ANATOMICAL AND BEHAVIORAL EFFECTS OF 5-FLUORO-2-DEOXYURIDINE ADMINISTRATION TO RATS AT DIFFERENT PHASES OF CENTRAL NERVOUS SYSTEM DEVELOPMENT By TED LaRUE PETIT A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1975 DEDICATION This dissertation is the culmination of a quarter of a century of education; for education is but life itself. It is dedicated to those who have taught me, and shared with me, the power and passion of life. ACKNOWLEDGMENTS It is very sad, looking back, to have to thank those that you are about to leave. Words often fall short of feelings. I hope that each of you knows how much I have cared if I am unable to fully express it on this page. Foremost on my mind are those professors who gave me guidance. Dr. Robert Thompson, who started me down this long path. . . . .Dr. Robert Isaacson who gave me the freedom to find my own way, and the guidance to help me along. A man who won my admiration for his love of life, for only through a true love of life are we able to pursue our dreams. . . . .Dr. Carol Van Hartesveldt, for her support and guidance, more as a friend than a colleague. .... Mrs. Virginia Walker, who has given more thoughtful- ness and understanding than ever I had expected. .... Mrs. Lillie HemanAckah for her help in preparing the histology and her jolly chats over morning coffee. My fellow graduate students who have helped me along. Especially Linda, Babs, and Jeanie, a very sincere and deep thanks for being who you are. And to the other members of the foursome: Dick, Lee, and very especially Sue. They have given me love, the one thing that makes all the rest worthwhile. Ill TABLE OF CONTENTS Page ACKNOWLEDGMENTS iii ABSTRACT V INTRODUCTION 1 METHOD 3 RESULTS 8 DISCUSSION 19 REFERENCES 24 BIOGRAPHICAL SKETCH 27 IV Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANATOMICAL AND BEHAVIORAL EFFECTS OF 5-FLUORO-2-DEOXYURIDINE ADMINISTRATION TO RATS AT DIFFERENT PHASES OF CENTRAL NERVOUS SYSTEM DEVELOPMENT By Ted LaRue Petit August, 1975 Chairman: Robert L. Isaacson Major Department: Psychology Pregnant Long Evans rats were injected subcutaneously with either 30mg/kg 5-f luro-2-deoxyuridine or water on Days 10 and 11, Days 13 and 14, or Days 16 and 17 of gestation. All groups were fostered to nontreated mothers at birth. During adulthood all animals were tested on a variety of behavioral tasks. Motor deficits were found in all three treated groups when tested for the ability to walk on parallel bars. None of the three treated groups were found to differ from controls in audiogenic seizure susceptibility. Activity decreases were seen in animals treated on Days 13 and 14 and Days 16 and 17. Although all groups learned a spatial discrimination in a T- maze with little difficulty, animals treated on Days 13 and 14 and Days 16 and 17 performed poorly on the position reversals, with the animals treated on Days 13 and 14 performing more poorly than those treated on Days 16 and 17. INTRODUCTION Cells of the central nervous system (CNS) are vulnerable to any agent or environmental condition which interferes with their proliferation, migration, or differentiation. All parts of the CNS do not develop simultaneously. Since each structure in the CNS has its own time table of maturation, at a given prenatal time some cell population will be especially vulnerable to factors which interfere with their development or existence. Other less active populations will be less affected. Agents interfering with cell proliferation on different days of development of the CNS will consequently result in cellular deficits and deformities in different re- gions of the adult brain. It would also be expected that different behavioral deficits arise as a consequence of inter- vention on different days of gestation because of the dif- ferent cell populations affected. The present study was an attempt to investigate this possibility by administering 5-f luoro-2-deoxyuridine (FUDR) during three different periods of pregnancy. Days 10 and 11, 13 and 14, or 16 and 17. FUDR inhibits the activity of the enzyme thymidine synthetase, thereby blocking DNA replication (Bosch, Harbers & Heidelberger , 1958; Taylor, Haut, & Tung, 1962; Reyes & Heidelberger, 1965; Conrad & Ruddle, 1972), and has been shown to inhibit cell division in the developing nervous system (Langman, Shimada, & Rodier, 197 2; Andreoli , Rodier, & Langman, 1973; Webster, Shimada, & Langman, 1973). In an