EFFECTS OF PARA- CHLORQPHENYLALANINE AND 5~ HY§ROXYTRYPTOPHAN ON THE SELECTTON 0F ETHANOL AND OTHER SOLUTIONS BY RATS Dissertation for the Degree of Ph. D. MlCHIGAN STATE UNIVERSITY JAMES KARL WALTERS 19 7 5 LIBRARY Midfigan Sum Um'mitr This is to certify that the thesis entitled EFFECTS OF PARA-CHLOROPHENYLALANINE AND 5-HYDROXYTRYPTOPHAN ON THE SELECTION OF ETHANOL AND OTHER SOLUTIONS BY RATS. presented by James Karl Walters has been accepted towards fulfillment of the requirements for Ph .D. degree in Psychology W Major professor DateLMLZZZL 0-7639 1 3’ BINDING BY "' ‘llllAG & SUNS 800K BINDERY INC. LIBRARY BINDERS SPRTNGPORT, HINTS]: N ‘ ABSTRACT EFFECTS OF PARA—CHLOROPHENYLALANINE AND S-HYDROXYTRYPTOPHAN ON THE SELECTION OF ETHANOL AND OTHER SOLUTIONS BY RATS By James Karl Walters Within the past several years, experimental evidence has accumulated which suggests that an association may exist between the neurochemistry of brain serotonergic systems and the ethanol self- selection behavior of rats. This evidence is primarily of a pharma- cological nature and most investigators have studied the effects on ethanol selection of manipulating whole-brain serotonin level with drugs. Para-chlorophenylalanine (pCPA), a serotonin depletor, and S-hydroxytryptophan (S-HTP), a serotonin precursor, have been used for this purpose. Questions have remained, however, as to whether these drugs are influencing only the selection of ethanol solutions and whether their effects are truly due to altered brain serotonin level. The present series of three experiments was therefore designed to further investi- gate the effects of pCPA and S-HTP on the selection of ethanol and other solutions by rats. In Experiment I, 50, 100 or 200 mg/kg pCPA or 25 mg/kg S-HTP (plus a peripheral decarboxylase inhibitor) were intragastrically James Karl Walters administered to rats for 10 consecutive days to determine if this treatment influenced ad_libitum water intake or body weight. Rats of Experiment 11 received 10 daily intragastric doses of either 100 mg/kg pCPA or 20 mg/kg S-HTP (plus a peripheral decarboxylase inhibitor) during or between 8-day ethanol preference test sequences. Preference testing with saccharin, glucose or sodium chloride solutions was carried out in Experiment III while animals were intubated for 10 days with 100 mg/kg pCPA. Results showed that pCEA, in most cases, and S-HTP (plus peripheral decarboxylase inhibitor) both caused significant body weight decreases and water intake increases. The effects of pCPA on water intake were greatest early in treatment and also were positively related to dose. With regard to ethanol, both pCPA and S-HTP produced significant reductions in selection, despite the fact that they should have had Opposite effects on brain serotonin level. It was found that the one dose of S-HTP employed failed to reverse the effects of pCPA on ethanol consumption and that pCPA had little influence on ethanol choice behavior when its administration did not coincide with ethanol drinking. The selection of saccharin solutions was suppressed during pCPA intubation, while the intake of glucose and sodium chloride solutions increased. Taken together, these data may be interpreted to support the hypothesis that the effects of pCPA are not specific to the con— sumption of ethanol, and that pCPA and S-HTP are most likely influ- encing ethanol self-selection through the formation of a conditioned taste aversion due to noxious drug effects. EFFECTS OF PARA-CHLOROPHENYLALANINE AND S-HYDROXYTRYPTOPHAN ON THE SELECTION OF ETHANOL AND OTHER SOLUTIONS BY RATS BY James Karl Walters A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Psychology 1975 ACKNOWLEDGMENTS I wish to express my appreciation to my committee members, Dr. Glenn 1. Hatton (chairman), Dr. Lawrence 1. O'Kelly, Dr. Richard H. Rech, and Dr. James L. Bennett, for their very helpful guidance of this dissertation project from its inception. I also extend my gratitude to Ken Ollila for invaluable assistance in conducting various phases of these experiments and to Patti for helping with part of the typing and continuing to have faith in me. Special thanks go to my sister and brother-in-law, Jan and Bill, for showing me the light at the end of the tunnel at a time when it was looking very dim. Support for this research was provided by the Department of Psychology and N.I.N.C.D.S. grant #09140 to Dr. Glenn 1. Hatton. Para- chlorophenylalanine and the peripheral decarboxylase inhibitor known as HMD were kindly supplied by Mr. Nathan Belcher of Pfizer Inc. and Mr. Clement Stone of Merck, Sharp 8 Dohme Co., respectively. ii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. INTRODUCTION. EXPERIMENT 1. Method . Subjects. Procedure . . . . . . . Drug Preparation and Administration. Results and Discussion. EXPERIMENT 11 Method . Subjects. Procedure . . . . . . . Drug Preparation and Administration. Results and Discussion. EXPERIMENT III Method . Subjects. Procedure Results and Discussion. Saccharin Glucose . . Sodium Chloride iii Page vi 10 11 13 31 32 32 32 34 34 69 69 69 7O 71 71 77 83 Page GENERAL DISCUSSION . . . . . . . . . . . . . . . 90 LIST OF REFERENCES . . ,. . . . . . . . . . . . . 95 APPENDICES A. Drugs, Supplies, and Equipment. . . . . . . . . 100 B. Summary Data-—Experiment I . . . . . . . . . . 102 C. Summary Data--Experiment II. . . . . . . . . . 106 D. Summary Data--Experiment III . . . . . . . . . 109 iv LIST OF TABLES Table . Page 1. Mean water intake in g/kg during each day of baseline, drug treatment and post-drug period for rats of Experiment I housed in standard cages . . . . . . 