(3x mm
wammimis
THE
UNIVERSITY OF
ALBERTA
RELEASE FORM
NAME OF AUTHOR . Mary- Jane Turtle
TITLE OF THESIS . Effect of Urophysectomy and Pre¬
optic Nucleus Lesioning on Ionic
and Osmotic Regulation in the
Goldfish (Carassius auratus)
DEGREE FOR WHICH THESIS WAS PRESENTED . M.Sc.
YEAR THIS DEGREE GRANTED . 1974
Permission is hereby granted to THE
UNIVERSITY OF ALBERTA LIBRARY to reproduce single
copies of this thesis and to lend or sell such
copies for private, scholarly or scientific re¬
search purposes only.
The author reserves other publication
rights, and neither the thesis nor extensive ex¬
tracts from it may be printed or otherwise repro¬
duced without the author's written permission.
THE UNIVERSITY OF ALBERTA
EFFECT OF UROPHYSECTOMY AND PREOPTIC NUCLEUS
LESIONING ON IONIC AND OSMOTIC REGULATION IN
THE GOLDFISH (Carassius auratus )
by
MARY- -JAILS TURTLE
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES AND RESEARCH
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE
OF MASTER OF SCIENCE
DEPARTMENT OF ZOOLOGY
FALL , 1974
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■
THE UNIVERSITY OF ALBERTA
FACULTY OF GRADUATE STUDIES AND RESEARCH
The undersigned certify that they have read, and
recommend to the Faculty of Graduate Studies and Research
for acceptance, a thesis entitled "Effect of Urophysectomy
and Preoptic Nucleus Lesioning on Ionic and Osmotic Regu¬
lation in the Goldfish (Caras si us auratus ) submitted by
Mary-Jane Turtle in partial fulfilment of the requirements
for the degree of Master of Science.
ABSTRACT
The effects of urophysectomy and preoptic
nucleus lesioning on the osmotic and ionic regulation
of Caras sius auratus L. were investigated.
Goldfish were maintained at 20 °C and ambient
photoperi.od throughout the study. An experimental group
consisted of urophysectomized , preoptic nucleus lesioned
or combined preoptic nucleus lesion/urophysectomized fish
and their respective sham-operated and intact control fish
Blood samples were taken at postoperative time
periods of five, ten and twenty days and plasma Na+, Cl
++ + —
and Ca concentrations determined. Urine Na , Cl and
++
Ca concentration and urine flow were measured at five
and ten day postoperative recovery times on preoptic nu¬
cleus lesioned and urophysectomized fish only.
Urophysectomy caused a reduction in plasma Na+
concentration and urine flow at five days postoperatively .
Urine Na , Ca and Cl excretion rates were reduced at
five days following urophysectomy as a result of the reduc
tion in urine flow in these fish. Plasma Na+ and urine
flow had returned to near normal control values by ten
days . Urophysectomy did not alter plasma Ca or Cl le-
•f •f'f — “ ,
vels, or urine osmolality, Na , Ca , Cl concentrations
iv
.
.
B
*
at any of the postoperative times.
Lesioning of the preoptic nucleus caused reduc¬
tion in plasma Na+ concentration at five and ten days post-
. ++ —
lesion. Plasma Ca and Cl were not changed at any post¬
operative time period. Urine Na+, Ca"1 + and Cl” levels
were increased at both the five and ten day sampling
periods , whereas urine flow was decreased at both of these
sampling times.
The reduction in urine flow compensated for the
+ ++ —
increase in urine Na , Ca and Cl concentrations, hence
electrolyte excretion was not altered in the five day le-
sioned fish. However, at the ten day postoperative time
period the electrolyte excretion rates were increased. In¬
completely lesioned fish did not show a reduction in plas¬
ma. Na+ level at any postoperative sampling time.
Simultaneous removal of the preoptic nucleus
•j-
and the urophysis resulted in a decrease in plasma Na
level equivalent to that produced by either operation
alone. Plasma C.a++ and Cl” concentrations were not changed
by the combined operation.
The results indicate that both the urophysial
and the neurohypophysial peptides have a diuretic effect
on the teleost kidney. This diuresis is most likely due
In addition, there appears to be an in-
v
to changes in GFR.^
.
d
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crease in tubular reabsorption of water in the preoptic
nucleus lesioned fish. Plasma Na+ balance is also affected
by both the urophysis and the neurohypophysis, as removal
of either of these glands caused hyponatria. However, the
mechanisms by which this hyponatremia was produced was
different between urophysectomy and preoptic nucleus le-
sioning. In the urophysectomized fish, the gill was pro¬
bably the major site of Na+ loss, while in the preoptic
nucleus lesioned fish, the kidney probably played a more
-j-
important role in Na loss.
vi
.
ox^Cfoasq add rr Jr oLtlti* s aaol to $^ia 'tc t*#i sift ^Idad
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to the
following :
Dr. R. E. Peter, for his support and supervision
throughout this study, for his time spent in performing
the stereotaxic lesion, and for his assistance in the
preparation of the manuscript;
Dr. W. C. Mackay, for his guidance and for his
critical review of the manuscript;
Dr. R. J. Christopherson , for his review of the
manuscript ;
Dr. A. Neil Cuthbertson, for his assistance in
the development of a technique for urophysectomizing fish;
V. E. Gill, for her help in sectioning the brain
tissue ;
My fellow graduate students who provided many
stimulating ideas and moral support;
The University of Alberta and the Department of
Zoology who provided financial support in the form of
graduate teaching assistantships and intersession bursuries.
• •
Vll
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TABLE OF CONTENTS
Page
ABSTRACT . iv
ACKNOWLEDGEMENTS . vii
LIST OF FIGURES . X
INTRODUCTION . 1
MATERIAL AND METHODS . . 7
Care of Study Animals . 7
Experimental Protocol . 3
Operative Procedures . 3
Anesthesia . . 3
Urophysectomy . . . 9
Preoptic Nucleus Lesion . 13
Preoptic Nucleus Lesion/Urophysec-
tomy . 13
Sampling Techniques . j.8
Plasma Electrolyte Study . j.8
Renal Study . 19
Analytical Procedures . 27
Statistical Tests . 28
RESULTS . 29
Urophysectomy . 29
Plasma Electrolyte Levels . 29
Renal Study . 33
Preoptic Nucleus Lesion . 53
Plasma Electrolyte Levels . 53
Renal Study . 5 2.
Preoptic Nucleus Lesion/Urophysectomy . . 79
Plasma Electrolyte Levels . 79
DISCUSSION . 88
Urophysectomy . . .
viii
TABLE OF CONTENTS (continued)
Page
Preoptic Nucleus Lesioning . . . . . 95
Preoptic Nucleus Lesion/Urophysectomy . 106
General Discussion . . 108
LITERATURE CITED . 116
APPENDIX . . . 125
ix
LIST OF FIGURES
FIGURE Page
la A dissection of the peduncle region of
the goldfish . 11
lb The peduncle region of the goldfish after
removal of the urophysis . 11
2 Cross section through the mid nucleus
preopticus (NPO) region of a control
animal . 15
3 Cross section through the mid nucleus
preopticus region of a partially (incom¬
pletely) lesioned goldfish . 15
4 Cross section through the mid nucleus pre¬
opticus region of a five day completely
lesioned goldfish . 17
5 Cross section through the mid nucleus pre¬
opticus region of a ten day completely
lesioned goldfish . 17
6 Cross section through the mid nucleus pre¬
opticus of a twenty day completely lesioned
goldfish . 17
7 The plexiglass container used to hold the
goldfish while inserting the catheter ... 21
8 Urinary system of the goldfish . 24
9 Experimental chamber used to contain the
goldfish during urine collection . 26
x
.
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■
10
11
12
13
14
15
16
17
18
19
20
21
22
Page
4.
Effect of urophysectomy on plasma Na
concentration .
Effect of urophysectomy on plasma Cl
concentration .
++
Effect of urophysectomy on plasma Ca
concentration .
Effect of urophysectomy on urine flow rate 38
Effect of urophysectomy on urine osmo¬
lality . 41
Effect of urophysectomy on Na+ concentra¬
tion . 43
Effect of urophysectomy on the Na+ excre¬
tion rate of the goldfish . 46
Effect of urophysectomy on urine Cl con¬
centration . 4 8
Effect of urophysectomy on the Cl excre¬
tion rate of the goldfish . 51
++
Effect of urophysectomy on urine Ca con¬
centration . 53
Effect of urophysectomy on the Ca++ excre¬
tion rate of the goldfish . 55
Effect of lesioning the NPO of the gold¬
fish on plasma Na+ concentration . 58
Effect of lesioning the NPO of the gold¬
fish on plasma Cl- concentration .
xi
61
24
25
26
27
28
29
30
31
32
33
34
63
66
69
71
73
76
78
81
83
86
89
92
Effect of lesioning the NPO of the gold¬
fish on plasma Ca++ concentration .
Effect of lesioning the NPO of goldfish on
urine flow . . .
Effect of lesioning the NPO of goldfish
on the urine osmolality . . .
Effect of lesioning the NPO of the gold¬
fish on urine Na+ concentration .
Effect of lesioning the NPO of the gold¬
fish on urine Na+ excretion rate
Effect of lesioning the NPO of the gold¬
fish on urine Cl” concentration .
Effect of lesioning the NPO of the gold¬
fish on Cl" excretion rate .
Effect of lesioning the NPO of the gold¬
fish on Ca++ concentration .
Effect of NPO lesioning of the goldfish
on Ca'H" excretion rate .
Effect of the combined operation of NPO
lesioning and urophysectomy on plasma Na+
concentration .
Effect of the combined operation of NPO
lesioning and urophysectomy on plasma Cl"
concentration .
Effect of the combined operation of NPO
lesioning and urophysectomy on the plasma
Ca+_,‘ concentration .
xii
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INTRODUCTION
The relationship of the caudal neurosectory cells
(the Dahlgren cells) and the urophysis (the neurohemal
organ) forming a neurosecretory system was first des¬
cribed by Enami (1955) . Since this time a caudal neuro¬
secretory system has been described in all teleosts,
elasmobranchs , holosteans and chondrosteans investigated
(Fridberg, 1962; Fridberg and Bern, 1968; Bern, 1969)
and it has been the subject of numerous histological,
morphological and pharmacological studies (see Bern et
al. , 1967 ; Bern, 1967; Bern, 1969 ; Lederis et_ 'al. , 1970;
Berlind , 1973) .
There is considerable evidence that the urophysis
plays a role in the hydromineral balance of teleost fish
(see review Bern, 1969; Berlind, 1973). Although urophy-
sial extracts have been shown to produce diuresis, in¬
creases in glomerular filtration rate, changes in branchial
Na+ flux and elevation in blood pressure in various species
of teleosts (Maetz et al. , 1964; Bern et al. , 1967; Chan et
al. , 1969 ; and Chester Jones et al_ . , 1967 , 1969b); uro-
physectomy has failed to produce any effects on osmoregu¬
lation (Takasugi and Bern, 1962; Chester Jones et. al. ,
1969; Berlind, 1973). Thus, the emphasis of previous in-
1
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2
vestigations has been on the chemical identities and the
pharmacological properties of the urophysi al factors
(Lederis , 1973). Their physiological activities have,
in the past, been largely ignored (Berlind, 1973).
The lack of a common reference preparation has
caused confusion in regard to the physiological role and
the number and nature of active principles which may occur
in the urophysis (Bern and Lederis, 1969). To date, at
least four urophysi al factors (Urotensins) have been
identified (Berlind, 1973) . Urotensin I consists of two
rat hypotensive components which have long and short term
effects and are separable by chromotography (Zelnik and
Lederis, 1973). Preliminary observations suggest that
in eels Urotensin I has pressor activity similar to but
not as potent as Urotensin II, and that it also causes a
decrease in glomerular filtration rate (Lederis, 1973).
Urotensin II causes an elevation in eel blood pressure
(Chan et al. , 1969; Zelnik and Lederis, 1973) and increases
the frequency of contractions of trout bladder, mudsucker
intestine, guppy oviduct (Lederis, 1970a, b, c) and the
mudsucker sperm duct (Berlind, 1972). A preliminary re¬
port indicates that Urotensin II also causes an increase
in glomerular filtration rate in eels (Lederis, 1973).
Urotensin III stimulates branchial Na+ influx in goldfish
(Maetz et al. , 1964a). Urotensin IV, the hydrosmotic fac¬
tor, has been demonstrated by Lacanilao (1972a, b) to
■
.
probably be arginine vasotocin (AVT) .