attempt to find behavioral correlates of the pre- dicted anatomical disruptions, we tested the animals using several techniques which have been shown to be sensitive indications of level of functioning of the CNS after terato- logical intervention. Changes related to prenatal interven- tion have been found in activity (Furchtgott & Echols, 1958a; Werboff, Havlena & Sikov, 1962; Petit & Isaacson, in press), audiogenic seizure susceptibility (Geller, 197 3; Petit & Isaacson, in press), walking on parallel bars (Werboff, Goodman, Havlena & Sikov, 1961; Furchtgott & Echols, 1958b), and T-maze learning and reversal learning (Haddad, Rabe , Laquer, Spatz , & Valsamis, 196 9; Furchtgott, Jones, Tacker & Deagle , 1970). Thus, on the basis of prior research, we selected these procedures for the study of animals born to FUDR treated mothers. METHOD Timing of Pregnancy Female Long Evans hooded rats (Rattus norvegicus) were placed with males and vaginal smears were taken every morning. The day on which sperm were found in the smear was considered Day 0 of pregnancy (EO) . Animals were then randomly assigned to either three experimental groups or three control groups and housed singly. Mating was continued until two mothers and two corresponding foster mothers for each experimental and control condition were obtained. Experimental and Control Treatments All injections were made subcutaneously . On ElO and 11, two mothers received 30mg/kg FUDR (dissolved in distilled water) , while two mothers received distilled water injections as a control. On E13 and 14, another two mothers received 30 mg/kg FUDR while two mothers received distilled water injections. On E16 and 17, an additional two mothers were given 30mg/kg FUDR and two mothers received distilled water. Thus, each experimental and control mother received one injection on each of two successive days of pregnancy. Fos- ter mothers were left undisturbed throughout pregnancy. Fostering and Postnatal Rearing Within 12 hrs after birth, litters from all experimental 3 and control mothers were culled to 12 pups and fostered to normal mothers which had delivered no more than 24 hrs earlier. All offspring were then left undistiirbed until Postnatal Day 23 (PN23) , when they were earmarked and separately housed. From the two litters comprising each experimental and control group, 16 animals were randomly selected for testing, one half of the animals in each group coming from each litter. Apparatus and Testing Procedure Beginning at PN45 the animals were handled for 5 min daily for 3 days. On PN48 , 49, and 50 the animals were tested for locomotor activity in an automated activity arena for 5 min each day (see Lanier & Isaacson, 1974) , for details of the apparatus. Starting on PN51, the animals were placed on a 23-hr water deprivation schedule. Starting on PN54, the animals were allowed to explore a T-maze for 3 days in groups of eight, The alleys of the apparatus were 11 cm wide and 14 cm high with a hinged plexiglas top. The stem was 90 cm long with a 27 cm start box at the end. Each arm was 47 cm long, with a water spout at the end protruding 1 cm into the arm. Clear guillotine doors were located between the start box and the stem, and in each of the arm.s adjacent to the choice point. Preliminary exposure lasted 1/2 hr for each group and con- sisted of handling, apparatus exploration, and drinking in the goal compartments. During preliminary exposure, water was available in both arms of the maze. On the fourth day, the animals began training on a spatial discrimination in the T- maze. The animals were trained to go to the right arm of the maze for water reinforcement; the left arm contained a spout from an empty water bottle. After entering the correct arm the plexiglas guillotine door was lowered and the animal was allowed to drink for 10 sec before being returned to the start chamber to begin the next trial. If the animal entered the incorrect arm, the guillotine door was lowered to prevent a corrective response, and the animal was left for 10 sec before being returned to the start chamber. The animals were given 10 trials per day until a criterion of no more than two errors in two successive days was reached. Upon reaching criterion, each animal was begun on reversal training, during which water was available in the left arm of the maze. During the reversa] learning the animals were given 10 trials a day until reaching a