23 2. Daily body metabolism data (X :_S.E.) during baseline, pCPA treatment and post-drug period for six animals . 26 3. Drug treatments, mean daily preference ratios (:_S.E.), and mean daily g/kg alcohol consumed (:_S.E.) for each group of Experiment II . . . . . . . . . 36 4. Drug treatments, mean daily preference ratios (:_S.E.) and mean daily g/kg alcohol consumed (:_S.E.) for each group of Experiment III. . . . . . . . . . . . . 72 LIST OF FIGURES Mean water intake in grams as a function of days during baseline, drug and post-drug periods for animals in standard cages receiving 50, 100, or 200 mg/kg pCPA or CMC vehicle alone during the lO-day drug period . Mean water intake in grams as a function of days during baseline, drug and post-drug periods for animals in standard cages receiving 75 mg/kg HMD + 25 mg/kg S-HTP or two saline vehicle injections during the lO-day drug period. . . Mean percent body weight as a function of days during baseline, drug and post-drug periods for animals in standard cages receiving 50, 100, or 200 mg/kg pCPA or CMC vehicle alone during the lO-day drug period . Mean percent body weight as a function of days during baseline, drug and post—drug periods for animals in standard cages receiving 75 mg/kg HMD + 25 mg/kg S-HTP or two saline vehicle injections during the lO-day drug period . Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving pCPA during the second of three preference test sequences. Mean prOportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving CMC vehicle during the second of three preference test sequences. Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving pCPA during the second of three preference test sequences . vi Page 15 17 19 22 4O 4O 42 Figure 8. 10. 11. 12. l3. 14. 15. l6. 17. Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving CMC vehicle during the second of three preference test sequences . Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving pCPA during the second and HMD + S-HTP during the third of three preference test sequences. . . . Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving pCPA during the second and HMD + NaCl during the third of three preference test sequences. . . . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving pCPA during the second and HMD + S-HTP during the third of three preference test sequences. . . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving pCPA during the second and HMD + NaCl during the third of three preference test sequences. . . . . Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving HMD + S-HTP during the second of three preference test sequences . Mean pr0portion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving HMD + NaCl during the second of three preference test sequences . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving HMD + S-HTP during the second of three preference test sequences . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving HMD + NaCl during the second of three preference test sequences . Mean prOportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving pCPA during the first of three preference test sequences . vii Page 42 48 48 50 50 S3 53 56 56 59 Figure 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving CMC vehicle during the first of three preference test sequences . . . . . . . . . . . . . . . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving pCPA during the first of three preference test sequences. . . . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving CMC vehicle during the first of three preference test sequences . . . Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving pCPA between two preference test sequences. . . . . . Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving CMC vehicle between two preference test sequences . . . . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving pCPA between two preference test sequences. . . . . . . . . Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving CMC vehicle between two preference test sequences . . . . . . . . Mean proportion saccharin solution to total fluids consumed as a function of saccharin concentration for animals receiving pCPA during the second of three preference test sequences . . . . . . . . . . . . . Mean proportion saccharin solution to total fluids consumed as a function of saccharin concentration for animals receiving CMC vehicle during the second of three preference test sequences. . . . . . . . . . . Mean g/kg saccharin consumed as a function of saccharin concentration for animals receiving pCPA during the second of three preference test sequences . . . Mean g/kg saccharin consumed as a function of saccharin concentration for animals receiving CMC vehicle during the second of three preference test sequences . . . viii Page 59 61" 61 65 65 67 67 74 74 76 76 Figure 29. 30. 31. 32. 33. 34. 35. 