3
In teleosts there are two octapeptides in the
neural lobe of the pituitary, AVT and isotocin (4 Ser -
8 lie - oxytocin) (Heller and Pickering, 1961; Sawyer,
1966; Perks, 1969). The cell bodies of the secretory
neurons whose axons form the neurohypophysis are located
in the nucleus preopt.icus (NPO) situated in the hypothal-
mus on either side of the preoptic recess just posterior
to the anterior commissure (see Perks, 1969).
AVT has been shown to cause diuresis and increased
glomerular filtration rate (GFR) and increased paramino-
hippuric acid (PAH) clearance in freshwater teleosts
(Carassius auratus : Maetz , 1963; Maetz et al. , 1964b;
Lahlouh and Sawyer, 1969; Lahlouh and Giordan, 1970; Salmo
gairdneri : Holmes and McBean, 1963; Amia and Protopterus
aethiopicus : Sawyer, 1966, 1970, 1972, 1973). However,
AVT does not cause diuresis in the aglomerular kidney of
the marine teleost Opsanus tau even though it does have a
pressor effect (Lahlcuh et al. , 1969a) . Although anti¬
diuretic effects of AVT on the teleost kidney have been
reported (Salmo gairdneri , Holmes, 1961; Salvelinus namay-
cush , Hammond, 1969) most of the attention has been focused
on the strong diuretic response which follows injections of
AVT. At the gill isotocin stimulates the influx of Na+
while AVT enhances both Na+ influx and efflux in freshwater
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4
teleosts (Maetz , 1963 ; Maetz et aJL. , 1964b). In the sea¬
water adapted flounder, Platichthys flesus, oxytocin will
stimulate branchial efflux of Na+ , while in freshwater it
stimulates influx of Na+ (Motais and. Maetz, 1964). Oxyto¬
cin and isotocin will also cause increases in urine flow
and inulin clearance in teleosts (Maetz and Julien, 1961;
Maetz ejt a]L. , 1964b; Butler, 1966 ; Sawyer, 1966 ;
Chester Jones et ctL. , 1969) .
There have been no studies on the effects of
lesioning of the NPO on osmotic or ionic regulation of
Carassius auratus L. . Chan (1969) has electro-cauterized
the preoptic area in Anguilla anguilla and measured plas-
*4” -f**!* * ■
ma Na , Ca and PO^ composition. The sham operation,
however, consisted of the removal of the forebrain. The
lesioning technique was not decribed nor was the extent
of lesion reported. Thus the study cannot be regarded
as conclusive in this respect.
With the exception of the aforementioned study,
physiological studies on the role of the teleost neuro¬
hypophysis in osmotic or ionic regulation have been li¬
mited to total or partial hypophysectomy . Hypophysectomy
removes both the neurohypophysial and adenohypophysial
peptides, thus normal hormonal balance is severely dis¬
rupted. And as there are several hormonal systems, such
as the adrenocorticoids , prolactin and AVT , believed to
....
.
*
5
be involved in ionic and osmotic regulation (Ol.ivereau
and Ball, 1970) , it is difficult to ascertain which system
is causing the observed effect. Also, there is consider¬
able evidence that hypophysectomy does not cause the
NPO to cease functioning. Instead, there can be
a regeneration of the neurosecretory axons and the infun¬
dibular stalk forming a Mneurohypophysis-like" organ
(Sathyanesan , 1966, 1969; Belsare, 1970). When regenera¬
tion of the stalk is obvious the neurosecretory neurons
appear normal thus suggesting that some of the neurohypo¬
physial functions could be maintained (Sathyanesan, 1970) .
Therefore, ablation of the NPO, the source of the neuro-
hypophysical peptides, is a more desirable approach to the
study of neurohypophysial function.
Investigations of the physiological activities of
the urophysial and neurohypophysial peptides in teleosts
have largely been confined to replacement therapy and the
study of the immediate effects of such treatments. There
has been no attempt to relate changes in kidney function
with passage of time following preoptic nucleus lesioning or
urophysectomy . Although there is evidence that the urophvsis
as well as the neurohypophysis secrete AVT (see above) ,
there have been no physiological studies where the effect
of simultaneous removal of these organs has been examined.
Thus, the objectives of the present study were.
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6
firstly, to determine what effect, if any, ablation of
the preoptic nucleus (NPO) and/or urophysectomy have on
plasma and urine Na+, Ca++ and Cl concentration and on
urine flow in the freshwater, stenohaline teleost Caras-
sius auratus L. . The second objective was to determine
if there are any compensatory changes in the osmoregularity
capacity in these fishes with respect to postoperative re¬
covery time.
'
MATERIALS AND METHODS
Care of Study Animals
Mature goldfish, Carassius auratus L. (common and.
comet varieties) were commercially obtained from Grassy-
forks Fisheries Co. (Martinsville, Indiana), and shipped
by air to the University of Alberta. Upon arrival, the
fish were held in dechlorinated Edmonton tapwater at 20°C
in large flow through holding tanks (15391) in the main
aquatic facilities of the Department of Zoology. The
fish were fed to excess daily with commercial fish food
(5/32 pellets, Silver Cup Fish Feed, Ferguson Feeds Ltd.,
Drinkwater, Saskatchewan).
Two weeks prior to experimentation randomly se¬
lected fish were moved to a private research room where
they were divided into groups and placed into smaller
(1361) continuous-flow holding tanks at 20°C. Operated .
fish and their respective sham and intact controls were
kept in the same holding tank. The light regime followed
the ambient photoperiod throughout the study. The fish
were fed to satiation twice daily. However, fish used in
the renal excretion studies were not fed while cathe-
terized. Postoperative maintenance was as described for
7
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8
preoperative care.
Experimental Protocol
Three major experimental groups were established?
urophysectomized fish, preoptic nucleus lesioned fish and
the combined operation of preoptic nucleus lesion/urophy-
sectomy. The above experimental groups were each used
for the plasma composition and renal excretion studies,
with the exception that the combined preoptic nucleus le-
sion/urophysectomized fish were used in only the plasma
electrolyte study. Plasma composition was measured at
the postoperative time periods of five, ten and twenty
days, while renal excretion was measured at only five and
ten days postoperatively . Included in each experimental
group were sham-operated and intact control fish.
OPERATIVE PROCEDURES
Anesthesia
Prior to operative procedures, the fish were anes¬
thetized by immersion in a 0.1 percent solution of tricaine
methanesulphonate (Kent Laboratories Ltd., Vancouver, B. C.)
in dechlorinated tapwater. Anesthesia was to the point
where the fish had lost righting ability and opercular
movement was barely detectable. Following anesthetization
the fish were weighed, wrapped in damp paper towelling to
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9
prevent desiccation and marked by fin clipping. The
operation was then performed (see below). In most cases,
the fish recovered immediately from the anesthetic when
placed in their holding tanks following the operative
procedures. Fish that did not immediately recover were
revived by perfusing the gills with oxygenated water. It
was necessary during the urophysectomy and the double
operation (urophysectomy and preoptic nucleus lesion) to
alternately perfuse the gills with a 0.033 percent solution
of tricaine methanesulphonate and oxygenated dechlorinated
tapwater to maintain anesthesia.
Ur ophy sec t omy
In the goldfish, the urophysis is located in a
depression on the dorsal surface of the urostyle, the
last caudal vertebra, and is surrounded by bone on
three sides. The entire urostyle is heavily covered with
connective tissue and its ventral surface lies adjacent
to the caudal circulatory system (Figure la) . To accom¬
plish urophysectomy, as described below, it was necessary
to dissect out the entire urostyle containing the urophysis
and filament terminale (Figure lb) .
An anesthetized fish with only the tail region ex¬
posed was placed on its left side on moist paper towelling.
The initial incision was made with a sterile no. 15 scal¬
pel blade about 3 mm dorsal to the lateral line in the
‘
'
FIGURE 1A
A dissection of the peduncle region of
the goldfish showing the urophysis (UH)
in the last vertebral element, the uro-
style (US) . The spinal cord (SC) was
cut at the level of the second last
vertebral disc. The caudal circulation
(CC) was left intact.
FIGURE IB The peduncle region of the goldfish af¬
ter removal of the urophysis. The
dotted line bisects the last vertebral
disc, and outlines the tissue that was
removed during urophysectomy .
11
1b
12
caudal peduncle area. This longitudinal incision was from
2.0 to 2.5 cm in length in fish over 100 grams and approxi¬
mately 1.5 cm in length in fish between 45-100 grams. The in¬
cision was made 3-5 mm in depth in order to expose the
lateral surface of the caudal vertebrae. The wound was
held open with four sterile stainless steel insect pins
bent to form detractors. The connective tissue was
scraped away from all facets of the urostyle and the last
vertebral disc with the edge of the scalpel blade, expos¬
ing the urostyle in its entireity. The spinal cord was
then cut at the level of the second last vertebral disc.
A pentagonal section was made around the urostyle with the
base of the pentagon bissecting the last vertebral disc.
The urostyle with the urophysis intact and the spinal cord
fragment were then carefully removed with forceps.
The wound was stitched using a h inch half-circle
reverse cutting atraumatic needle with an attached 5-0 silk
suture (Opthalmic suture, Davis and Geek, Division of Ameri¬
can Cyanamid Company, Danbury, Connecticut). Two hemosta¬
tic stitches were made to reduce hemorrhage and three to
four skin stitches were used to finish closing the wound.
Barring complications, such as excessive bleeding, the time
required to complete the operation was 12 to 15 minutes.
Fish used in the plasma composition study were between 45-
75 grams while those used in the renal study were between
.
! i
n^. ;
.
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13
110-165 grams.
The same procedure was followed for the sham opera¬
tion. However, in this case, neither the spinal cord nor
the vertebral disc were severed, nor was the urophysis
(urostyle) removed.
Preoptic Nucleus Lesion
The operative procedures for electrolytically lesion-
ing the NPO were as described by Peter (1970) , and as modi¬
fied by Peter and Gill (1974) . The direct current anodal
lesions were made by passing 1 mA of current for 20 se¬
conds. The electrodes were no. 00 stainless steel insect
pins insulated with Insl-X (Insl-X Products Corp. , Yonkers,
New York) , as described by Peter (1970) . The coordinates
for electrode placement were +0.9,M,D 2.0 (Peter and Gill,
1974) .
Sterile gut (Davis and Geek) was used to seal the
skull cap on the ten and twenty day experimental animals ,
while silk suture (3-0) was used on the five day experimen¬
tal fish. Fish used in the plasma composition studies
were between 35 and 60 grams, while those used for the
renal study were larger, between 60 and 90 grams, due to
the problem of catheterizing the urinary duct of the
smaller fish. The sham-operated animals underwent the
same surgical procedures , with the exception that no
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14
FIGURE 2.
Cross section through the mid nucleus
preopticus (NPO) region of a control
animal. The NPO cells are intensely
stained with paraldehyde fuscliin.
Nucleus endopeduncularis , NE ; optic
tract, OT; preoptic recess of the III
ventricle, PR; telencephalon, T.
Cross section through the mid nucleus
preopticus region of a partially (in¬
completely) lesioned goldfish. There
is one stainable neurosecretory cell
stainable for neurosection remaining
in the section (arrow) .
FIGURE 3.
15
200u
3
16
FIGURE 4.
Cross section through the mid nucleus pre¬
opticus region of a five day completely
lesioned goldfish. The lesioned area is
outlined with arrows. No stainable neuro¬
secretory cells remain.
FIGURE 5. Cross section through the mid nucleus pre¬
opticus region of a ten day completely
lesioned goldfish. The lesioned area is
outlined with arrows. No stainable neuro¬
secretory cells remain.
Cross section through the mid nucleus pre¬
opticus region of a twenty day completely
lesioned goldfish. The lesioned area is
outlined with arrows. No stainable neuro¬
secretory cells remain.
FIGURE 6.
17
6
18
current was passed through the electrode. To determine
whether the lesions were complete serial sections of 8 pm
were made of the forebrain of each experimental fish
(Figures 2, 3, 4, 5, and 6). The sections were stained
with paraldehyde fuchsin and counterstained with fuchsin,
ponceau xylidine and fast green. A fish was regarded as
partially lesioned (incompletely lesioned) if one or more
stainable neurosecretory cells remained in the preoptic
area.
Preoptic Nucleus Lesion/Urophysectomy
In the joint operation of preoptic nucleus lesion
and urophysectomy , the same surgical procedures were
followed as described for the single operation. The fish
used in this experiment were between 45 to 75 grams. The
sham-operated fish were treated in the same manner as the
previously described sham groups.