criterion of no more than two errors in 1 day. When the animals reached criterion on the reversal, the posi- tion of the water was again switched to the right arm of the maze. They were run to criterion performance again. Once more a change in the location of the reward was made as the animal reached criterion, for a total of three reversals. Following reversal learning, all animals were maintained on 23-hr water deprivation and tested for their ability to walk on two horizontal bars 36 in long, set 1.5 in apart, and connected to a supporting panel at both ends. One end had a water tray placed against a dark surface and was designated the goal. The other end, the starting point, had a light svarface. The rods were marked off in inches. The animal was first placed at a distance of 6 in from the goal end of the rods with front and rear feet in position and facing the water tray. If the animal reached the water it was placed 12 in, then 18 in, or farther from the goal end until 120 sec of time on the bars had elapsed. Animals were never allowed to drink more than 5 sec each time they reached the water tray during the practice trials. Immediately following the practice period the animal was placed in position at the starting end with front and rear feet on the rods and pointed in the direc- tion of the water tray. The animal was then observed until it traversed the length of the rods. Time to traverse the rods was recorded, as well as complete or partial falls. The animals were then returned to ad lib. water, and 2 days later tested for audiogenic seiztire susceptibility, once that morning and once 36 hrs later (at night) . The apparatus was a modified Lehigh Valley Electronics operant chamber with the feeding mechanisms removed. A noise level of 10 3-105 dB was produced by placing a telephone bell in the space previous- ly occupied by the feeding mechanism. The animals were placed inside the apparatus for 15 sec, after which the sound was presented continuously for 120 sec. For scoring purposes, the 120 sec was divided into four, 30-sec segments. Behavior was scored during each 30-sec segment on seven counts: (1) quiet, (2) grooming, (3) walking, (4) running, (5) hopping, (6) clonic movements, and (7) tonic movements. Two days following audiogenic seizure testing, all animals were sacrificed with an overdose of ether and intracardially perfused with physiological saline followed by 10 percent formalin. The brains were removed, trimmed flush to the anterior extent of the cerebral cortex and posterior extent of the cerebellum. The brains were placed in 10 percent formalin and allowed to harden for 3 days and weighed. The dimensions of the brains were measured to the nearest .001 in {.0025cm) in three planes by use of a micrometer caliper. Anterior-posterior (A-P) measurements were taken from the most anterior tip of the isocortex to the most posterior tip of the isocortex closest to the midline. The medial-lateral (M-L) brain width was evaluated at the most posterior portion of the isocortex measuring the width across the entire extent of the brain (both hemispheres) . Dorsal-ventral (D-V) measurements were taken from the base of the brain to the top of the isocortex at its most posterior extent. The brains were then embedded in celloidin and cut coronally at 30 pm. Every eighth section was mounted and stained with thionin. RESULTS An initial analysis of variance was run on scores of the three control groups on all variables. No differences were found, so their scores were combined to form a single control group (Group C) for subsequent statistical analysis. An analysis of variance was used to analyze all data unless otherwise noted. When a significant F was found, a Dunnetts post-ANOVA test was used to compare the control group with the experimental groups (see Edwards, 1968). Only significant differences are reported. Anatomical Results The anatomical results of this study are summarized in Table 1. Brain Weight No differences were found between the brain weights of Group C, the group receiving FUDR on ElO-ll (Group 10-11) or the group receiving FUDR on E13-14 (Group 13-14) . The brains of animals receiving FUDR on E16-17 (Group 16-17) , however, weighed less than the control group (£_ < .01). Brain Size No differences were found in any measurement of brain size between Group C, Group 10-11, or Group 13-14. The Group 16-17 brains were smaller than the Group C brains in