36. Mean proportion glucose solution to total fluids consumed as a function of glucose concentration for animals receiving pCPA during the second of three preference test sequences . . . . . . . . . . . . Mean proportion glucose solution to total fluids consumed as a function of glucose concentration for animals receiving CMC vehicle during the second of three preference test sequences . . . . . . . . Mean g/kg glucose consumed as a function of glucose concentration for animals receiving pCPA during the second of three preference test sequences . . Mean g/kg glucose consumed as a function of glucose concentration for animals receiving CMC vehicle during the second of three preference test sequences. . . . Mean proportion NaCl solution to total fluids consumed as a function of NaCl concentration for animals receiving pCPA during the second of three preference test sequences. . . . . . . . . . . . . . Mean proportion NaCl solution to total fluids consumed as a function of NaCl concentration for animals receiving CMC vehicle during the second of three preference test sequences. . . . . . . . . . . . . . . . Mean g/kg NaCl consumed as a function of NaCl concentration for animals receiving pCPA during the second of three preference test sequences . . . . . . . . . . Mean g/kg NaCl consumed as a function of NaCl concentration for animals receiving CMC vehicle during the second of three preference test sequences . . . . . . . . ix Page 79 79 82 82 85 85 88 88 INTRODUCTION An enduring enigma for those concerned with determining the etiology of excessive alcohol intake in man is the contribution of central nervous system anatomy, physiology and biochemistry to this phenomenon. Within the past several years an association has apparently been demonstrated between the neurochemistry of brain serotonin systems and the consumption of alcohol by experimental animals (Myers 8 Veale, 1968; Veale 8 Myers, 1970; Geller, 1973; Ho, Tasi, Chen, Begleiter, G Kissin, 1974; Myers 8 Melchior, 1975). The import of this association for understanding human alochol abuse remains quite equivocal though, for no true animal model of alcoholism presently exists (Myers 8 Veale, 1970). Such knowledge may nonetheless provide clues to alcohol's interaction with the nervous system and may possibly help to elucidate some of the neurochemical processes which participate in determining alcohol consumption. Therefore, this proposed relationship deserves further close scrutiny. Great interest was aroused in the brain's serotonergic neuro- chemical systems, among others, by the pioneering demonstration of Dahlstrom and Fuxe in 1964- Using fluorescent histochemical techni- ques, they showed that most, if not all, of the nerve cell bodies containing the putative neurotransmitter (Rech G Moore, 1971, p. 108) serotonin (5—HT) are located within a number of nuclear groups in the brainstem. These raphe nuclei lie along the midline tegmentum through- out the medulla, pons and midbrain (Morgane G Stern, 1973). Axons from the serotonergic raphe neurons course both caudally and rostrally, ramifying to most areas of the central nervous system (Anden, Dahlstrom, Fuxe, Larsen, Olson, G Ungerstedt, 1966). Reviews of the anatomical, pharmacological, physiological and behavioral aspects of brain serotonin can be found in several recent books (Barchas & Usdin, 1973; Costa, Gessa, G Sandler, 1974a,b; Cooper, Bloom, 8 Roth, 1974). A major step forward in the study of the functional signifi- cance of brain serotonergic systems was made in 1966 when Koe and Weissman reported that the drug para-chlorOphenylalanine (pCPA) could selectively deplete S-HT neurons of their serotonin. It apparently does so by inhibiting tryptophan hydroxylase activity and/or impairing tryptophan transport. Experimentally, pCPA has been used extensively despite the complication that it also depletes blood and peripheral tissue of 5-HT. As far as whole-brain serotonin levels are concerned, one result which "remains quite stable within and across laboratories is the amount of brain S-HT depletion which results from pCPA" (Rechtscaffen, Lovell, Freedman, Whitehead, G Aldrich, 1973). Such depletion usually approximates 90% with only a slight 10 to 15% reduction in catecholamine levels early in treatment. Dosage and .Schedule of pCPA administration can, of course, make a difference (diolman, Hoyland, G Shillito, 1974). Extremely rapid reversal of both eatectr0physiologica1 and behavioral changes resulting from pCPA is uzsually accomplished by giving small doses of S-hydroxytryptophan (S-JETP), the immediate precursor of 5-HT (Weissman, 1973). Unfortunately though, the ubiquitous nature of the decarboxylating enzyme which converts S-HTP to S-HT results in S—HT being formed within neurons where it is not normally present (Moore, 1971). In 1968 Myers and Veale first applied the pharmacological technique of depleting brain serotonin with pCPA to the study of alcohol preference in experimental animals. Their initial study revealed that the ethyl alcohol preference of rats was significantly reduced or abolished by pCPA treatment. Later studies by Myers and his collaborators tended to confirm and extend these original findings (Myers 5 Cicero, 1969; Veale G Myers, 1970; Myers 8 Tytell, 1972; Myers, Evans, 8 Yaksh, 1972; Myers 6 Martin, 1973). They showed that pCPA, given in daily oral doses of 300 mg /kg, reduced not only the preference but also the absolute amount of alcohol consumed, in g /kg /day, by animals selecting ethanol solutions for various reasons. These included: (1) an initial predisposition, (2) acclimation to ethanol, and (3) stress induced by electric shock delivered randomly during a conditioned shock avoidance task. One unusual finding from these many studies was that an even more pronounced rejection of ethanol occurred in preference test sequences given up to two months after drug administration was dis- continued. Surprisingly, Veale and Myers (1970) reported preliminary assays carried out in a parallel fashion which showed S-HT content in (iiscrete brain regions to recover to levels even greater than control fi>1lowing chronic pCPA administration. This seems to shed doubt on the hypothesis that the changes in alcohol consumption actually did rGJE‘lect changes in central serotonin stores, since alcohol intake was depressed when serotonin