SAMPLING TECHNIQUES
Plasma Electrolyte Study
The operated fish (urophysectomized , preoptic
nucleus lesioned or urophysectomized and preoptic nucleus
lesioned) and their respective sham-operated and intact
controls were sampled at five, ten and twenty days post-
operatively . Individual fish were removed from the hold—
06
.
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■
ing tank with minimum disturbance to the other fish in the
tank, wrapped in paper towelling and weighed. A blood
sample was taken and the fish then terminated.
Blood samples of % to 1 cc were obtained by puncture
of the caudal circulation using a 1% inch, 23 guage needle
with a heparinized (ammonium heparin) 2h cc syringe, ac¬
cording to the technique of Mackay (pers. comm.). To avoid
hemolysis or coagulation the blood was immediately centri¬
fuged for two minutes. The plasma was then pipetted into
a 400 pi centrifuge tube and frozen. The samples were
stored at -15°C until analysis, at which time the plasma
"f* 4"!* •— ,
was analyzed for Na , Ca and Cl concentrations (see
Analytical Procedures) .
Renal Study
Individual fish were removed from the holding tanks
and anesthetized with tricaine methanesulphonate . The fish
were then weighed and a catheter inserted into the urinary
duct so that the tip of the catheter was in the urinary blad¬
der. The operation was carried out in a plexiglass operating
box (25 x 13 x 8 cm) that held the fish rigid while expos¬
ing only its ventral surface (tigure 7) . Urinary catheters
were fabricated from lengths of PE 50 and PE 90 tubing
(Intramedic, Clay— Adams Inc.) . A 5 cm length of PE 90 tub
moulded to the shape of the ventral body surface
ing was
-
. . {*aa
.
20
F I CURE 7.
The plexiglass operating box used to hold
the goldfish while inserting the catheter.
An anesthetized fish with its head region
wrapped in wet paper towelling was placed
on its side on the incline. The tail re¬
gion was exposed to allow for catheteri¬
zation.
21
10cm
22
of the fish by heating and the sides of the last 3 nun of one end
perforated to allow urine to entire from the entire circum¬
ference of the tubing (Figure 8) . One end of a 60 cm length
of PE 50 was inserted inside the unperforated end PE 90
collecting tube to form a tight joint fit. The catheter
was long enough to extend from the fish into the collec¬
tion cylinder. The catheter was made entirely of a 60 cm
length of PE 50 tubing when urine was collected from fish
under 100 grams.
To prevent leakage of urine after the catheter had
been inserted, a purse string suture was placed around the
posterior side of the rectum and around the opening of the
urinary duct posterior to the bladder, according to the
technique of Mackay (pers . comm.). To avoid having the
catheter pulled out, it was stitched to both the anal and
caudal fins (Figure 8) . The entire catheterization was
completed in five minutes. The fish was then transferred
to an experimental box (25 x 5 x 10 cm) (Figure 9) , where
it was allowed to recover.
To eliminate the effects of handling diuresis
(see Hunn and Willford, 1970) , urine was not collected
until twenty-four hours after catheterization and the
surrounding environment was kept as quiet as possible dur¬
ing the urine collection. Urine was then collected under
fni oil for 24 i 1 hours in an acid washed 25 cc gra—
.
'
■
FIGURE 8. Urinary system of the goldfish showing
the position and construction of the
catheter used to collect urine.
24
/: d
■
■
. .
'
FIGURE 9.
Experimental chamber used to contain the
goldfish during urine collection. The
plexiglass box was designed to allow only
limited movement so that the urinary
catheter could not be pulled out when
the fish struggled. Aerated water was
pumped to the chamber from the main
aquatic facilities supplies f where the
temperature was controlled.
26
10cm
27
ducted cylinder located 30 cm below the experimental cham¬
ber. The graduated cylinder was covered with parafilm to
further reduce evaporative loss during the collection
period. At the end of the twenty four hour collection
period, total urine volume was measured and the urine was
placed in an acid washed 10 cc plastic test tube and im¬
mediately frozen. The samples were stored at -15°C until
analysis, at which time total osmolality, Na+, Ca++ and
Cl levels were determined (see Analytical Procedures).
Three experimental boxes were available. Thus, an
intact control, a sham-operated and an operated fish were
run simultaneously.
ANALYTICAL PROCEDURES
4* 4*f
Na and Ca concentrations in urine and plasma
were determined by flame emmission on a Jarrel-Ash Flame
emmiss ion- atomic absorption Spectrophotometer (Model 82-
270 Atomsorb) using standard grade acetylene (Liquid Air
++
Canada Ltd., Edmonton, Alberta) as fuel. For Ca deter¬
minations a nitrous oxide-acetylene flame was used while
+ _
an air-acetylene flame was used for Na analysis. Cl
concentrations were measured by amperometric titrations
with silver ions usinq a Buchler - Cotlove Chlori-
dometer (Model 4-2000) . Urine osmolality was determined
by freezing point depression using a Fiske Osmometer (Model
C-66A) . The procedures outlined in the operators manual
.
,
■
■
if4i
28
for the above analytical instruments were followed.
All chemicals used throughout the study were analy¬
tical grade. Glassware was washed with sulphuric acid
saturated with potassium dichromate and then stored in
double distilled water. Duplicate determinations were
made on all samples for each ion. Electrolyte excretion
rates were calculated by multiplying urine flow by the
urine electrolyte concentration.
STATISTICAL TESTS
The Students' t~test for unpaired samples (Sokal
and Rohlof, 1969) was used to determine if there were
i
significant differences between the experimental groups.
Differences were considered to be statistically signifi¬
cant when the p value was less than 0.05.
'
■
RESULTS
Urophysectomy
Plasma Electrolyte Levels
The sham-operated and intact controls maintained
similar plasma Na+ concentrations throughout the study
(Figure 10) (Appendix, Table 1). The plasma Na+ concen¬
tration in the five day urophysectomized animals was
significantly lower than in the sham-operated and intact
control groups for that postoperative time period (Figure
10) (Appendix, Table 1). The ten and twenty day urophy¬
sectomized animals had plasma Na+ levels that were not
significantly different from their respective sham and in¬
tact controls (Figure 10) (Appendix, Table 1). The plasma
Na+ concentration was significantly lower in the five day
urophysectomized animals compared to the ten and twenty
day fish (p<0.01 and p<0.001 respectively). There was no
significant difference between the ten and twenty day
animals (Figure 10) (Appendix, Table 1).
The sham-operated animals maintained plasma Cl
levels which were not significantly different than the
intact controls at any of the postoperative times (Figure
11) (Appendix, Table 1) . The plasma Cl levels of the
29
'
.
30
■
FIGURE 10. The effect of urophysectomy on plasma
sodium concentration in goldfish at five,
ten and twenty days postoperatively . The
vertical bars represent ±SEM. The number
of individuals in each experimental group
(N) is shown at the base of each bar.
p < 0.01, comparing the means of opera¬
ted and control groups .
**
31
l/pxJUUUOpOJyJOSUOD lurnpos
Postoperative recovery time, days
32
. .
: •
.
FIGURE 11.
The effect of urophysectomy on plasma
chloride concentration in goldfish at
five, ten and twenty days postopera-
tively. The vertical bars represent
±SEM . The number of individuals in
each experimental group (N) is shown
at the base of each bar.
Postoperative recovery time, days
33
Chloride concentration
mM/l
6
~~t ~e
Fo
CJl
O CJl
o
C
“5
o
"D
ZT
*<
10
<T>
o
C+
o
3
N
<D
CL
00
IT
CD
3
I
o
T3
n>
0)
0)
CL
AA Intact control
.
34
urophysectomized fish did not differ from the sham or intact
control groups at any of the postoperative times (Figure 11)
(Appendix, Table 1). There also were no significant dif¬
ferences in plasma Cl levels between the five, ten and
twenty day urophysectomized fish.
-f-j-
Plasma Ca levels were not changed by urophysec-
tomy (Figure 12) (Appendix, Table 1). The plasma Ca++
concentrations of the sham-operated control groups were
not significantly different than the intact controls at
any of the postoperative times. The five, ten and twenty
day urophysectomized fish had plasma Ca levels that
were not significantly different from their respective
sham-operated and intact control groups , nor were there
any significant differences between these groups.
Renal Study
The effects of urophysectomy on urine flow (V)
are shown in Figure 13 (Appendix, Table 2) . There were no
significant differences between the sham-operated and the
intact control groups at either five or ten days post-
urophysectomy . There was a significant decrease in urine
flow rate in the five day urophysectomized fish compared
to the sham-operated and intact controls for that time
period. The urine flow of the ten day urophysectomized
animals did not differ significantly from the values ob—
■ta.in.ed for the sham and intact control groups. The urine
■
' ■
.
.
8J3W
.
35
■
’
i *
FIGURE 12. The effect of urophysectomv on plasma
calcium concentration in goldfish at
five, ten and twenty days postopera-
tively. The vertical bars represent
±SEM . The number of individuals in
each experimental group (N) is shown
at the base of each bar.
Intact control
36
TJ
0
-*->
03
L.
0
CL
O
i
£
OJ
_C
in
~o
0
N
£
o
u
0
</)
>.
_c
a
o
c
ZD
1/lAjlU
uoi^rji-U0DuoD uunpiOQ
Postoperative recovery time days
.
37
.
'
■ | ...
■
.
FIGURE 13. The effect of urophysectomy on the urine
flow of the goldfish at five and ten
days postoperatively . The vertical bars
represent ±SEM. The number of indivi¬
duals in each experimental group (N) is
shown at the base of each bar.
** P °*01, comparing the means of opera¬
ted and control groups.
Urine flow ml/kg -hr
.
39
f lov/ of the five day urophysectomized fish was significant¬
ly lower (p<0 . 01) than for the ten day urophysectomized
fish, indicating that the decrease was transient.
As shown in Figure 14 (Appendix, Table 2) , urine
osmolality w as not changed signif icantly by urophysectomy .
The sham-operated animals had an average urine osmolality
corresponding to the intact controls at both the five and
ten day postoperative periods. The average urine osmolali¬
ty for the five day urophysectomized fish was slightly, but
not significantly, lower than that of the sham and intact
control groups. There was no difference in the urine osmo¬
lality of the ten day urophysectomized fish and the respec¬
tive sham and intact controls, nor was there any signifi¬
cant difference between the five and ten day urophysectomized
fish .
Urophysectomy did not affect the urine Na+ concen¬
tration (U,7 ) of the goldfish in this study (Figure 15)
JN ci
(Appendix, Table 2). The urine Na+ concentration did not
vary significantly between the sham-operated and intact
control groups at either postoperative time periods. The
urine Na+ concentration of the five and ten day urophysec¬
tomized animals was not significantly lower than that of
their respective sham— operated and intact control groups .
The urine Na+ levels of the five and ten day urophysec¬
tomized groups were also not significantly different.
'
.
/'<. '' **, h&i'
C f > ,
.
.
,
■
FIGURE 14. The effect of urophysectomy on the osmo¬
lality (mOsm/1) of goldfish urine at
five and ten days pos toper atively. The
vertical bars represent ±SEM. The number
of individuals in each experimental group
(N) is shown at the base of each bar..
Wk Intact control
io7o° ° 1 Sham-operated
I I Urophysectomized
i - 1
o
O
O
O
•
o
LO
O
10
cn
cm
CM
LUSOW
A;!|B|OLUSO suun
Postoperative recovery time
days
•
■
„
■
•• • • •
■
FIGURE 15. The effect of urophysectomy on the urine
sodium concentration of the goldfish at
five and ten days postoperatively . The
vertical bars represent + SEM. The number
of individuals in each experimental group
(N) is shown at the base of each bar.
<Z/j Intact control
Sham-operated
I I Urophysectomized
LO
uo!;bj;u0duod uunjpos
Postoperative recovery time
days
■
.
44
As a reflection of the decreased urine flow, Na*"
excretion rate (V*U^ ) was also significantly lower in the
urophysectomized fish five days post-urophysectomy when
compared to the sham and intact control animals (Figure 16)
(Appendix, Table 2). The sham-operated animals maintained
Na+ excretion rates similar to the intact control fish
.f-
throughout this study. The average rate of Na excretion
in the five day urophysectomized fish was reduced to 31.5
±1.3 ^m/kg^hr, which was approximately fifty percent of
the Na excreted by the sham or intact controls. There was
no significant difference in Na+ excretion rate between the
ten day urophysectomized, sham-operated or intact control
groups. The rate of Na+ excretion was significantly lower
in the five day urophysectomized fish (p<0.001) compared to
the ten day urophysectomized animals.