the A-P 8 m < N •<-\ a •H u PQ 'O C (0 -P en ■H I c •H u ffl o M QJ 3 rH > c re QJ X m ro n •<* > o O o o 1 a • • • Q rH rH rH rH 0) N •H C/2 — Xt £ VD VO in fN c u ^q VD VO VO VO •H ^ 1 • • • • m S rH rH rH rH S-l 03 X! 00 ro ^ -{ rH +J ^ tT> ■H Xi (1) ^ in in in r-i S E ■^ ■^ ^ rH Cn • • • • c — CN CN (N (N •H m u a •-{ •* r- 1 1 1 1 o 1 1 VO U <-i <-i <-\ 00 V£> VO VO Qj'* 04 rH CUrH OirH D II 3 II 3 II 3 II 0 C 0 c 0 c 0 c )-i ^^ M u O O o o 0) > 1 rH -P C m o •H 14H •rH C 01 •H W II Xi 10 (£<.01) and M-L (£ <.01) dimensions but not in the D-V dimension. Histology Histological examination of sample brains revealed no obvious abnormality of cellular elements or structures in any of the groups (see Figures 1, 2, 3, and 4) . Behavioral Results The results of the various behavioral tests are summarized in Table 2. Activity The females were more active than the males in all groups (£< .01). The Group 10-11 animals were less active than the Group C animals (g^ x: CQ c o CO 0) 0 o CQ c. A) 0) S M (« CO (0 m (0 ffi M o ■^r (N o cn * 00 (0 c • • • • Oi 0 r^ in m CN o m >^ 00 r^ 0) tn r-i m (0 x; CO H CO m CN4 k <^ • • • • Q) fN t-i r« n > Q) « rH (0 nl (TJ to V£) H (U M « w N (t3 ■H S m XI CO (U « iH tjl (d C C -H U3 CN •"a" m •H C • • • • Cn ^^ 00 r^ r^ 00 ■H nj U Q) O J >i +J rtJ XI XI •H in n n o > • • • » •H ro CT^ in n 4J r- in o o O i-i rH < r-( ^ [~~ 1 1 1 1 o 1 1 U rH i-i rH 00 VD > Q) (U rH iH in iH o O • • (U fl) x: ^ +J +J +J +) fCj td u u a. Q* 3 3 0 0 u M o o e g o 0 M M 4-1 M-l +J +J c c 0) a) M M QJ cu <4-l i >i rH iH -P -P c c (d fd u u •H •H m U-J •r4 •H c c en Crv •iH -H to to H II Id XJ 16 9' 6- ^ — ^•^ Group C o—- . >~~.oGroup 10-11 ^^^^'^^ —'-■Group 13-14 A->arB< ■ — •A Group 16-17 V /\ \ \\ \ \ \ \ ,.'■ s. o s- s- 4- •O Original Learning Reversal 1 Task Reversal 2 Reversal ■ 3 Figure 5. Mean error scores on T-maze learning and rehearsal for all groups. 17 On all three reversals, Group 13-14 made more errors than Group C (£< .01) . Group 16-17 made more errors only on re- versal two (£< .01) and three (g< .05). While Group C and Group 10-11 showed a consistent drop in the number of errors made over the three reversals. Group 13-14 continued to make approximately the same number of errors as original learning throughout the three reversals. Group 16-17 showed a drop in the mean number of errors across the three reversals, but the drop was not as great as in Group C. Inspection of Figure 5 indicates that Group 13-14 per- formed more poorly on reversal learning than Group 16-17. Group 13-14 made more errors than Group C on all three revers- als, while the Group 16-17 error scores were higher only on the last two reversals. During the last two reversals. Group 13-14 made more errors than Group 16-17 (£<.01, Duncan's post-ANOVA test, Edwards, 1968). Parallel Bar Walking Performance on the parallel bars was measured in terms of seconds taken to transverse the bars and number of complete or partial falls. All three experimental groups were inferior to Group C on both measures. Audiogenic Seizure Susceptibility For purposes of evaluation, all animals were divided into two groups: those animals that had a score of 6 or 7 (clonic or tonic movements) on either of the two test trials, and those animals that never had a score higher than 5 (hopping) . 18 Using a Chi-Square analysis, no differences were found between the groups. In the control group, 25.5 percent of the animals had scores above 5 while 3 3.3 percent of Group 10-11, 33.3 percent of Group 13-14, and 7.1 percent of Group 16-17 had scores above 5. DISCUSSION - The results of this study indicate that administration of FUDR at three different points in pregnancy results in different behavioral consequences in the offspring. Activity decreases were seen in Group 10-11, while an increase in activity was seen in Group 13-14 and Group 16-17. Although all groups learned the spatial discrimination in the T-maze with little difficulty, animals in Group 13-14 and Group 16- 17 performed poorly on the position reversals, with Group 13- 14 performing more poorly than Group 16-17. Problems in motor performance were seen in all three experimental groups, while no differences in audiogenic seizure susceptibility could be detected in any of the experimental groups. Although a significant reduction in brain size and weight was found in Group 16-17, no anatomical anomalies could be detected in the brains of any of