levels were either depleted or elevated. With regard to S—HTP, Myers, Evans, and Yaksh (1972) found i.p. doses of 50 mg /kg injected every eight hours to also suppress ethanol intake. These effects persisted for over three months, despite the fact that S-HT levels determined shortly after treatment ended revealed no significant differences in S-HT content between the brains of control and experimental animals. Finally, in 1973 Myers and Martin reported that S-HTP also lowered ethanol consumption when it was infused directly into the lateral cerebral ventricle. In these animals, oral pCPA administration during S-HTP infusion reversed the lowered ethanol consumption. It also increased ethanol intake in a preference sequence given after infusion ended. In neither of these experiments did there seem to be a close correlation between preference behavior and brain serotonin levels. These phenomena, particularly with regard to the effects of pCPA, have proven quite ephemeral to other investigative teams. For instance, Frey, Magnussen, and Nielsen (1970) found a less pronounced effect from pCPA treatment and equivocal results with p-chloroamphetamine which produced less serotonin depletion than pCPA at the doses they used. Cicero and Hill (1970) found a marked distinction between results obtained from pCPA treatment when ethanol solutions were prepared with 95% versus 100% alcohol. Interestingly, the typical pCPA effect was found with solutions made from 100% (absolute) alcohol. This drug little affected the consumption of solutions prepared with 95% alcohol, however. They suggested that the pCPA treated rat may be more sensitive to the taste or odor of a contaminant, benzene perhaps, which azeotropic distillation imparts upon absolute alcohol. Another very recent experiment by Holman, Hoyland, and Shillito (1974) also questioned the hypothesis that the decreased ethanol consumption found by the Myers group was actually due to brain serotonin depletion. They gave pCPA to rats at intervals of three to four days and found it to be as effective as daily pCPA administration at depleting serotonin levels. Such intermittent treatment, however, did not produce a reduction in voluntary alcohol consumption. They speculate, as Nachman, Lester, and Le Magnen (1970) had done several years earlier, that a learned aversion or the route and time of pCPA administration may be responsible for the drug effects. Results in direct contradiction to those of Myers and his collatorators have been obtained by both Geller (1973) and Hill (1971). Hill found pCPA to produce no rejection of ethanol, but rather an increased intake of 3% and 5% solutions. Geller's results were even stronger. According to him, pCPA nearly always increased alcohol intake when given in daily oral doses ranging from 75-300 mg /kg. Daily intraperitoneal doses of 50-100 mg /kg S-HTP consistently reduced intake. The use of extended periods of daily drug administra- tion (up to four weeks) and a debatable interpretation of results render Geller's study questionable. The final studies concerning serotonergic involvement in ethanol preference have used the relatively new technique of lesioning central serotonin neurons by injecting 5,6-dihydroxytryptamine (5,6-DHT) intracisternally or intraventricularly. Two such studies have both shown 5,6-DHT to increase alcohol consumption within a few days after injection (Ho, Tasi, Chen, Begleiter, G Kissin, 1974; Myers 6 Melchior, 1975). Ho 33.31: found the effect to last from the fifth to the eleventh day post-treatment, while Myers and Melchior found ga is an; ex; increased consumption to persist 60 days after drug administration ended. Are any of these effects specific to the consumption of alcohol solutions? Unfortunately, few of the many studies implicating serotonin in ethanol self-selection also included preference testing for other solutions. Cicero and Hill, in 1970, reported preliminary findings of an increased saccharin intake due to pCPA treatment, but no detailed account of that study has apparently been published. Concerning sucrose consumption, Nance and Kilby (1973) showed pCPA to increase both preference and absolute amount consumed. They suggest that S-HT neurons may participate in regulating the amount of carbohy-- drate ingested. Suppression of quinine intake by pCPA has been observed by Brody (1970) in a study of the "hyper-reactivity hypothe- sis" of serotonin depletion effects. Suppression was especially evident when the quinine solution was novel to the rats. As an aside, Brody reported increases in dextrose intake due to pCPA treatment but gave no details of the experiment. Strikingly surprising to me in regard to most of these studies is the fact that little attention was paid to the effects which pCPA and S-HTP might have on the health and water consumption of the experimental animals. Although little quantitative data are available concerning water intake, present results are conflicting. Brody (1969) found a single dose of 300 mg /kg pCPA to increase drinking; a second dose given to the same animals five days later again facilitated water consumption. Holman, Hoyland, and Shillito (1974) also reported increased total fluid intake during an ethanol preference test sequence when pCPA was administered; other investigators have not reported such to be the case. In contrast to these findings, Panksepp and Nance (1974) found 20 days of pCPA treatment to reduce water intake by 28%. They also showed, as have others, that prolonged pCPA administration reduces food intake by 25% to 50% while lowering body weight 10% to 20%. The deleterious