KAUFMANN and CHAUvVEAU: Archives de physiologie normale et patho- logique, 1892, p. 283. 16 BOWEN: Joc. cit., p. 60. Muscular Exercise on the Activity of Bulbar Centres 317 not yet been demonstrated. Masing ?’ has shown that the cuta- neous vasodilation occurring in prolonged exercise appears only when there is sweat secretion, suggesting a common, perhaps non- nervous, cause for the two phenomena. The increased activity of the respiratory centre in exercise would seem, from the work of Geppert and Zuntz,!8 to be wholly explicable upon a non-nervous basis, as due to the presence of metabolites in the circulating blood. Recapitulating the evidence thus far cited we note that the bulbar centres controlling the heart rate are influenced nervously during exercise so as to cause acceleration; that direct evidence for nervous action upon the vasoconstrictor and vasodilator centres is wanting, and the indirect evidence indicates no very striking influence; and that the respiratory centre is apparently unaffected during exercise by nervous influences. So meagre a play of nerve impulses upon the medulla as here indicated seems strange when we consider on the one hand the demonstrated great susceptibility of the bulbar centres to afferent impulses in general, and on the other the great volume of nervous activity called into play during muscular exercise. Such nervous influence upon the medulla as does accompany muscular exercise may possibly be of two sorts, associated innerva- tion from the motor cortex, or reflex from excitation of organs of muscle sense in the active muscles. The possibility of associated innervation of the bulb during exercise seems to have been con- sidered thus far chiefly with reference to cardiac acceleration. Johannson !° believed that the immediate acceleration accompany- ing exercise is due chiefly to associated innervation. He based his view on the observation that experimental animals showed much more marked acceleration during voluntary struggling than during vigorous passive moments. Athanasiu and Carvallo, on the other hand, concluded from experiments on human beings suffering from paraplegia, in whom powerful but ineffective efforts toward movement brought about 17 Mastnc: Deutsches Archiv fiir klinische Medicin, 1903, lxxiv, p. 253. 18 GEPPERT and ZuNvTz: loc. cit., p. 189. 19 JOHANNSON: Joc. cit., p. 20. 20 ATHANASIU and CARVALLO: Archives de physiologie, 1898, xxx, p. 552- 318 E. G. Martin and C. M. Gruber no acceleration, that muscular exercise acts only reflexly in its effect upon the cardiac centres. They cite in support of their view the observation of Asp *! that stimulation of the central end of nerves from skeletal muscles causes cardio-acceleration. Her- ing * considered both possibilities without arriving at any con- clusion in favor of one over the other. Bowen concluded that the increased pulse rate is partly cortical in origin and partly reflex. We have undertaken the present investigation in the hope of throwing additional light upon the general problem of the reaction of the bulbar centres to muscular exercise and particularly in the attempt to determine whether or not the influence of muscular exercise is uniform in its effect upon the different centres. The suggestion which we wish to offer as the result of our work may be stated in brief as follows: The immediate effect upon the bulbar centres of muscular exercise is due in the main to associated innervation from the motor cortex. This innerva- tion acts to depress the cardio-inhibitory centre, the vasoconstrictor centre, and the respiratory centre. The Depression of the Cardio-inhibitory Centre by Associated Innervation. — That the acceleration of the heart in exercise is due to depression of the inhibitory centre, rather than to stimu- lation of the augmentor centre, was well established by Hunt *4 on the basis of the short latent period of the acceleration as com- pared with the long latent period shown when the accelerator nerves are stimulated directly. Hering’s earlier observation that the acceleration fails when the accelerator nerves are cut” is satisfactorily explained by Hunt *® as showing the necessity for constant tonic activity of the augmentor centre to make depres- sion of the inhibitory centre effective. In the attempt to decide whether this depression of the inhibi- tory centre is cortical or reflex we have to consider the conflicting *1 Asp: Ludwig’s Arbeiten, 1867, p. 182. HERING: loc. cit., p. 483. *s BOWEN: Contributions to Medical Research, Ann Arbor, 1903, p. 462. 74 Hunt: loc. cit., p. 464. 25 HERING: Joc. cit., p. 483. 26 Hunt: loc. cit., p. 464. Muscular Exercise on the Activity of Bulbar Centres 319 evidence of Johannson and of Athanasiu and Carvallo already cited. The position taken by these latter investigators seems to us to be not justified by their evidence. Powerful efforts toward move- ment on the part of paraplegic individuals do not necessarily result in a flow of impulses as far as the bulb, and in the absence of positive proof that impulses do reach the bulb, the experiment does not invalidate positive evidence on the other side. We have attacked the problem of associated innervation vs. muscle reflexes, as accounting for the cardio-acceleration of exer- cise, in three different ways. Our first experiment was a repetition of Johannson’s *’ observation on the influence of passive move- ments on heart rate. To avoid possible complications from the cortex we performed the experiment on a decerebrate cat. The form of exercise used was vigorous passive flexion and extension of both hind lmbs, continued for about thirty seconds. We obtained acceleration of the heart in four of eight periods of exer- cise. The acceleration did not exceed 14 per cent in any case, and did not appear until after the exercise had been in progress at least five seconds. This latter