Urophysectomy had no significant effects on urine
Cl” levels (Ucl) (Figure 17) (Appendix, Table 2) . The sham-
operated group had urine Cl” levels which were not signifi¬
cantly different from the intact control groups. The urine
Cl” concentration of the five day urophysectomized fish,
though somewhat lower, was not significantly different than
that of the five day sham and intact control fish and the
ten day urophysectomized animals. The urine Cl levels of
the ten day urophysectomized fish were also not signifi¬
cantly different from their respective sham-operated or
*
1 1 l j L Jl ’ * 1 mlyti I
v'-I
45
’
'
FIGURE 16.
The effect of urophysectomy on sodium
excretion rates of the goldfish at five
and ten days postoperatively . The ver¬
tical bars represent ±SEM. The number
of individuals in each experimental
group (N) is shown at the base of each
bar.
** p < 0.01, comparing the means of
operated and control groups.
//A Intact control
\ ° Col Sham-operated
I I Urophysectomized
46
O
in
o
o
o
o
o
6
o
o
6
o
00
CD
CM
Jl)
uo|;0JDX0 Lunipos
Postoperative recovery time
days
.
FIGURE 17. The effect of urophysectomy on urine
chloride concentrations of the gold¬
fish at five and ten days postopera-
tively. The vertical bars represent
±SEM. The number of individuals in
each experimental group (N) is shown
at the base of each bar.
47
YYX Intact control
°°°o \ Sham-operated
48
l/HLU
uoi;bj;u9duod £>puo|io
Postoperative recovery time
days
49
intact control groups,
Urophysectomy resulted in a significant decrease
in the total amount of Cl excreted (V'U^) in the five day
urophysectomized fish compared to their sham and intact
control groups (Figure 18) (Appendix, Table 2) . This re¬
flects the decrease in urine flow rate for the urophysec¬
tomized fish. There was no significant difference in the
Cl" excretion rate between the sham-operated animals and
the intact controls throughout this study. The ten day
urophysectomized fish had a Cl excretion rate similar to
that of the sham and intact controls. Cl excretion was
significantly lower in the five day urophysectomized fish
(p<0.05) compared to the ten day urophysectomized fish,
again reflecting the decreased urine flow in the five day
operated fish.
J.J. t ■j-'j"
The urine Ca concentration (uCa) an(^ Ca ex“
cretion (V*U„ ) rate of the five and ten day sham and in-
Ca
tact control groups were not significantly different (Fi¬
gures 19 and 20) (Appendix, Table 2) . There was a signi—
ficcLnt decrease in both urine Ca concentration and the
rate of Ca++ excretion in the five day urophysectomized
fish compared to the sham and intact control groups for
that postoperative time period. There was, however, no
difference in the urine Ca concentration or Ca ex
cretion rate of the ten day urophysectomized fish and their
■
-
■
50
FIGURE 18. The effect of urophysectomy on the urine
chloride excretion rates of the goldfish
at five and ten days postoperatively .
The vertical bars represent ±SEM. The
number of individuals in each experimen¬
tal group (N) is shown at the base of
each bar.
** p < 0.01/ comparing the means of
operated and control groups.
51
I - 1
o
o
O
O
6
•
o
6
•
o
xr
00
C\J
jij-6>i/[A|rf
UOH-GJDXG 0pUO|LJO
Postoperative recovery time
days
'
'
'
.
FIGURE 19. The effects of urophysectomy on the urine
calcium concentration of the goldfish at
five and ten days postoperatively . The
vertical bars represent ±SEM. The number
of individuals in each experimental group
(N) is given at the base of each bar.
* p < 0.05, comparing the means of
operated and control groups.
Sham-operated
Urophysectomized
N
CO
in
oo
6
6
6
6
6
I /[AjLU
UOi;ej;U03UOD UUnpjBO
Postoperative recovery time
days
<
■
.
I _ _ _ 1
FIGURE 20. The effect of urophysectomv on the calcium
excretion rate of the goldfish at five and
ten days postoperatively . The vertical
bars represent ±SEM. The number of indi¬
viduals in each experimental group (N) is
shown at the base of each bar.
** P < 0.01 , comparing the means of
operated and control groups.
55
O
c
o
u
T5
0
-f->
05
C
0
Q.
O
i
E
03
n
00
13
N
E
o
-*— *
o
O
_c
CL
o
L.
JL|-6>j/[A|rf
UOj^OJDXO LunpiBO
Postoperative recovery time
days
.
56
respective sham and intact control groups. The urine Ca++
level and the rate of Ca+^ excretion of the five day uro-
physectomized fish were significantly lower than the ten
day urophysectomized fish (p<0.01 and p<0.01 respectively).
Preoptic Nucleus Lesion
Plasma Electrolyte Levels
The effects of lesioning the NPO on the plasma
-f-
Na concentration are shown in Figure 21 (Appendix, Table
3) . The sham-operated and intact control fish maintained
similar plasma Na levels at all postoperative sampling
times. The plasma Na+ levels in the five and ten day NPO
lesioned fish (complete lesion) were significantly lower
than in the sham and intact controls at the same postopera¬
tive time periods. The twenty day completely lesioned
animals had plasma Na+ concentrations similar to the sham
and intact control values. A significant difference in
-j.
the plasma Na concentration was found between the com¬
pletely and those partially lesioned at both five and ten
days postoperatively (p<0.01) but not at the twenty day
sampling period. The five and twenty day partially le¬
sioned fish had plasma Na+ levels similar to their respec¬
tive sham and intact control groups. However, the ten
■f
day partially lesioned group had a plasma Na level signi¬
ficantly higher (p<0.05) than for the ten day sham-operated
-4-
and intact controls. The plasma Na levels of the five and
’
■
’
FIGURE 21. The effect of lesioning the preoptic
nucleus of the goldfish on plasma
sodium concentration at five, ten
and twenty days postoperatively .
The vertical bars represent ±SEM.
The number of individuals in each
experimental group is shown at the
base of each bar.
p < 0.05, ** p < 0.01, comparing
the means of operated and control
groups .
*
Y/s Intact control
„° o ° j Sham-operated
£zz| Partial preoptic nucleus lesioned
1 1 Preoptic nucleus lesioned
58
1/l/SjUJ rUOnrEJ}.U0DUOD lunjpos
Postoperative recovery time days
'
59
ten day completely lesioned fish were not significantly
different. Both the five and ten day completely lesioned
fish were, however, significantly lower than the twenty
day completely lesioned fish (p<0.01 and p<0.01 respective¬
ly) .
Plasma Cl levels were quite variable (Figure 22)
(Appendix, Table 3). There was a large range in plasma
Cl concentrations among the individuals within an experi¬
mental group. No significant differences were detected
between the sham-operated and intact controls at any of
the postoperative recovery times. There were no signifi¬
cant differences in plasma Cl concentration between the
five, ten and twenty day completely lesioned groups and
their corresponding sham and intact controls. There also
was no significant differences in plasma Cl levels between
the five, ten and twenty day completely lesioned groups,
although there was some decrease in the Cl level of the
twenty day lesioned animals. The partially lesioned ani¬
mals showed no differences in plasma Cl concentrations
from one postoperative sampling time to another and were
not significantly different from the completely lesioned
animals for the same postoperative time periods.
++
NPO lesioning did not significantly alter plasma Ca
concentrations (Figure 23) (Appendix, Table 3). The sham-
operated and the intact control animals were not
* k
'
'
FIGURE 22. The effect of lesioning the preoptic
nucleus of goldfish on the plasma
chloride concentrations at five, ten
and twenty days postoperatively .
The vertical bars represent ±SEM.
The number of individuals in each
experimental group is shown at the
base of each bar.
Intact control
Fq?°| Sham-operated
61
O
CM
in
l/ksiuu
uoi;ej|U0duod opuoipo
Postoperative recovery timeJ days
62
_
Postoperative recovery time, days
FIGURE 23. The effect of lesioning the preoptic
nucleus of the goldfish on plasma
calcium concentrations at five, ten
and twenty days postoperatively .
The vertical bars represent ±SEM.
The number of individuals in each
exDerimental groun is shown at the
base of each bar.
YAA intact control
Sham-operated
Partial preoptic nucleus lesioned
63
73
0
C
O
</)
0
0
<J
D
C
o
'■&
o
0
L.
CL
l/Hcu
uoj;Bj;u3ouoD uunpieo
Postoperative recovery time, days
64
significantly different at any of the postoperative samp¬
ling times. Plasma Ca levels of the five, ten and twenty
day completely lesioned animals were not significantly
different from their respective sham and intact control
group values, nor were they significantly different from
each other. There also were no significant differences
between the partially lesioned animals at the five, ten and
twenty day postoperative recovery periods and their respec¬
tive sham and intact controls.
Renal Study
Lesioning of the NPO produced a dramatic antidiure¬
tic effect on both the five and ten day lesioned fish (Fi¬
gure 24) (Appendix, Table 4) . The sham-operated groups
had urine flow rates (V) that were not significantly dif¬
ferent than the intact controls at either postoperative
time period. The average urine flow in the five day le¬
sioned fish was reduced to approximately forty percent of
that of the sham-operated and intact controls. The ten
day lesioned fish had an average urine flow approximately
fifty percent of that of the sham and the intact control
groups. Urine flow was significantly lower (p<0.05) in
the five day lesioned fish compared to the ten day lesioned
fish. However, urine flow in the ten day lesioned animals
was comparable to that found for the five day urophysec-
tomized fish (see above) . There were no incompletely
'
t Ua* an ill'll q BdJ abewi+ti
'
.
.
.
• ■
■
.
- •
.
FIGURE 24. The effect of lesioning the preoptic
nucleus of goldfish on urine flow at
five and ten days postoperatively .
The vertical bars represent ±SEM.
The number of individuals in each ex¬
perimental group is shown at the base
of each bar.
** p < 0.01, comparing the means of
operated and control groups.
YSA Intact control
E -° °1 Sham-operated
I 1 Preoptic nucleus lesioned
66
1
o
o
o
o
o
o
06
cd
CM
Jl) • 6>j/|LU MO|J. 0UUfl
o
Postoperative recovery time
days
67
lesioned animals in the renal study.
Urine osmolality significantly increased as a re¬
sult of lesioning the NPO (Figure 25) (Appendix, Table 4).
There were no differences in urine osmolality between the
sham-operated fish and the intact controls at either five
or ten days postoperative ly . Urine osmolality was, however,
significantly higher in both the five and ten day lesioned
groups compared to the respective sham and intact control
groups. No significant differences in urine osmolality
between the five and ten day lesioned groups were found.
There was a significant increase in urine Na+
concentration (U ) as a result of lesioning the NPO (Fi-
N cl
gure 26) (Appendix, Table 4). There was no difference in
urine Na+ concentration between the sham-operated and the
intact controls at either the five or ten day postoperative
sampling times. Urine Na+ concentration was significantly
increased in the lesioned fish at both five and ten days
pos toperatively compared to the respective sham and con¬
trol groups. There was, however, no difference between the
lesioned groups.
+
No significant differences in the Na excretion
rates (V*U ) were found between the sham-operated and the
Na
intact control groups in this experiment (Figure 27) (Ap¬
pendix, Table 4). While the rate of Na+ excretion in the
'
68
■
■
-s : b
'
.
■
FIGURE 25. The effect of lesioning the preoptic
nucleus of the goldfish on urine os-
molality at five and ten days post-
operatively. The vertical bars re¬
present ±SEM. The number of indivi¬
duals in each experimental group is
shown at the base of each bar.
**
p < 0.01, comparing the means of
operated and control groups.
69
UUSQW
A}(|B|OlUSO 0UUf|
Postoperative recovery time
days
70
'
■
• . - •
•
FIGURE 26. The effect of lesioning the preoptic
nucleus of the goldfish on urine sodium
concentration at five and ten days post-
operatively. The vertical bars repre¬
sent ±SEM. The number of individuals
in each experimental group is given at
the base of each bar.
** p < 0.01, comparing the means of
operated and control groups.
Sodium concentration
mM/l
I L
V/A Intact control
1° t o J Sham-operated
i i Preoptic nucleus lesioned
...
.
• ...
■
FIGURE 27.
The effect of lesioning the preoptic
nucleus of the goldfish on urine
sodium excretion rates , at five and
ten days postoperatively . The ver¬
tical bars represent ±SEM. The num¬
ber of individuals in each experi¬
mental group is shown at the base of
each bar.