effects of pCPA on an animal's health might hamper data interpretation. In addition, drug-induced changes in water intake alone, if grams of alcohol consumed are not considered, could lead to erroneous conclusions. Also obvious in this hodge-podge of studies is the lack of appreciation for variability due to differences in the species or strain of animals tested. As pointed out by Tilson and Rech (1974) and others (Miller, Cox, 8 Maickel, 1968; Rosecrans 8 Schechter, 1972), sex, strain or supplier differences in whole brain or regional distri- bution of 5-HT may sometimes account for contradictory results. Two experiments by Ahtee and Eriksson (1972, 1973) illustrate this crucial fact. They discovered whole-brain 5-HT and S-hydroxyindoleacetic acid (S-HIAA) contents to be 15% to 20% higher in Wistar rats genetically selected for high ethanol preference than in Wistar rats selected for low preference. Additionally, they demonstrated that the distribution of 5-HT in various brain regions was not the same in each strain. As should be obvious from this brief overview, considerable evidence has accumulated which suggests that the consumption of ethanol, by experimental animals at least, may possibly be influenced by ‘Variations in the activity or anatomy of the brain's serotonergic DEHarochemical systems. The data are extremely contradictory, however, Particularly in regard to the effects on ethanol intake of the perripherally administered drugs, pCPA and S-HTP, which have been used to manipulate brain serotonin content. Often there has been little correspondence between the consummatory behavior itself and actual brain serotonin levels in those instances where these drug treatments did prove effective. Therefore, three interrelated experiments were carried out to further assess the effects on preference behavior of pharmacological manipulations, especially pCPA treatment, known to alter brain serotonin levels. In the first, three doses of pCPA and one of S-HTP were administered to rats to determine whether the drugs themselves or the resulting brain serotonin depletion had an effect on body weight and ad_libitum water consumption. The second was intended to determine whether decreasing brain serotonin with pCPA and increasing it with S-HTP would affect ethanol self-selection in opposite directions, while giving both treatments to the same animals would produce a cancellation of effects. In addition, procedural variables were investigated to help provide insight into whether any changes in preference behavior resulting from pCPA treatment were actually due to brain serotonin manipulation or if they could have been due to drug administration itself. To assess this possibility, pCPA was given during the first of three preference test sequences and also between preference test sequences. Administration of pCPA at these times had not previously been attempted. Finally, the third experiment was aimed at discovering whether pCPA treatment would affect the selection of three other solutions (saccharin, glucose, and sodium chloride), or if its effects were specific to the consumption of ethanol. EXPERIMENT I The purpose of this experiment was to determine quantitatively the effects of lO-day periods of pCPA or S-HTP treatment on the water consumption and body weight of rats. Primary interest was focused on the effects of pCPA, since this drug would be employed extensively in later preference testing experiments. No previous researchers have systematically varied the dosage of pCPA and made quantitative deter- minations of resulting changes in water intake or body weight. Three dose levels of pCPA and one of S-HTP were employed. The peripheral decarboxylase inhibitor, L—a-hydrazino-a-methyl-B-3,4-dihydroxyphenyl propionic acid (HMD), was used in conjunction with S-HTP to reduce its side effects. To help shed some light on how pCPA might be influencing water intake and body weight, the intermediate dose level (100 mg/kg) was administered to rats housed in standard metaboloism cages. This allowed monitoring of such variables as food intake, urine volume and urine electrolytes. Method Subjects Thirty-six male Sprague-Dawley albino rats supplied by Holtzman Co. of Madison, Wisconsin served as subjects. Thirty animals 10 were housed individually in a standard Wahman laboratory rack. Each had Wayne Mouse Breeder Blox and tap water freely available. Twelve of the 30 were 118 days old and 18 were 137 days old at the start of experimentation. The remaining six rats were 135 days old when experimentation began, and they were housed in standard metabolism cages supplied by Acme Metal Products Co. They had powdered Mouse Breeder Blox and tap water available ad_libitum. All 36 animals were maintained in rooms kept at 22-25°C which were on 14:10 light-dark cycles. Procedure At least two weeks of adaptation to the laboratory preceded the commencement of all baseline readings. Following adaptation, 24-hour measures of water intake and body weight were begun for the 30 animals in standard cages; food intake and urine volume were additionally recorded for the six rats in metabolism cages and a urine sample saved each day. The 3-bottle method of Myers and Holman (1966) was used to dispense water to the animals in standard cages. Three 125 ml. bottles with stainless steel drinking spouts were attached to the front of each cage. Two were filled with tap water while the third always remained empty. Bottle positions were changed daily according to the random schedule provided by Myers and Holman. Animals in metabolism cages had just one water bottle attached to the