the treated animals. Anatom- ical effects frequently can not be detected in offspring treated with teratogens, despite great behavioral differences (Butcher, Brunner , Roth & Kimmel, 197 0; Butcher, Vorhees, & Kimmel, 197 2; Hutchings, Gibbon, & Kaufman, 197 3; Vorhees, 1974; Butcher, Hawver , Burbacker , & Scott, 1974; Hutchings & Gaston, 1974) . Proliferating cells can partially restitute cell populations which are destroyed or prevented from forming by a teratogen (Altman & Anderson, 1971; Andreoli , Rodier , & Langman, 197 3) . Therefore, cells depleted by FUDR treatment 19 20 could have been replenished at least in part after the FUDR treatment had been terminated. Butcher et al. (1974) have suggested three progressive effects of increases in terato- genic drug dosage: functional, anatomical, and lethal levels. They postulated that at low doses there could be functional, i.e., behavioral, effects of teratogens without producing anatomical effects. Hutchings & Gaston (1974) have also em- phasized the lack of correlation between teratologically pro- duced brain damage and behavioral impairment. Noting the similarity in behavioral, but not anatomical, impairments found after treatments with a variety of agents, the authors suggested a common underlying mechanism, possibly of a bio- chemical rather than structural nature. Activity changes in offspring subjected to teratogens appear to be correlated with the time of prenatal inter- vention. Treatment during early prenatal CNS formation with hypervitaminosis A on E8, 9, and 10 (Vorhees, 1974), X-irradi- ation on ElO (Werboff, Havlena, & Sikov, 1962), or FUDR on ElO and 11 (present study) leads to reduced activity levels in the offspring. Intervention during the middle of the prenatal period of CNS formation with methylazoxymethanol on E14, 15, or 16 (Haddad, Rabe , Laquer , Spatz, & Valsamis, 1969), X-irra- diation on E15 (Furchtgott , Tacker , & Draper, 196 8; Furchtgott & Echols, 1958a; VJerbof f , Havlena, & Sikov, 1962) or FUDR on E13-14 (present study) causes hyperactivity in the offspring. Activity differences are also found following intervention during late prenatal CNS formation. Animals treated with 21 FUDR on E16-17 (present study) were found to be hyperactive. Furchtgott & Echols (1958b) irradiated animals on E13 through 17 and tested them in tilting cages and an open field. They found maximal activity enhancement in animals treated on E15- 16. Irradiation on E17 produced increased activity levels, whereas neonatal irradiation produced decreased activity levels. Petit & Isaacson (in press) tested animals treated with col- chicine on E17, 18, and 19 in an open field on Days PN25, 26, and 45. The animals were not found to differ from the controls on PN25 or 45, but were hypoactive on PN26 . It appears that the hyperactivity found from intervention during mid-prenatal CNS formation is seen after intervention during the early part of this period. As intervention time nears birth, however, a drop in activity level of the treated animal is seen. Animals in Group 13-14 and Group 16-17 made more errors during spatial reversal learning in the T-maze than the con- trols. Previous researchers have reported that anim^als treated With methylazoxymethanol (Rabe & Haddad , 1972) , or X-irradiation (Furchtgott, Jones, Tacker, & Deagle , 1970) on E15 learned a spatial discrimination in a T-maze as quickly as controls; but when reversal learning was required the treated animals per- formed significantly worse than controls. However, in a WGTA adapted for rats, a series of visual pattern discriminations of increasing difficulty and their reversal were mastered by the treated as well as control rats (Rabe & Haddad, 1972). This is in contrast to the reversal learning deficits in the T-maze shown by these same animals. These authors concluded 22 that the animals had a deficit in spatial reversal learning, rather than a general cognitive impairment. The deficits found in reversal learning in this study are consistent with earlier findings and indicate that animals treated with FUDR during early prenatal CNS formation, ElO-ll, do not show deficits in reversal learning, while animals treated with FUDR during late pregnancy, E16-17, although showing a spatial reversal deficit, may not perform as poorly on this task as animals treated during mid-prenatal CNS formation, E13-14. Animals treated on ElO-ll, E13-14 , or E16-17 showed