**
p < 0 . 01 , comparing the means of
operated and control groups.
y//\ Intact control
73
jg -6>]/iAjrf
uopajoxo Lunipos
Postoperative recovery time
days
■
74
ten day lesioned fish increased significantly compared to
the ten day sham-operated and intact controls , the Na+
excretion rate in the five day lesioned animals was not
significantly different from the control groups. This
is a result of the combined effect of a reduced urine
f low and an increased urine Na+ concentration in the five
day lesioned fish. Na+ excretion in the five day lesioned
fish was significantly lower (p<0.01) than the ten day
lesioned group. This was due to an increase in urine flow
in the ten day lesioned group.
There were no differences in the urine Cl levels
(U ) between the sham and intact control groups (Figure
V-* -L
28) (Appendix, Table 4) . The urine Cl concentration was
significantly higher in the five day and ten day lesioned
animals than in the respective sham-operated and intact
control groups. No significant differences in the urine
Cl” levels between the two lesioned groups were observed.
The Cl” excretion rates (V*UC^) °f t^ie sham and
intact controls were not significantly different at any
postoperative sampling time (Figure 29) (Appendix, Table
4) . There were also no significant differences in Cl
excretion between the five day lesioned fish and the sham-
operated and intact control groups. The ten day lesioned
Oi 6»i*q«oo ^IlneotSinel* b^aaioni rt.il b*nc,U*l nsi
, ,; ,; ■ L a ■ 3 3: ; . W ■• '• '""1
'
~10 ni aaoadxslllt }reer iilo?i» on be <■ aia*
75
.
'
FIGURE 28. The effect of lesioning the preoptic
nucleus of the goldfish on urine
chloride concentrations at five and
ten days postoperatively . The ver¬
tical bars represent + SEM. The number
of individuals in each experimental
group is shown at the base of each
bar.
** p < 0.01, comparing the means of
operated and control groups.
76
O
■+->
c
o
u
u
03
+-»
c
o
1/lAjLU
uoj;ejt.u0DUOD apuoipo
Postoperative recovery time
days
■
.
■
*
.....
FIGURE 29. The effect of lesioning the preoptic
nucleus of the goldfish on urine
chloride excretion rates at five and
ten days postoperatively . The ver¬
tical bars represent + SEM. The number
of individuals in each experimental group
is shown at the base of each bar.
* p < 0.05, comparing the means of
operated and control groups.
Y/\ Intact control
Fo^l Sham-operated
1 1 Preoptic nucleus lesioned
78
jL)-6>]/lAjrr
UOIT.0JDX0 0pUO|LQ
Postoperative recovery time
days
79
animals did, however, have a urine Cl excretion rate that
was significantly higher than in either the sham-operated
or the intact control fish. The Cl excretion rate was
also significantly higher in the ten day lesioned animals
(p<0.01) compared to the five day lesioned animals. Again,
this is a reflection of the changes in urine flow.
The sham and intact control fish did not have urine
++
Ca concentrations (U ) that were significantly different
at either postoperative recovery times (Figure 30) (Appen-
++
dix. Table 4) . Urine Ca levels were significantly higher
in both the five and ten day lesioned fish compared to their
respective sham and intact control groups. There was no
significant difference between the lesioned groups.
There were no significant differences in Ca++ ex¬
cretion (V'U^ ) between the sham-operated and the intact
controls at either five or ten days postoperatively (Fi¬
gure 31) (Appendix, Table 4) . The five day lesioned fish
had Ca++ excretion rates significantly lower than the
sham-operated and intact control groups. Ca excretion
was also significantly lower in the five day lesioned fish
(p<0 . 01) than in the ten day lesioned animals. Again,
this is a result of the reduction in urine flow in spite of
an increase in Ca concentration of the urine.
, 3 • r. '
,B i-u, av.rt ioa bib rtaxl L#**x> iosial bm MB ^
-1%) tl+v a*>b » 9Vil *staU ie " *
to 9iiqE rtl woii eai™ «1 wti . «««•* n ei aifU
.
■
■
JJ1 ■ . ■ ' JJn .
FIGURE 30. The effect of preoptic nucleus lesioning
of the goldfish on the urine calcium con¬
centration at five and ten days postopera-
tively. The vertical bars represent ± SEM.
The number of individuals in each experi¬
mental group is shown at the base of each
bar.
* p < 0.05, comparing the means of
operated and control groups.
'//\ Intact control
r°°.i Sham-operated
81
l/WW
UOH.EJIU0DUOD UJnjD|EO
Postoperative recovery time
days
■
FIGURE 31. The effect of preoptic nucleus lesioning
of the goldfish on urine calcium excre¬
tion rates at five and ten days post-
operatively. The vertical bars represent
+ SEM. The number of individuals in each
experimental group (N) is shown at the
base of each bar.
** p < 0 . 0 1 , comparing the means of
operated and control groups.
Y//\ Intact control
Rol Sham-operated
83
T3
0)
c
o
O)
o
</)
D
o>
u
D
C
U
V>
d
O
CD
i_
CL
□
O
ID
o
o
o
o
O
cd
in
'd-
rd
C\J
Jtj-6>|/tAirf
UOI}0J0X0 LUniDjEO
Postoperative recovery time
days
84
Preoptic Nucleus Lesion/Urophysectomy
Plasma Electrolyte Levels
The combined operations of NPO lesioning and urophy-
-I-
sectomy caused a significant reduction in the plasma Na
levels of the five day completely lesioned/urophysectomized
fish and partially lesioned/urophysectomized fish compared
to the sham-operated and intact control groups (Figure 32)
(Appendix, Table 5) . The five day completely lesioned/
urophysectomized animals and partially lesioned/urophysec¬
tomized animals showed no differences in plasma Na+ con¬
centration. The extremely low plasma Na+ levels in the ten
day completely lesioned/urophysectomized and the lowered
levels in the sham and intact controls can be correlated
with an accidental failure of the aquatic facilities three
days prior to the ten day sampling time. High mortality
of both experimental and control fish occurred as a result
of the break-down. (The trauma and mortality was attri¬
buted to supersaturation of the water with oxygen and ni¬
trogen and a sudden temperature increase.) Plasma Cl
levels were also reduced in these same experimental groups
(Figure 33) . Results from the ten day postoperative samp¬
ling period should, therefore, be disregarded. By the
-f* •
twentieth postoperative day the plasma Na concentration
of the completely lesioned/urophysectomized fish was simi¬
lar to the sham and intact control levels. There were no
ii039* ,, a* be^ooo d.« tcx.no, bn6 t**«wtaoq*«. lo
.
.
'
-•
-
.
■
■
FIGURE
32. The effect of the combined operation of pre-
optic nucleus lesioning and urophysectomy
on the plasma sodium concentrations
in goldfish. The vertical bars repre¬
sent ±SEM. The number of individuals
in each experimental group is shown at
the base of each bar.
Partial - PON/UX
PONX/UX
partially preoptic
nucleus lesioned and
urophysectomized
completely preoptic
nucleus lesioned and
urophysectomized
* p < 0.05
** p < 0.01/ comparing the means of opera¬
ted and control groups.
86
O
L_
TJ
d)
•+-*
05
i
+■> c
L> c
05 H3
-+-> _C
c oo
X
Z>
X
z
£ <y o x
o CL Q. 3
u O ' ^
05 X
£ z
O
CL CL
inn
o
CM
O
ID
o
o
O
O
o
00
CM
pi
o
r—
1/lAjUJ
uojibjiusduod tunipos
Postoperative recovery time, days
'
87
significant differences in plasma Na+ levels between the
twenty day completely lesioned/urophysectomized and par¬
tially lesioned/urophysectomized fish. With the exception
of the ten day groups the sham-operated and the intact
control groups were not significantly different.
Plasma Cl levels were not changed by the simul¬
taneous removal of the NPO and the urophysis (Figure 33)
(Appendix, Table 5) . The plasma Cl concentration for
the five day completely lesioned/urophysectomized group was
not significantly different from the five day sham and in¬
tact controls. There also were no significant differences
between the five day completely lesioned/urophysectomized
and the partially lesioned/urophysectomized animals. As
previously stated, the Cl levels of all the ten day ex¬
perimental groups should be disregarded. There were no
significant differences between the twenty day completely
lesioned/urophysectomized and the sham-operated control,
the intact control or the partially lesioned/urophysec¬
tomized fish. Plasma Cl” levels were also not significant¬
ly lower in the five day completely lesioned/urophysec¬
tomized fish compared to the twenty day completely lesioned/
urophysectomi zed animals. There were no differences in
plasma Cl concentration between the partially lesioned/
urophysectomi zed fish at any of the postoperative recovery
times .
(tc Willi) miaxdttotu sitt bn* <*K »tt) lo miooaaS
\fasnoiaoi *X^siq«OT oris a* b^t.qmoo tlax? bosimoS
,banoi33l aria n.oaoood noJ3***W*>«M SO smaolq
88
.
• ■■ . •
.
. .
.
.
FIGURE
33. The effect of the combined operation
of preoptic nucleus lesioning and
urophysectomy on the plasma chloride
concentration in goldfish. The ver¬
tical bars represent + SEM. The num¬
ber of individuals in each experi¬
mental group is shown at the base of
each bar.
Partial - PONX/UX - partially pre¬
optic nucleus
lesioned and
urophysectomized
PONX/UX - completely pre¬
optic nucleus
lesioned and uro¬
physectomized
* p < 0.05 / comparing the means of
operated and control groups.
89
O
-*->
c
o
u
T3
0)
+-»
CO
L.
<D
Q.
O
i
£
OJ
jC
(/>
X
3
X
o
0.
j_
co
+-»
L.
fO
CL
X
3
X
z
O
CL
If)
O O O O
CXJ *= o 0)
I/Huu
UO!^EJ;U0DUOD 0piJO|L|O
Postoperative recovery time, days
.
90
The combined operation of the NPO lesion/urophy-
+ +
sectomy did not alter the plasma Ca levels (Figure 34)
(Appendix, Table 5). There were no significant differences
in the plasma Ca++ concentrations between completely
lesioned/urophysectomized and partially lesioned/urophy-
sectomized fish at any of the postoperative sampling times,
nor were there any differences compared to the sham and
intact controls for the same recovery periods. Plasma Ca++
concentrations for the sham-operated and intact controls
were not different and were in accordance with those ob¬
tained in the singly operated animals (Figures 12 and 23) .
There also were no significant differences between the five,
ten and twenty day completely lesioned and urophysectomized
fish .
fiicrfa
91
.
.
. .
.
.
■
'
.
... .
.
FIGURE 34. The effect of the combined operation of
preoptic nucleus lesioning and urophy-
sectomy on the plasma calcium concentra¬
tion of goldfish. The vertical bars
represent ±SEM. The number of indivi¬
duals in each experimental group is
shown at the base of each bar.
Partial - PONX/UX - partially preoptic
nucleus lesioned
and urophysectomized
completely preoptic
nucleus lesioned
and urophysectomized
PONX/UX
T//X Intact control
[° °n 1 Sham-operated
92
X
=>
X
z
o
CL
i
oj
’+-»
L.
03
CL
X
z>
X
z
o
CL
o
O
O
O
oo
CXJ
V-
o
l/HLU
o
CVJ
LO
UOI}EJ}U3DUOD Ujnp|BO
Postoperative recovery time, days
'
DISCUSSION
Urophysectomy
Plasma and urine electrolyte levels , urinary ex¬
cretion rates, osmolality and urine flow rates of the
sham-operated and intact control fish are in compliance
with values obtained for goldfish by other workers (Maetz,
1963; Maetz et a]^. , 1964a; Maetz et al. , 1964b; Bourget et
al . , 1964; Donaldson et al. , 1968; Ogawa, 1968; Motais et
al. , 1969; Lahlouh and Sawyer, 1969 ; Lahlouh and Giordan,
1970; Mackay, 1974).
Urophysectomy altered the ability of the goldfish
to regulate plasma Na levels. The plasma Na levels,
which were significantly depressed at five days postopera-
tively, returned to control values by the tenth postopera¬
tive day. Maetz et aiL. (1964a) observed intraperitoneal
(I.P.) injections of urophysial extracts stimulated bran-
+ +
chial Na influx in goldfish causing a net gain of Na .
Although they found no significant effect on Na+ efflux,
their results were variable (Maetz et. cCL. , 1964a) . The
Na+ stimulating factor of the urophysis, later to be called
Urotensin III (Lederis et al. , 1969; Bern and Lederis, 1969;
Lederis, 1970c; Berlind, 1973), is apparently a separate
93
.
* i
.