rear of the cage. Following eight days of baseline measures, the 30 rats in standard cages were divided into groups matched for mean daily water consumption during baseline. Each of the six groups of five animals contained two younger and three older rats. Beginning on Day 8 and continuing for 10 consecutive days, each of the groups received one of 11 the following drugs and dosages administered approximately three hours after taking daily water intake and body weight readings: (1) 50 mg/kg pCPA (4) vehicle (2) 100 mg/kg pCPA (5) 75 mg/kg HMD + 25 mg/kg 5-HTP (3) 200 mg/kg pCPA (6) vehicle + vehicle Upon completion of 10 days of drug administration, water intakes and body weights continued to be measured for approximately three weeks. The six rats in metabolism cages underwent four days of base- line readings. Beginning on Day 4 and continuing for 10 consecutive days, each animal received 100 mg/kg pCPA approximately three hours after water intake, food intake, urine volume and body weight were recorded. All measures continued to be recorded for four days post- drug. Urine samples from each animal were saved and frozen on all 18 days of treatment. Later determinations of urine specific grativty were made by refractometry; urine sodium and potassium concentrations were determined by flame photometry. Drug Preparation and Administration DL-Para-chlorOphenylalanine (Charles Pfizer Co.) was prepared as a suspension in 0.5% carboxymethylcellulose (CMC) solution and maintained under constant stirring prior to each administration. It was prepared at a concentration of 20 mg/ml and then the proper dosage was diluted with additional 0.5% CMC vehicle if necessary so that each animal received a constant fluid volume of 5 ml. The pCPA vehicle group received 5 ml. of CMC alone. Both pCPA and vehicle were administered by stomach tube under very light ether anesthesia. Animals were placed in a covered glass jar (25 cm. high X 22 cm. diameter) which was half 12 filled with ether-saturated cob meal. Thirty to 60 seconds elapsed, in most instances, before they were removed and intubation began. The peripheral decarboxylase inhibitor L-a-hydrazino-a-methyl- B-3,4-dihydroxyphenyl prOpionic acid (Merck, Sharp 8 Dohme Co.) was prepared as a suspension in 0.9% saline at a concentration of 20 mg /ml and maintained under constant stirring until administered. The proper dosage of HMD was diluted with 0.9% saline if necessary so that each animal received a constant fluid volume of 3 ml. The vehicle group received 3 m1. of 0.9% saline. Both HMD and saline were administered intraperitoneally under very light ether anesthesia. The chief reason for using HMD was to eliminate some of the undesired peripheral side effects of the decarboxylated products of S-HTP. HMD inhibits aromatic L-amino acid decarboxylase without crossing the blood brain barrier. By so doing, it allows S-HTP to be decarboxylated mainly in the central nervous system (Moore, 1971; Swonger G Rech, 1972) and potentiates the central action of a dosage of S-HTP by four to six times. Approximately 45-60 minutes after injecting HMD or its vehicle, DL-S-hydroxytryptOphan (Sigma Chemical Co.) was administered. It was dissolved in 0.9% saline with gentle warming and prepared as a 10 mg/ml solution. Dilution with additional 0.9% saline followed if necessary, so that each animal received the proper dosage in a 2 ml. fluid volume. The vehicle group received 2 ml. of 0.9% saline, making a total of 5 m1. Both S-HTP and vehicle were administered under very light either anesthesia. All drugs were prepared fresh daily, and every animal received a total of 5 ml. of fluid on each day of treatment. 01‘ th the 3115 13 Results and Discussion Figure 1 shows the mean water intake in grams during baseline, drug and post-drug periods for animals in standard cages receiving pCPA or CMC vehicle. The groups did not differ significantly during base- line. An analysis of variance did reveal differences in water intake among the groups during the lO-day drug period (F = 7.14, df 3/16, p <.005). A Duncan's test showed that the vehicle group drank signi- ficantly less than the pCPA-100 group (p <.05) and the pCPA-200 group (P <.005). The pCPA-50 group also drank significantly less than the pCPA-200 animals (p <.005). In comparing other pairs of groups, no significant differences in water intake were found between the vehicle and pCPA-50 groups, pCPA-50 and pCPA-100 groups, or the pCPA-100 and pCPA-200 groups. Plotted in Figure 2 are the mean water intakes for animals in standard cages receiving HMD + S-HTP or saline vehicle injections. No differences in intake existed during baseline, but the HMD + S-HTP group drank significantly more than the control group during the 10 days of drug treatment (P = 23.7, df 1/18, p <.001). Mean body weight for animals in standard cages receiving pCPA or CMC vehicle is plotted as a percentage of each group's weight on the last day of baseline in Figure 3. There were no differences among the groups during baseline. During the drug period, however, an analysis of variance showed the groups to be significantly different (F = 17.94, df 3/16, p <.001). A Duncan's test found the vehicle group to be significantly different from the pCPA-50 (p <.005), the pCPA-100 (p <.001) and the pCPA-200 (p <.001) groups. The pCPA—50 group also differed in body weight from the pCPA-200 group (p <.01). 14 .BOMHQQ wasp xmwlofi oz» mcfluSp macaw oaoflno> 020 no wouou momma pudendum :fl mamaficm Mom mwofiuom mspwuumom was msnv .ocflfiommn mcfinsw mxmc mo :oauocsm a ma madam cw oxmucfi Roam: awe: .H mpswflu 15 hm mm mm nm _N t m. m L _ _ P r L P . P b F _ _ Um Jrom 9.4 /. 