an increase in falls and time taken to traverse the parallel bars. This is consistent with earlier reports. Similar results on this same task were found by Werboff , Goodman, Havlena, & Sikov (1961) in animals irradiated on ElO, 15, or 20, and by Furcht- gott & Echols (1958b) in animals irradiated on E13. Thus deficits in motor performance can be produced in animals after teratological intervention at several points in pregnancy. Audiogenic seizure susceptibility was not found to be affected by treatment with FUDR at any of the three points in pregnancy. Geller (197 3) has shown that audiogenic seizure susceptibility was not altered in irradiated animals regard- less of the time of administration. Butcher, Smith, Kazmaier, & Scott (197 3) were unable to find differences in this measure in animals treated with hydroxyurea on E12. Petit & Isaacson (in press), however, found a decrease in audiogenic seizure susceptibility in animals treated with colchicine on E17 , 18, and 19. The results of the latter authors may be specific to 23 the drug colchicine. Thus, although X-irradiation and FUDR administration during different periods of pregnancy may not affect seizure susceptibility, results on this test may be specific to the teratogen used. In conclusion, this study indicates that while certain behaviors are affected in a similar fashion after FUDR inter- vention at any point in pregnancy (motor behaviors) , some behaviors are not affected at any point in pregnancy (audio- genic seizure susceptibility) , and other behaviors are differ- entially affected depending on the time of intervention (activ- ity and reversal learning) . FUDR intervention during early prenatal CNS formation (ElO-ll) produces hypoactivity and no deficits in reversal learning. Intervention during mid- prenatal CNS formation (E13-14) results in hyperactivity and large deficits in reversal learning. Intervention later in pregnancy (E16-17) results in a mild deficit in reversal learning and hyperactivity. The results of this study indicate that different be- havioral syndromes are associated with teratological inter- vention on different days of gestation. Additional studies using a variety of agents administered at different gestation- al ages are needed to fully understand the principles involved in this area of research. If it were possible to identify the behavioral consequences of interference with cell prolifera- tion at different stages of gestation and postnatal life, it might be possible to specify the etiology of some brain damage syndromes . REFERENCES Altman, J., and Anderson, W. J. (1971). Irradiation of the cerebellum in infant rats with low level X-ray: Histo- logical and cytological effects during infancy and adulthood. Exp. Neurol. , 30: 492-509. Andreoli, J., Rodier , P. M. , and Langman, J. (1973). Influ- ence of prenatal trauma on formation of Purkinje cells. Am. J. Anat . , 137: 87-102. Bosch, L., Harbers, E. , and Heidelberger , C. (1958). 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Effects of prenatal X-irradiation on activity, emotionality and maze learning abilities in the rat. Rad. Res. , 16 : 444-452. BIOGRAPHICAL SKETCH Ted LaRue Petit was born October 20, 1949 in Bogalusa, Louisiana. He received his Bachelor of Science degree from Louisiana State University in 1971. In 1972 he received his Master of Arts degree from the same university. In the Fall of 197 2 he entered the University of Florida where he is presently a candidate for the degree of Doctor of Philosophy. 27 I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree ofiDoctor of Philosophy. {'■• Robert L. Isaacson, Chairman Professor of Psychology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ^12^ 1 1 (kiOH^^ Frederick A. King Professor of Neuroscieiyce I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Merle E. Meyer Professor and Chairman of Psychology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Carol Van Hartesveldt Associate Professor of Psychology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. y - I W. Keith Berg / Assistant Professor of Psychology This dissertation was submitted to the Graduate Faculty of the Department of Psychology in the College of Arts and Sciences and to the Graduate Council, and was accepted as partial ful- fillment of the requirements for the degree of Doctor of Philosophy. August, 1975 Dean, Graduate School UNIVERSITY OF FLORIDA 3 1262 08553 9475