XOU19 BO ias’ii* sn bnuoi Y- &* dguodilA
'
94
entity from the other urophysial factors (Geshwind, e_t al. ,
1968; Lederis, 1969). Thus, the temporarily reduced plasma
Na concentration observed in the present study could
either be due to an increase in branchial Na+ efflux or
to a reduction in the branchial Na+ influx due to the ab¬
sence of this urophysial factor. Furthermore, since
+ +
urine Na concentration and renal Na excretion was de¬
creased, the decrease in plasma Na+ was most likely due to
Na+ loss across the gills.
Plasma Cl levels were not affected by urophysec-
tomy in the present study. Maetz et a]L. (196 4a) state that
in a preliminary experiment I.P. injections of urophysial
— +
extracts increased Cl influx concurrently with Na influx.
The experimental procedure was not described, however.
Takasugi and Bern (1962) reported a decrease in serum Cl
with urophysectomy in the euryhaline teleost Tilapia mos-
sambica maintained in freshwater. The fact that these fish
were starved and handled daily for a period of ten days
suggests that any difference was probably due to surgical
and handling procedures, especially as the decrement was
not significantly lower than in the sham-operated group.
Because Cl” excretion was lowered as a result of urophy¬
sectomy, and plasma and urine Cl concentrations were not
altered, there had to have been either an increase in Cl
efflux or a decrease in Cl" influx to compensate for the
I IIMUnBili sub od «■
§M | H ?%r.
-
bf^^fsiroqo -tr'/lS £ffct ni ^viol •••’•;;• ninpla fact
■
95
reduction in renal Cl loss.
++
There were no changes in plasma Ca levels in the
urophysectomized goldfish compared to the sham-operated
and intact control fish in this study. Chan (1969) found
++
that urophysectomy did not alter the overall Ca balance
in the European eel. No other studies have been done that
indicate that urophysectomy alters Ca+T balance in teleosts
++
Thus , it appears unlikely Ca is regulated by the urophy-
sis .
Urophysectomy did, however, produce an antidiure¬
sis in the goldfish used in this study. This antidiuresis
was , as with the drop in plasma Na+ , transitory. Recovery
in urine flow to near control values occurred by ten days
postoperatively . Although intraperitoneal and intravenous
(I.V.) injections of urophysial extracts elicit an imme¬
diate rise in PAH, free water (CR Q) and inulin clearances,
and urine flow in goldfish (Maetz e t al . , 1964a) and in the
freshwater adapted eel (Bern et al . , 1967; Chan et al . ,
1969; Chester Jones et al. , 1967, 1969), urophysectomy has,
up until the present study , failed to produce any altera¬
tion in renal function (Berlind, 1973). The same operation
performed on Fundulus kansae and T i lapi a mos s amp i ca had no
effect on urine volume or urinary Na excretion (Imai et al
1965) . However, the urine was collected at more than three
weeks postoperatively in Imai ' s study and the fact that
.
.
'
j , s ) f i, a 4 \ a > i ov r.i/ t -j oalla
96
urine flow had returned to normal values by ten days in
the present study could account for the difference between
the results of his study and the present one. Chester
Jones et_ cCL. (1969) also found that urophysectomy did not
+
cause a significant decrease in urine flow or Na excre¬
tion in the freshwater adapted eel, Anguilla anguilla.
There was, however, a decrease in urine flow from 38.0
ml/kg body weight -day during the initial control period
to 31.7 ml/kg b. wt.-day on day seven. The mean value
obtained on day five was 28.0 ml/kg b. wt. *day. Also, a
significant reduction in GFR (inulin clearance) occurred
by day seven between the urophysectomized eels and the
sham animals but not between the urophysectomized and in¬
tact control group. Although not statistically signifi¬
cant, there was, in the above study, a trend in the urophy¬
sectomized eels to have a reduced urine flow up to seven
days postoperatively . This reduction in urine volume is
similar to the antidiuresis observed in the five day uro¬
physectomized goldfish in the present study . It is pos¬
sible that in the study by Chester Jones et al. (1969) that
diuresis due to handling stress could account for the
variability of the data, since their fish were handled
daily in order to take measurements.
The reduction in urine flow seen in the present
study could be due to either a decrease in GFR or to an
.
.
.
.
97
increase in tubular reabsorption of water. It is suggested
that the antidiuresis observed in this study was due to a
reduction in GFR. This hypothesis is substantiated by Chester
Jones et al. (1969) who found a reduction in GFR in eels fol¬
lowing urophysectomy and by preliminary studies by Lederis
which indicate that the urophysial principles Urotensin I and
II affect GFR in these fishes (See Introduction). Recovery,
then, could be due to an increase in AVT production from the
neurohypophysis resulting in an increase in the GFR.
+ —
Urine osmolality and Na and Cl levels were not
changed significantly by urophysectomy in the present study.
Urine Ca+ concentration was, however, significantly decreased
by urophysectomy at five days postoperatively but returned
to near control levels by ten days. As a result of the re¬
duction in urine flow in the five day postoperative fish,
electrolyte excretion rates were significantly decreased.
Because urine flow had increased in the ten day urophysec-
tomized fish, electrolyte excretion rates were similar to
control animals.
Imai et al. (1965) found no effect of urophysectomy
on urinary Na"*" concentration in F. k ansae , or in the abi¬
lity of T. mossambica to excrete Na+ after injection of a
hypertonic NaCl solution. In the urophysectomized freshwater
adapted eel, A. anguilla , Na+ excretion was not significant¬
ly changed although there was an initial rise in Na+ ex¬
cretion on the first postoperative day (Chester Jones et
.
*
■
■
la trails Qfl bauoi i2 *****
.
98
al. , 1969). I.V. injections of urophysial extracts,
+
however, caused an immediate rise in urinary Na excretion
in freshwater eels (Bern et al. , 1967; Chester Jones et al. ,
1967, 1969). Maetz et al. (1964a) found that the natriure-
•f.
sis as well as the increase in relative Na clearance that
occurred with I.P. injections of urophysial extracts in
goldfish were followed by a reduction in these parameters
to below normal values in the second and third hours after
treatment. It was suggested that this reduction corres¬
ponded to an increase in tubular reabsorption of Na+. Be¬
cause urine Na+ concentration did not change the reduction
in renal Na+ excretion found in the urophysectomized gold¬
fish in the present study was due to the reduction of urine
output.
Urine Cl levels with respect to urophysectomy have
not been reported in any foregoing work. However, urophy¬
sectomy did cause a decrease in Cl excretion rates in the
goldfish at five days postoperatively . The decrease in Cl
excretion was, however, proportional to the decrease in
•Q^ine flow. Maetz et al. (1964a) did not find a change in
Cl excretion following I.P. injections of urophysial ex¬
tracts, however, he stated that branchial Cl influx was
increased (see above) . In order for the five day urophy¬
sectomized fish to have maintained normal plasma Cl con¬
centration, compensatory changes in branchial Cl flux
» t ^ I* 9 3 *1 B 3
*'■ X|| ' ' " ^ I
{
*
■
99
would have had to occur. This could have been either a
decrease in influx or an increase in efflux of Cl". A
decreased Cl influx would be expected because urophysial
peptides will increase Cl~ influx.
In A. anguilla , Chan (1969) found that urophysec-
tomy caused an immediate calciuresis in the first and second
++
postoperative days. This Ca loss was subsequently com-
-f-
pensated for by renal Ca retention. Chester Jones et al.
(1969) also found an immediate increase in Ca++ excretion
after urophysectomy in A. anguilla which later declined to
very low levels by the fourth postoperative day. The fact
that there was Ca retention in the eels in the above studies
lend support for the reduction in urine Ca concentra¬
tion and excretion rates in the five day urophysectomized
goldfish in the present study. The fact that the plasma
Ca++ concentration of the fish used in this study did not
•j'4’
change suggests there could be renal retention of Ca in
order to maintain normal plasma balance. Conservation of
Ca++ could be important at this time in order to repair
bone tissue that was damaged due to the removal of the uro-
style during surgery. Bone loss could create a hypocal-
++
cemia, and thus, renal reabsorption of Ca would be stimu¬
lated.
The effects observed following urophysectomy are
most likely due to the absence of more than one urophysial
-*oo 'fXSB'»«p8adu» .a <* ■»»* -c^c-aoq
ri:: ,': ‘->a- ' * ' ,>«l*i-5 9
'*
100
principle. Thus, it is possible that the reduction in
urine flow was due to the absence of an entirely different
urophysial peptide than that which caused the reduction
■ . -f*
m plasma Na level. Therefore, one factor, such as Uro-
tensin IV, could be affecting onlyGFR, reducing total,
urine output, and at the same time another principle,
such as Urotensin III, could be affecting ion transport
across the gill.
The temporary effects of urophysectomy are probab¬
ly not due to the regenerative property of the urophysis,
but rather to pituitary (specifically the neurohypophysis)
intervention (see General Discussion) .
Preoptic Nucleus Lesioning
Lesioning of the NPO caused a decline in the
plasma Na+ levels in Caras sius auratus at both five and
ten days postoperatively . This hyponatria appears to be
of short term duration as normal Na balance returns by
twenty days. Electro-cautery of the NPO in freshwater
adapted eels, A. anguilla and A. japonica , also caused a
reduction in plasma Na+ in these fishes (Chan, 1969).
It is well documented in the literature that hypo-
physectomy causes a reduction in plasma Na in a variety
of species (A. anguilla, Chan et al. , 1968a; Chan, 1969;
Chan et al. , 1969; Fundulus species, Pickford et al. ,
'
■
. -
ioiwv e nl +b* E»si« ftl aoitoubo? i *»*>* ***»•&*
101
1966; Stanley and Fleming, 1967b; Fleming and Ball, 1972;
Pang et al. , 1973; Poecilia latipinna , Ball and Ensor, 1967;
C. auratus , Lahlouh and Sawyer, 1969; Lahlouh and Giordan,
1970; Donaldson et al. , 1968; Ogawa, 1968). However, the
values that Ogawa (1968), Lahlouh and Sawyer (1969) and
' , +
: Lahlouh and Giordan(19 70 ) report for plasma Na levels in
hypophysectomized goldfish were much lower than the plasma
Na+ levels in the NPO lesioned goldfish in the present
study. Plasma Na+ in hypophysectomized goldfish, six days
postoperatively , dropped to 87.7 mEq/1 from the sham and
intact control values of 133 and 142 mEq/1 respectively
(bahlouh and Giordan, 1970). While plasma levels were 119
mEq/1 in the hypophysectomized goldfish at three weeks
(Lahlouh and Giordan, 1970). Lesioning of the NPO of the
goldfish in the present study resulted in a decrease in
plasma Na+ to 122.5 and 121.5 mM/1 at five and ten days
respectively with a return to near control values of 136
mM/1 at twenty days. These data suggest that, firstly,
total removal of pituitary function has a greater effect
on plasma Na+ level (the drop in plasma Na+ was of greater
magnitude) than does just lesioning the NPO. Secondly,
there is partial recovery in plasma Na concentration in
the hypophysectomized animals by three weeks. The recovery
could possibly be due to neurohypophysial peptides, as
hypophy s ectomy is believed not always to eliminate the
functioning of the NPO (see Introduction). Thus, Na+
.... . V. e .
- i*. J t .3 Si-
96. t 50 8«/.XoT Cottsaoo 3£«t o* * *»iw Vl»v
.
ytavoosw sr!T .sfcsw saicO yd 69s ar.oiosayriqogYn arid
102
balance in teleosts is probably normally mediated through
a balance in both the adenohypophysial and neurohypophy¬
sial systems (see General Discussion) .
The phenomena of incomplete ablation of a hypo-
thalmic nucleus not producing identical results as total
lesioning has also been found in other studies. Chan
(1969) found that, in the European eel, an incomplete
lesion of the NPO did not result in the same reduced plas-
+ ++
ma Na and Ca levels observed in the totally lesioned
fish. Inconsistent results related to electrode deposits
++
of Fe following D. C. current lesioning of the hypothal-
mic area have been seen in mammals (Everett et. al . , 1961;
Rabin, 1972). Thus, the increase seen in plasma Na+ in
the partially lesioned fish in the present study could
possibly be due to an irritation caused by ionic residue
from the electrode.