10m -0» .zm> a on (non I oo. face 0 CON (con 0 tom oacu-hmoa oacv eczomon fiO: UBLVM NVBW (6) HWINI 16 .cofiuom want xmplofi ecu mcfiusw mcoflpomhcfl oaoflno> mafiamm ozu no mhzlm wx\me mm + azm mx\me mm mcfl>fiouon momma wumwcmum :« mfimeflcm How mVOHuom wshw-umom cam wasp .ocfifiommn mcwpsp mxmw mo cowuoczw m mm mewhm ca oxwpcfi seam: :66: .N opswfim 17 oscuu.moa .Im> 4 china 4 oacv eczeman 10h 10mm IO.— HBLVM NVBW (5) axvml 18 .vauom mane xmnuoH ecu mcfiusw macaw ofioflzo> 020 so flooon momma pudendum ca madeflam pom mnofinom mshvuumom use make .ocfifiomma Meagan mxmv mo :ofiuucsm m we ucmwoz xwon ucoouom :mo: .m whamfim 19 pm mm mm nm .N .2 n. m _ F r _ F _ F r r _ c _ _ _ V j CON (can 0 tom 00. (can a on .20.. I .:w> 0 tom now ..,\_.u 56‘. t”\.\ .00. Inc. 3.3-32. 9?... 2.5.2:. (7o) .LHOIBM ACIOQ NVBIN 20 Figure 4 shows a similar plot of percent body weight for animals receiving HMD + S-HTP or saline vehicle injections. The groups did not differ during baseline, but the HMD + S-HTP group weighed significantly less than controls during the lO—day drug treat- ment period (P = 11.3, df 1/18, p <.005). The water intake data for animals in standard cages were also analyzed in terms of g/kg water consumed as shown in Table 1. Analyses of variance revealed the pCPA-50, pCPA-100, pCPA-200, and pCPA vehicle groups to differ significantly on this measure during baseline (F = 4.3, df 3/28, p <.025), drug treatment (F = 24.3, df 3/36, p <.001) and the last nine days post-drug (F = 8.2, df 3/32, p <.001). During baseline the pCPA vehicle group was significantly different from the pCPA—50 (p <.05) and pCPA-100 (p <.05) groups. Also, the pCPA-50 animals differed significantly from the pCPA-200 animals (p <.05) and the pCPA-100 rats were reliably different from the pCPA-200 group (p <.05). While drugs were being administered, the pCPA vehicle group was significantly lower than the pCPA-50 (p <.Ol), pCPA-100 (p <.001) and pCPA-200 (p <.001) groups. The pCPA-50 rats were significantly below the pCPA-100 (p <.Ol) and pCPA-200 (p <.001) rats at this time also. For the last nine post-drug days the pCPA vehicle group was significantly different from the pCPA-50 (p <.001) and pCPA-100 (p <.005) groups; the pCPA-50 group was also reliably different from the pCPA-100 subjects (p <.01). Analysis revealed that the HMD + S-HTP group drank signifi- cantly fewer g/kg water than its vehicle control group during baseline (p <.05), drug treatment (p <.001), the first 10 days post-drug (p <.001) and the last nine post-drug days (p <.001). 21 .BOnnom wane xmwnoH may wannsv mGOnnooncn enunco> mannmm 03» no mnzum mx\wa mm + as: mx\mE mm wcn>nooon momma unmucmum an mHmEncm now meannom wanplumom cam mane .ocnnommn wannzp mxmv mo :Onpocsm m we unmnoz xvon ucounom :moz .v onswna 22 o:.u-.moa 95.70 4 .zm> d oacu oc=omon :Auo_ .umn: ('lo) .LH9I3M A008 NVBW 23 Table l.--Mean water intake in g/kg during each day of baseline, drug treatment and post-drug period for rats of Experiment I housed in standard cages. pCPA Vehicle Group Baseline--72, 81, 87, 80, 90, 77, 86, 78 Drug Treatment--64, 72, 66, 71, 77, 65, 73, 67, 65, 67 First 10 Days Post-drug--83, 81, 80, 78, 75, 81, 84, 78, 80, 83 Last 9 Days Post-drug--8l, 78, 81, 81, 79, 75, 94, 80, 78 pCPA-50 Group Baseline-—78, 103, 89, 90, 87, 80, 91, 89 Drug Treatment--78, 108, 110, 95, 94, 89, 88, 93, 111, 121 First 10 Days Post-drug--94, 93, 84, 87, 79, 89, 9s, 91, 92, 94 Last 9 Days Post-drug--109, 89, 84, 103, 93, 86, 109, 100, 99 pCPA-100 Group Baseline--98, 101, 87, 88, 92, 82, 87, 87 Drug Treatment--88, 141, 178, 143, 135, 120, 127, 127, 138, 130 First 10 Days Post-drug-—120, 122, 95, 73, 78, 78, 80, 88, 91, 86 Last 9 Days Post—drug--92, 82, 83, 102, 89, 89, 102, 99, 87 pCPA-200 Group» Baseline--88, 80, 77, 83, 87, 77, 84, 78 Drug Treatment--96, 208, 178, 150, 140, 136, 126, 114, 118, 128 First 10 Days Post-drug--107, 102, 88, 77, 74, 72, 77, 82, 84, 85 Last 9 Days Post-drug--83, 82, 83, 100, 84, 91, 88, 82, 83 24 Table l.--Continued. S-HTP Group Baseline-—95, 90, 87, 87, 87, 82, 80, 78 Drug Treatment—-66, 73, 82, 82, 96, 89, 87, 90, 84, 88 First 10 Days Post-drug--115, 112, 98, 92, 90, 97, 90, 92, 83, 93 Last 9 Days Post—drug--90, 86, 82, 91, 84, 83, 100, 96, 84 S-HTP Vehicle Group Baseline-—80, 85, 81, 77, 78, 78, 86, 76 Drug Treatment-~72, 76, 65, 63, 66, 66, 70, 68, 68, 68 First 10 Days Post—drug-—80, 82, 78, 76, 76, 83, 83, 77, 75, 86 Last 9 Days Post-drug--83, 82, 83, 100, 84, 91, 88, 82, 83 25 Table 2 provides the mean body metabolism data during baseline, pCPA treatment and post-drug period for the six animals which were housed in metabolism cages. It was expected that the data from these six animals might help to elucidate the causes for the pCPA-induced increases in water intake shown in Figure 1. As it turned out, the large transient increases in water consumption found previously to result from pCPA treatment did not occur among all six of these animals. Inspection of the individual drinking data revealed four quite different responses to 100 mg/kg daily oral doses of pCPA. Three animals actually showed slight decreases in water intake; one changed his intake little; one increased considerably and continued to drink excessively throughout the 10 days; and one other animal responded very similarly to the pCPA-100 group shown in Figure 1. Therefore, the mean data presented in Table 1 do nothing to explain the pCPA-induced water consumption portrayed in Figure 1. It seems unwise to attempt to explain results which, for the most part, failed to be replicated by using