observation we consider significant in view of the great promptness with which acceleration occurs in ordinary voluntary activity. ”% Although passive movements of the joints give rise, undoubtedly, to considerable streams of afferent impulses, the objection may be offered that the impulses generated by passive movements are not necessarily equivalent to those aroused: in the muscles during active contraction. Our second series of experiments was designed to overcome this possible objection. In these experiments we ob- tained vigorous active movements in two decerebrate cats by the use of strychnine. Our strychninized cats showed a rapid heart rate, ranging between 35 and 4o beats in ten seconds, but not by any means a maximal rate for the cat’s heart; we have repeated observations of rates exceeding 44 beats in ten seconds. In twelve observations of the effect of strychnine convulsions on the heart rate we got acceleration in only three cases; not exceeding in any of them 9g per cent, and coming on more than ten seconds after the beginning of the convulsions. 27 JOHANNSON: Joc. cit., p. 20. 2 See BOWEN: loc. cit., p. 462. 320 E. G. Martin and C. M. Gruber : . Our third series of experiments was a repetition of Johannson’s original ones, except that we used human beings as subjects. The procedure was as follows: The subject lay flat on his back with legs extended. At intervals of one minute the pulse was counted for twenty seconds with a stop-watch. The subjects in these tests had been having their pulse counted regularly for several weeks, and were, therefore, presumably free from disturbing psychic re- actions. After four or five minutes of preliminary pulse-counting the subject flexed his legs forcibly at the hips a designated number of times, leaving them extended again at the end of the exercise. The pulse was counted for twenty seconds beginning within two seconds after the body came to rest, and at minute intervals there- after. For the passive exercise the subject’s feet were grasped by an assistant and the legs alternately flexed and extended as vigor- ously as possible. The results obtained were as follows: Subject G. had for five minutes a pulse rate not exceeding 24 beats in twenty seconds. He flexed his legs four times; in the succeeding twenty seconds there were 26.5 beats. Three minutes later the rate was 23.5; two leg movements raised it to 26. Two minutes later, with the rate at 22, a single flexion of the leg brought about a rate of 25 in twenty seconds. Four minutes after this last read- ing the rate was 23.5; the legs were flexed passively one hundred times; the rate immediately afterward was 22.5. Two minutes later, with the heart rate at 22, the passive movements were re- peated. The rate rose to 23. Two minutes after this last reading the rate had fallen to 21.5. A single active flexion of one leg raised the rate to 24. A second subject, M., showed precisely simi- lar results. Prolonged passive exercise brought about no significant increase in heart rate, while one to four active leg flexions increased the rate three to four beats in twenty seconds. The striking features of these experiments on human subjects were the marked acceleration resulting from very moderate amounts of active exercise, and the total failure of acceleration from vigorous passive exercise. Unless we deny absolutely the possibility that effective afferent impulses may be generated by passive movements, we must admit that these experiments point strongly toward associated innervation as the chief, if not the only, cause for the immediate acceleration of exercise. Our observa- Muscular Exercise on the Activity of Bulbar Centres 321 tions on decerebrate animals seem to us to point the same way, since neither vigorous passive movements nor the violent convul- sions of strychnine brought about increases in rate at all com- parable, either in amount, in promptitude, or in uniformity of occurrence, with the increases observed by Johannson and by our- selves in consciously active organisms. An argument apparently in favor of the reflex source of the acceleration is that afforded by the well-known effect of posture on the heart rate, the erect posture being accompanied by a more rapid heart than is the recumbent. That this change of rate is not dependent on the increased muscular effort involved in main- taining the erect posture, but is due to the increased flow of blood to the lower parts of the body under gravity, was shown by Erlan- ger and Hooker.”® In corroboration of their conclusion we can report the observation that even so marked a heightening of postural tonus as appears in decerebrate rigidity is without marked effect on heart rate. In two experiments on cats in which we compared the heart rate before decerebration with the rate after decerebration we had average rates of 18 and 15 beats in five seconds before, and of 16 and 15 in five seconds after the opera- tion, and after rigidity had manifested itself. The Response of the Vasoconstrictor Centre to Muscular Exer- cise. — The rise in blood pressure which accompanies muscular exercise is to be explained, as already noted, as due to mechanical effects of the exercise, together with the augmented heart beat. Whether direct nervous or chemical influences dependent upon muscular activity exert any effect upon the vasomotor centre has not been certainly determined. A fact noted by Lowsley * sug- gests that the metabolites poured out into the blood during exer- cise may depress the vasoconstrictor centre, as they were supposed by Johannson to depress the cardio-inhibitory centre. Lowsley observed that shortly after the cessation of activity blood pressure falls to a point lower than that obtaining before the exercise began. Since this lowered blood pressure cannot be referred to a diminished heart beat it signifies depression of vasomotor tone. The explana- 29 ERLANGER and Hooker: Johns Hopkins Hospital Reports, 1904, xii, P. 332. 