Lesioning of the NPO had no influence on the plasma
Cl" concentrations of goldfish in this study. Ogawa (1968)
and Lahlouh and Sawyer (1969) found a decrease in plasma Cl
in hypophysectomized goldfish. Pickford and Phillips (1959)
found that hypophysectomized killifish, F. heteroclitus died
of severe hypochloremia. Furthermore, Pickford et al. (1965)
1966) found the neurohypophysial hormones failed to increase
-
(i X I ' > " r. O l »f i ' >) '0 ‘ sir:0 ■ 0
10 3
the Cl levels in hypophysectomized F. heteroclitus . Hypo
physectomy also reduced plasma Cl levels in the eel,
A. rostrata (Butler, 1973). Pang et al. (1973), however,
did not find a change in Cl concentration in the serum
of hypophysectomized F. heteroclitus.
There is no direct evidence in the present study
that indicates that plasma Cl levels are affected by the
NPO. However, in order for the ten day lesioned fish to
maintain normal Cl levels, an increase in Cl uptake
would have to occur to compensate for the increase in Cl
excretion for these animals. Because prolactin will stimu
late Na+ uptake (Olivereau and Ball, 1970), there could
be a concurrent increase in Cl uptake, thus, allowing
prolactin to maintain normal plasma Cl levels.
Lesioning of the NPO did not change the plasma
Ca++ levels of the goldfish in the present study. How¬
ever, Chan (1969) found a decrease in plasma Ca++ in both
hypophysectomized and NPO lesioned freshwater eels com¬
pared to the respective sham-operated fish. Although Chan
reported a significant decline in plasma Ca due to hypo-
physectomy , he did not indicate whether the decline in
plasma Ca++ level due to electro-cautery of the NPO was
statistically significant. He did not find any signifi¬
cant differences between the sham— operated (forebrain re¬
moval) and those with partial removal of the NPO. Reduc—
'
.
.
104
++
tion in plasma Ca due to hypophysectomy has also been
observed by Chester Jones et al. (1968) and Chan et al.
(1968) in A. anguilla , by Pang (1973a, b) in Fundulus
species, and by Ogawa (1968) in Carassius auratus. The
results in the present study suggest that the neurohypo-
physis does not have a role in Ca regulation. Therefore,
the reduction in plasma Ca observed following hypophy¬
sectomy is most probably due to the absence of some factor
released from the adenohypophysis (see General Discussion) .
In the present study, a marked reduction in urine
flow and a concomitant rise in the urine osmolality, and urine
-f- “K •“
Na , Ca and Cl concentrations resulted after lesionmg the
NPO. There is general agreement that removal of the pitui¬
tary gland is followed by a decrease in urine flow rates
and an augmentation in urine osmolality and/or urine Na+
levels (Chester Jones et. al . , 1965; Butler, 1966; Stanley
and Fleming, 1966, 1967b ; Lahlouh and Sawyer, 1969; Lahlouh
and Giordan, 1970; Butler, 1973).
The urine flow rates of the ten day lesioned gold¬
fish in the present study (6.05 ml/kg-hr) are consistent
with rates obtained in goldfish three weeks after hypophy¬
sectomy (Lahlouh and Sawyer, 1966; 6.04 ml/kg-hr; and Lah¬
louh and Giordan, 1970, 6.0 ml/kg-hr). Lahlouh and Giordan
(1970) obtained urine flow rates of 3.1 ml/kg-hr in hypo-
phy sectomi zed goldfish on the sixth postoperative day which
.. ,r ■ t : •' • • ■ ■ °di
'
■
.
-rteJ on* -^ri-eAXm MU iMCI ,i»YWa 6» tluolOnJi ymo^oee
105
is comparable to the urine flow rate exhibited by the five
day lesioned fish (3.8 ml/kg«hr) in the present study.
Thus, it appears that there is a similar time effect on
urine flow rates following hypophysectomy and lesioning
the NPO . Lahlouh and Sawyer (1969) and Lahlouh and Giordan
(1970) found that although prolactin increases urine flow
in hypophysectomized fish, it does not cause diuresis in
intact goldfish. The neurohypophysial peptide AVT will,
however, cause diuresis in both hypophysectomized and in¬
tact goldfish (Lahlouh and Giordan, 1970; Sawyer, 1972).
Therefore, it is likely that the reduction in urine flow
following hypophysectomy is, at least in part, due to the
removal of neurohypophysial function. The compensatory
changes in urine flow seen by ten days in the NPO lesioned
goldfish could be due to prolactin involvement in restoring
homeostasis. However, owing to the evidence presented in
this paper and the diuretic effect of urophysial extracts
found by other workers, it is quite possible that the increase
in urine flow rate of the ten day lesioned fish over the five
day lesioned fish could be due to changes in urophysial func¬
tion.
It was observed in the present study that the le¬
sioned fish had larger weight gains and a bloated appearance
compared to the sham-operated and intact control fish.
These differences in weight gain were not, however, signi¬
ficant. This might be a general hydration due to reduction
■
.
106
in glomerular filtration or an increase in tubular reab¬
sorption of water , as a result of the lesion. Owing to
the augmentive effect of AVT on glomerular filtration
(Maetz et al. , 1964b; Sawyer, 1970, 1972), one would sus¬
pect that it could be due to a reduction in GFR. Chan
(1969) also observed a rise in body water content and hemo-
dilution when the NPO was cauterized in the freshwater
eel/ ^.* anguilla. A gradual increase in body weight which
reflected an increase in body water was also observed in
the hypophysectomized goldfish (Lahlouh and Giordan, 1970).
However, the increase in urine concentration seen in the
lesioned fish in this study strongly suggests that there was
increased tubular reabsorption nf water. This could have
been in addition to a decrease in GFR. To determine whether
the antidiuresis produced by urophysectomy and NPO lesioning
was glomerular in oripin, further studies measuring the ef¬
fects of these operations on inulin and PAH clearance would
he necessary.
Urine Na+ levels of the goldfish used in the pre¬
sent study were significantly increased to 15.1 and 14.7
mM/1 in the five and ten day lesioned animals respectively
from mean sham-operated and intact control values of 6.9
and 6.4 mM/1 respectively. Lahlouh and Sawyer (1969) also
found an increase in urine Na+ concentration from 6.0
mEq/1 in the intact goldfish and 8.5 mEq/1 in the sham-
'
'
OB s (MW) «Y?»» Oftfi fUioXrfBj .^9V^O‘.-qs9l t\Mm >.3 Drtfi
107
operated controls to 15.0 mEq/1 in hypophysectomized fish.
Although the results in the study by Lahlouh and Sawyer
(1969) were not statistically significant, the values they
obtained are quantitatively comparable to those presented
in the present work. The goldfish in the present study
were not subjected to the stress of daily handling which
likely made the results less variable than those of Lahlouh
and Sawyer (1969). Lahlouh and Giordan (1970) found that
the urine Na+ concentration of goldfish increased progres¬
sively from 3.6 mEq/1 in intact goldfish to 27.0 mEq/1
in the hypophysectomized fish by six days post-hypophysec-
tomy. However, there was no difference in urine Na+ con¬
centration between the hypophysectomized and intact control
4-
fish at three weeks. Urine Na concentrations were also
elevated in F. kansae (Stanley and Fleming, 1966, 1967a, b);
and in A. rostrata (Butler, 1973) following hypophysectomy .
The increase in urinary electrolyte concentration following
hypophysectomy and a decrease in urine flow are the only
consistent effects of hypophysectomy on renal function in
teleosts (Butler, 1973). In the goldfish, prolactin and
cortisol were only partially able to restore the urine Na+
concentrations which were elevated by hypophysectomy,
while AVT was able to return urine Na+ concentration to
control values in these fish (Lahlouh and Giordan, 1970).
In the same study, neither prolactin or cortisol affected
urine Na+ levels in these fish (Lahlouh and Giordan, 1970).
' . ;
A3 bfu;ol (o^ei) rtfibaoia bna .luo I rlaJ .(ed?!) I9Y'jb3
pniwcXIo? :toi3»33nsonoo viot-foal* • 9e«*ao|- odT
.
108
Therefore, it is possible that the increase in urine Na+
concentration due to hypophysectomy is due, in part, to
the loss of neurohypophysial function. This is supported
by the elevated urine Na concentration found in the NPO
lesioned fish in the present study.
At ten days postoperatively , the NPO lesioned
goldfish used in the present study had Na+ excretion rates
that were significantly higher than control values. Lah-
louh and Sawyer (1969) reported that urine output of Na+
was not altered by hypophysectomy in goldfish three weeks
after the operation. This was later substantiated by
Lahlouh and Giordan (1970) where Na+ excretion in.aoldfish
increased until the sixth or seventh day post-hypophysec-
tomy, but returned to the control rate by three weeks.
The ratio of renal Na+ output to plasma Na+ concentration
in the study by Lahlouh and Sawyer (1969) was, however,
significantly higher in hypophysectomized goldfish, there¬
fore not eliminating the contribution of the kidney to the
loss of plasma Na+. This ratio was also higher in the NPO
lesioned fish in this study. Thus, the resultant hyponatria
could be attributed, at least in part, to changes in renal
filtration or reabsorption of Na+. As further support for
this interpretation, hypophysectomy has been shown to cause
increased Na+ loss in F. kansae (Stanley and Fleming, 1966,
1967a, b) and an increase of 30-50% in the fraction of fil-
bsmolzal 0 m : “'i®6 n9 ;
i3>r«wei , <*v ( •' 3 ?£) is1 wsE ,6r > duo d* e £•« ■ .
.
.
109
tered Na excreted in A. r os trat a (Butler, 1973) . Also, hypo—
physectomized F. k ansae showed reduced renal Na+ reabsorption
compared to control fish (Stanley and Fleming, 1966).
Because the NPO lesioned fish were losing Na*11, com¬
pared to the sham and intact control groups , regulatory ad¬
justment in Na uptake would have had to occur in order for
the twenty day lesioned fish to have normal plasma Na+ le¬
vels. Prolactin is able to increase branchial (Dharamamba et
a^l. ) and intestinal (Bern, et al^. , 1974) Na+ influx and could,
therefore, have elevated the plasma Na+ level. Some regula¬
tion may have come from the urophysis, as Urotensin III is also
believed to increase Na uptake.
Goldfish urine Cl concentrations in the present study
were elevated by lesioning the NPO. This was possibly due to
an increase in tubular reabsorption of water. Cl excretion
had not changed by the fifth postoperative day but was signi¬
ficantly increased by the tenth postoperative day. Changes in
urinary Cl excretion between the lesioned fish at the two
postoperative times are a result of changes in urine flow, as
there was no difference in either plasma or urine Cl levels
between the two lesioned groups. Maetz et a_l. (1964b)
found that urine Cl” concentrations in goldfish remained un¬
changed with I.P. injections of AVT. These authors did
find a transient increase in urinary Cl excretion which
was proportional to the diuresis produced by the AVT.
■
- U, {,«#«**« rf.--.iUNM* «* *m#&9**** ~i0 *nla* 4telt* aat'°
110
Butler (1973) found that the urine concentration of
Cl , Cl excretion ratef and the rate of Cl excreted rela¬
tive to the amount of Cl filtered were significantly
elevated by hypophysectomy in the freshwater adapted eel,
A. ros trata . Because urine Cl levels and excretion rate
(in the ten day fish) were elevated following lesioning of
the NPO, a change in branchial Cl influx must have oc¬
curred for plasma Cl to remain at normal values.
Urinary Ca++concentrations were also elevated by
NPO lesioning. Again, this was probably due to reabsorp¬
tion of water in the distil tubule. Hypophysectomy also
resulted in an elevated urine Ca++ concentration in F. kan-
sae (Stanley and Fleming, 1967b) . Ca++ excretion was sig¬
nificantly lowered in the goldfish five days after lesion¬
ing due to the reduced urine flow. At ten days, the ele¬
vation in urine concentration counterbalanced the reduction
in urine flow in the lesioned fish, resulting in a Ca
excretion rate that was comparable to that of the control
animals .
Because neither the plasma Ca++ concentrations or
the • Ca++ excretion rate were altered following the le¬
sioning of the NPO of the ten day fish, the increase in
jj£-j_ni0 Ca++ concentration was probably due to increased
tubular reabsorption of water , causing a concentration of
the urine. Therefore, the ten day lesioned fish were in
-OO »V*li n« to U : ' *0*“'*'
■
-al Ljzv^nl >ftl ,(4ai* ««*» ^ ":p Bnxn°ie
Ill
Ca balance. The difference between the five and ten day
lesioned fish was an increase in urine flow in the latter
group. This increase in urine flow could have been due to
an increase in GFR mediated by either a urophysial principle
or by prolactin, or due to a decrease in tubular reabsorp¬
tion of water.