30 LOWSLEY: Joc. cit., p. 451. 322 E. G. Martin and C. M. Gruber tion proposed by Lowsley,*' that this lowered blood pressure is due to fatigue of the centre following its great activity during the period of exercise, does not commend itself, in view of the prob- ability that there is, as a matter of fact, little such activity. The observation of Bowen, already cited,” of cutaneous vasodilation during later stages of prolonged exertion, counts against the notion that the vasomotor centre is active during exercise, and may be looked upon, perhaps, as additional evidence pointing toward a depressor function for fatigue products. While we have in metabolites carried by the blood a probably adequate mechanism for the vasomotor effects which follow exer- cise, these are too slow in operation to explain any reactions of the vasomotor centre that may occur at the outset of activity. If any such are normal accompaniments of exercise they are due to the operation of one or both the nervous mechanisms already noted as possible agents in bringing about bulbar responses, namely associated innervation, and muscle-sense reflexes. A procedure which might be indicative of the existence of nervous influences affecting the vasoconstrictor centre during exer- cise would be stimulation of the motor cortex. Vasomotor re- sponses to such stimulation might be supposed to represent the normal results of associated innervation during voluntary muscular activity. The earlier investigators who studied the effects of cortical stimulation on blood pressure obtained contradictory results.** Usually vasoconstriction with rise of blood pressure was observed, but in a number of cases a fall of pressure occurred instead. These observations were made upon curarized animals. Howell and Austin,** repeating the experiment, found that the effect varied with the anesthetic used. They obtained with dogs rise of pressure uniformly when morphia and curare were used, and fall of pressure when morphia and ether were used. We stimu- lated the motor cortex in several cats, using ether and morphia, and ether alone, and obtained uniformly a fall of carotid pressure. 31 LOWSLEY: loc. cit., p. 465. ® BowEN: This. journal, 1904, xi, p. 69. 8 For early literature see TIGERSTEDT: Physiologie des Kreislaufes, Leip- zig, 1893, p. 536. 34 HOWELL and AustTIN: This journal, 1900, p. xx. Muscular Exercise on the Activity of Bulbar Centres 323 The percentage drop varied from 16.7 to 35, averaging in fourteen observations 23.7. That this drop was due to vasodilation and not to diminished heart action is shown by the fact that in all but two of more than twenty-five observations the heart was slightly accelerated during the period of falling pressure. To determine whether the dilation was the result of depression of the constrictor centre or stimulation of the dilator mechanism we clamped the abdominal aorta, below the renal arteries, and also both axillary arteries, thus shutting the extremities out of the circulation. Repe- tition of the cortical stimulation gave a fall of carotid pressure as before, and the percentage change equalled that of our previous experiments. In another cat, whose splanchnic nerves had been cut sometime previously, in connection with another research, we stimulated the motor cortex repeatedly, recording blood pressure throughout. A slight drop in pressure accompanied each stimu- lation, not exceeding in any case eleven per cent, whereas in ani- mals with intact splanchnics the least drop observed exceeded sixteen per cent, and the average was above twenty-three per cent. Since these procedures show the splanchnic area to be predominant as the seat of pressure changes, and since dilators to the splanchnic area have not been conclusively demonstrated, the evidence for splanchnic vasodilators depending at present solely on the observa- tions of Dale,*° we interpret our results as indicating an associated innervation from the motor cortex, depressor to the vasomotor centre. A criticism which might be urged against this conclusion is that we have accepted the results of cortical stimulation with ether anesthesia, and rejected contrary results obtained with cura- re-morphia anesthesia, because the former fit our theory and the latter do not. In reply to such a criticism we would state that our laboratory experience with ether and with curare, together with some observations to be published in due time, indicate that in ether anesthesia the behavior of reflex mechanisms corresponds in kind, although not in degree, with their behavior in decerebrate unanesthetized animals, whereas under curare the responses are often different in kind as well as in degree. The very fact that diametrically opposite results are obtained from cortical stimula- tion under these two drugs shows that one or both of them brings 3 Date: Journal of physiology, 1913, xlvi, p. 291. 324 E. G. Martin and C. M. Gruber about profound modifications in the nervous mechanisms involved. In our opinion curare probably has this effect, and for that reason we are inclined to question the soundness of many observations on vasomotor reactions in which curare was employed. As a means of determining whether a definite immediate effect of exercise on the vasomotor sys- tem can be demonstrated in ani- mals in which cortical influences have been excluded, we made a number of observations on decere- brate cats. One method of in- ducing vigorous activity in these animals was by the use of strych- nine. Decerebrate cats dosed with strychnine (.3 mg. in 3 c.c. saline) show typical convulsions. The blood-pressure changes observed during these convulsions were in some of our experiments such as to suggest more than mere me- chanical effects from the strongly contracted muscles.