To support the hypothesis that changes in prolactin
secretion could have compensated for the reduction in Ca++
excretion in the five day lesioned fish, hypophysectomized
F. kansae had greater renal loss of Ca++ than either sham
or normal controls two weeks after the operation (Stanley
and Fleming, 1967b) .
Preoptic Nucleus Lesion/Urophysectomy
The plasma Na+ concentrations of the completely and
partially lesioned/urophysectomized fish at five days post-
operatively, and of the completely lesioned/urophysectomized
fish at ten days postoperatively were significantly lower
than in the sham and intact control groups. Because the par¬
tially lesioned fish at ten days postoperatively did not have
reduced plasma Na levels (Figure 21) , the reduction in the
partially lesioned/urophysectomized five day fish was most
likely due to loss of urophysial function. This confirms re¬
sults of the effects of urophysectomy along on plasma Na+
levels .
'
’
Hi -ic ,.w»u6*i »it» ,(CS w«»W) -I bmbbXj bsou
112
There was no additive effect of the combined opera¬
tions, since the Na+ levels found in the doubly operated
fish were the same as in the single operated (urophysec-
tomized or NPO lesioned) fish. Lacanilao (1972a) found
that the effects of submaximal doses of urophysial extracts
(4 ^pg/ml) and oxytocin (100 juU/ml) on water loss in isolated
toad bladders were synergistic, but at maximal doses (20 pg
and 500 juU respectively) their combined effect was no
greater than either alone. This offers an explanation as
to why the plasma Na+ concentration in the doubly operated
fish was not lower than singly operated fish and suggests
that similar sites (i.e., the gill) were affected by the
operations. The fact that isotocin will stimulate branchial
Na+ influx (Maetz et al. , 1964b) as will Urotensin III
(Maetz et al. , 1964a) supports the above explanation.
The reduction in plasma Na+ in the present study
does not appear to be chronic as normal Na+ balance (no
differences between experimental and control groups) was
attained by the twentieth postoperative day. As regenera¬
tion of the urophysis is probably not a factor in this
experiment (see General Discussion) , the recovery of the
plasma Na+ levels could be due to changes in the endo¬
genous prolactin secretion (see General Discussion) .
Plasma Cl" and Ca++ levels were not affected by the
combination of the operations. This supports the findings
Il 1 ' ' "T: 5ao:|
uj a it ms *<3 nf 1 ^ *Dt*® <3 o mN
t- ,•: v-.c s<» [noiax uoniO tJ5*en*S > ->• ; '
113
of the present study where no effects on plasma Cl and
Ca levels were found following urophysectomy or NPO
lesioning.
General Discussion
There has been considerable evidence in recent
years that the corpuscles of Stannius and pituitary gland
++
control Ca metabolism in teleosts (Chan, 1968; Chan et
al. , 1968a; Chan and Chester Jones, 1968; Chan et. al. ,
1969; Pang, 1971, 1973; Pang et al. , 1973). Although Chan
et al . (1968b) and Chan (1969) have suggested that prepara¬
tions of calcitonin from the ultimobranchial body have a
hypocalcemic effect in A. anguilla and A. j apcnica , subse¬
quent workers have failed to confirm this in other species
(see Pang, 1973). The current hypothesis (Pang, 1973;
Pang et al., 1973) concerning the endocrine control of cal¬
cium metabolism postulates that the pituitary, mediated by
prolactin, has a distinct hypercalcemic function which is
manifested in low Ca++ environments regardless of the Na+
level and osmotic conditions of the environment. On the
other hand, the corpuscles of Stannius function in a hypo¬
calcemic manner to allow regulation in environments high in
Ca++ (Pang, 1973; Pang et al . , 1973). Pang et al. (1973)
found that neurohypophysial hormones would not raise plasma
Ca++ in hypophysectomized fish in a hypocalcemic environ¬
ment, but prolactin could.
! '
'
-OQYrt b ni XO&BMti suimteiB «*toei*j*oo Brfs tsriio
CCTCX) . 2 Pne<* 't£Tet » -i* ®n*a ‘€*8X 83
114
Urine Ca concentration was, however, elevated
following NPO lesioning in the present study. This could
have been due to an increase in tubular reabsorption of
water. On the other hand, the decrease in urine Ca++
concentration that occurred following urophysectomy was
due to renal retention of Ca . The changes observed in
•f “I" ,
Ca excretion thus, were probably not the primary effects
of these operations. Renal retention of Ca++ in the uro-
physectomized fish could have been caused by an increase in
prolactin.
Although prolactin has been hypothesized as being
the major hypophysial hormone involved with maintenance of
Na+ homeostasis in hypophysectomized teleosts (Pickford
and Phillips, 1959 ; Pickford et a_l. , 1966 ; Pickford and
Pang, 1966; Fleming and Ball, 1972; Ball and Ensor, 1967),
other workers have found that injections of prolactin are
not able to entirely prevent the fall in plasma Na+ or
osmolality in hypophysectomized freshwater fish (Dharamambo
et al. , 1967; Donaldson et al. , 1968; Chan, 1968; Chan et.
al. , 1968a; Lahlouh and Sawyer (1969). Prolactin also is
not diuretic in the intact goldfish (Lahlouh and Giordan,
1970). Thus, it is possible that other hypophysial hor¬
mones such as AVT, isotocin or ACTH could account for the
Na+ deficit in hypophysectomized fish in the above studies.
As ACTH has no effect on plasma Na+ in hypophysectomized
'
l
-
■
115
goldfish (Lahlouh and Giordan, 1970) , it is possible to
speculate that the neurohypophysial hormones also affect
Na+ regulation. This is supported indirectly by the fact
that the drop in plasma Na in the NPO lesioned fish is
not due to a change in prolactin secretion because the
preoptic area has been shown to have no influence on the
plasma or pituitary prolactin levels (Peter and McKeown,
1974). However, the return to normal plasma Na+ levels
could be due to compensatory adjustments in prolactin secre¬
tion. Prolactin increases branchial permeability (Lahlouh
and Giordan, 1970) and stimulates branchial Na+ uptake
(Stanley and Fleming, 1967a; Fleming and Ball, 1972) ;
therefore; prolactin could have restored the plasma Na+
levels in the NPO lesioned fish.
The results from this study indicate that urophy-
sectomy also has a hyponatremic effect on the goldfish,
which is similar to that seen by ablation of the NPO. Re¬
covery from this hyponatria can be accounted for by pitui¬
tary control of Na+ balance either through a functional
NPO or through prolactin secretion.
Though regeneration of the urophysis has been re¬
ported in some species (Fridberg et al. , 1966) , this does
not begin until about two weeks after urophysectomy . At
this stage, in the species investigated, there was no
■
‘ 1 < *« -t C **
- '
.
116
stainable neurosecretory material nor was there any contact
between the neurosecretory axons and capillaries. There
were only a few cells that showed rudimentary signs of be¬
coming neurosecretory cells. By 22 days, however, these
subependymal cells could be identified as Dahlgren cells.
However, there was still no contact between the neurosecre¬
tory axons and the capillaries. It was not until 5-6
months that structural regeneration of the neurohemal
organ was complete (Fridberg et al. , 1966) . Regeneration
is, therefore, probably not an influential factor in the
present study. Both the neurohypophysial peptides, Iso-
tocin and AVT, will stimulate Na+ influx across the gills
(Maetz, 1963; Maetz et a_l. , 1964b) as will Urotensin TII
(Maetz et al. , 1964a) . Because of the similarity of the
effects of urophysectomy and NPO lesioning on plasma Na
levels in the present study the recovery seen in the
plasma Na+ levels of the ten day urophysectomized fish was
probably due to an increase in neurohypophysial function.
Maetz _et _aj. (1964a) found that, in goldfish, the
response of Na+ movement to I.P. injections of urophysial
extracts was more accentuated on the gill than on the kid¬
ney. Conversely, Maetz _et _al. (1964b) found that the
neurohypophysial peptides affected the kidney more tnan
the gills. These observations support the findings in
the present data that renal Na+ loss was greater in the NPO
It '
'
■
Mia YMtbjfjt &**>»«* .***Hpf lMSvriWVrtOt»M
117
lesioned fish than in the urophysectomized fish.
In the present study, there were no changes in
urine Na concentration following urophysectomy . Therefore,
the hyponatria caused by urophysectomy could be due to
branchial Na+ loss, indicating that the gill is an impor¬
tant target organ for the urophysial principles.
Recovery of plasma Na+ in the doubly operated fish
was most likely due to changes in prolactin secretion,
because the NPO was destroyed and, therefore, unable to
+
contribute in Na regulation. In addition, the regenerat¬
ing urophysis is not believed to be functional in this time
period.
As urophysectomy did not alter urine electrolyte
concentrations , the decrease in electrolyte excretion was
due to a reduction in urine output. This antidiuresis
could be due to the absence of such urophysial principles
as the hydrosmotic and/or the trout bladder contracting
urophysial factors, Urotensin IV and II, respectively (see
Introduction). The hydrosmotic factor, Urotensin IV, has
chromatographic properties and a pharmacological profile
similar to that of AVT (Lacanilao, 1972a, b) . If such is
the case , then it is possible that the antidiuretic effect
of lesioning the NPO and urophysectomy could be due to
the absence of AVT. The transitory decrease in urine flow
**A*$*fPW ;- niqu^oJ &moido
Jostle asU ***te aldia«d$ mi ' °*f>° QflJ
118
as a result of urophysectomy can be explained by hypothal-
mic control resulting in an increased secretion of AVT
from the neurohypophysial tissue. This hypothesis is sup¬
ported by the observation by Takasugi and Bern (1962) that
urophysectomy resulted in a hypertrophy of the NPO. The
hydrated state of the NPO lesioned fish (see above) could
have been caused by increased renal tubular reabsorption
of water , thus explaining the increase in urine concentra¬
tion. This hydration was not observed in the urophysec-
tomized fish.
This study has shown that there is a time sequence
in the events following lesioning of the preoptic nucleus
and urophysectomy. In both cases urine flow was higher
at ten days postoperatively than at five days. This in¬
crease in urine flow resulted in increased urine electro¬
lyte loss in the ten day lesioned group. Urine flow and
electrolyte excretion could possibly return to near normal
levels by twenty days as other pituitary factors became in¬
volved in osmotic and ionic regulation. This was suggested
(see above) to be the case in the return of plasma Na+ to
control levels by twenty days post-lesion. The changes in
urine flow and urine electrolyte excretion rates over time
in hypophysectomized F. k ansae (Stanley and Fleming, 1966)
and in the hypophysectomized goldfish (Lahlouh and Sawyer,
1969; Lahlouh and Giordan, 1970) lend support to this hypo¬
thesis .
.
*
'
’
119
The responses seen following the injection of uro-
physial and neurohypophysial extracts are analogous to the
effects of artificially lowering concentrations of plasma
electrolytes (Bourget et al. , 1964). These facts, combined
with the observations of Lahlouh and Giordan (1970) that AVT
caused a decrease in branchial water permeability and is
strongly diuretic, suggests that the urophysis and the neuro¬
hypophysis respond to an internal hypoosmotic stimulus , thus
allowing a freshwater teleost to eliminate excess water.
Lahlouh and Giordan (1970) suggest that normal water balance
in the teleost is maintained by the opposing actions of
prolactin and AVT. Prolactin, although it will cause diure¬
sis, is believed mainly to increase the permeability of the
skin and gills to water, thus resulting in an osmotic in¬
flux of water (Stanley and Fleming, 1967a; Lahlouh and Saw¬
yer, 1969; Lahlouh and Giordan, 1970). AVT would then be
released in response to the waterload created by prolactin
to restore normal hydration of the tissues.
Further physiological studies are required in order
to determine what physiological role the neurohypophysis
and the urophysis play in ionic and osmotic regulation. In
addition to the present study , the effects of urophysectomy
and NPO lesioning on branchial salt and water flux in con¬
junction with inulin clearance studies would resolve whether
it was renal or branchial compensation that occurred due to
these operations.
. s I O 3 ' J*' C 6 ' 3 1
120
The simultaneous lesioning of the hypothalmic
control centers for the neurohypophysial hormones and pro¬
lactin, and the comparison with the effects observed by
ablation of either one of these centers alone would define
the roles that these peptides have in osmotic and ionic
regulation.
i
'
* „■
-
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■
APPENDIX
Table 1. The effect of urophysectomy on the Concentration of Na , Cl and Ca
in goldfish plasma at five, ten and twenty postoperative time periods
131
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Table 3. The effect of lesioning the preoptic nucleus of goldfish on plasma Na , Cl and
Ca concentrations at five, ten and twenty postoperative time periods
133
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