data from a single animal who responded in the expected fashion. This unsuccessful attempt to replicate previous results was quite unexpected, since eight other animals housed earlier in metabolism cages and subjected to the same (irug regimen all responded very much like the pCPA-100 group of IFigure 1. Unfortunately, urine samples were not being saved at that time. Only two known differences in procedure occurred during the urnsuccessful attempt to replicate the water intake findings. They Inere: (a) the three-bottle method was not employed so animals had only one water bottle attached to the rear of the cage and (b) the pCPA 26 NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH HH HH HH NH NN NN NN NN NN NN NN NN NN NN NN 4N NN NH NN NH NH H.HEN N23HN> NzHNN NHH NHH HNH NNH NHH NHH NH NHH NHH NHH NH HHH NHH NH NH NH NH NH NN NN NN NN NN NN NN NN NN NN NN NN NN NN No NN No NN HNNNNN Nxnuumnmon 659 we mnones: oonnn umNH can xncoN .oanommp mo see ume esp co pawnoz can mo owwncoonom m we co>nm ma pawnoz atoms NNH NNH NNH NNH NHH oHH oHH NHH NHH NHH oHH NHH NHH NH NHH NH NHH NHH NNH NNH NNH NHH NN NN NN NN NN NN NN NN NN HN NHH NNH NNH NNH H.H\.NNEN onnHHoNHHoHN nnH>HH Nee .00 I I I I 1 N4 ETHANOL CONCENTRAT ION 60 Figure 19. Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving pCPA during the first of three preference test sequences. Figure 20. Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving CMC vehicle during the first of three preference test sequences. MEAN G/ KG ALCOHOL CONSUMED . pCPA I Post-drug 1 A Post-drug 2 O CMC D Post-drug l A Post-drug 2 NI #4 I 6 I 8 V T 10 12 16 20 ETHANOL CONCENTRAT ION 62 treatment. No significant differences among the tests on this measure were found for the CMC vehicle control animals (Figure 18), despite a trend toward higher preferences on later tests, eSpecially at higher concentrations. Figures 19 and 20 provide data on mean g/kg alcohol consumed for pCPA and CMC vehicle groups, respectively. Differences among the tests were significant for each group (F = 11.30, df 2/12, p <.005 for the pCPA group; F = 6.12, df 2/14, p <.025 for the CMC vehicle group). Animals receiving pCPA consumed significantly greater g/kg alcohol on the third test than during either the drug sequence (p <.001) or the second sequence (p <.01), the latter two of which did not differ significantly. A significant increase above the first sequence in g/kg alcohol consumed was found for the CMC vehicle group during the test immediately following treatment (p <.05) and during the third sequence (p <.01), which did not differ from each other. Data from only seven animals were analyzed for the pCPA group, since one animal died on the day after pCPA treatment ended. Interpretation of this information is difficult for two reasons. First, these are the only two groups which were intubated without light ether anesthetization, and the pCPA animals, in particular, did not adapt at all well to the procedure. What influ- ence, if any, this had on their ethanol selection remains unclear. Second, since the treatments were given during the first preference sequence, there is no baseline behavior with which to compare a group's later performance. Direct comparison between experimental and control groups is dubious, since their initial ethanol preference functions could well have been different. Individual differences in ethanol preference are great among rats (Myers 8 Veale, 1970). 63 It does appear though that during drug treatment pCPA animals had a lower preference and consumed less alcohol than controls at the three lowest ethanol concentrations. At higher and thus later con- centrations, both measures are quite low for each group, possibly indicating an effect of the intubation procedure. The tendency for mean increases in selection during the post—pCPA period (Figures 17 and 19) conflicts with the trend for further decreases following pCPA administration when animals got the drug on the second preference sequence (Figures 5 and 7). This again fails to support correlations between ethanol selection and brain serotonin. The very long-lasting depression in ethanol selection caused by pCPA which Veale and Myers (1970) reported was not found when pCPA was given on the first preference test. This may indicate that procedural variables such as animal‘s prior exposure to ethanol may influence the effects produced by pCPA. Finally, Figures 21 and 22 show the mean proportion of ethanol consumed by rats receiving either pCPA or CMC vehicle, respectively, between two preference sequences. No significant changes in preference occurred for either group, although both showed a slight acclimation effect with increased mean preference at the middle concentrations on the second test sequence. Mean g/kg alcohol consumed for the same groups is plotted in Figures 23 and 24. Treatment with pCPA did not result in significantly different amounts consumed during the two tests (Figure 23). On the other hand, animals receiving CMC vehicle between tests significantly increased their consumption of alcohol in g/kg on the second preference sequence (F = 8.58, df 1/7, p <.025). 64 Figure 21. Mean proportion ETOH to total fluids consumed as a function of ethanol concentration for animals receiving pCPA between two preference test sequences. Figure 22. Mean proportion ETOH to total fluids consumed as a ' function of ethanol concentration for animals receiving CMC vehicle between two preference test sequences. MEAN PROPORTION ETOH TO TOTAL FLUIDS CONSUMED 65 1.00 . . Pre-pCPA A Post-pCPA .75 . .50 .. .25 .. 1.00 . O Pre-CMC A Post-CMC .75 d ‘ .50 . .25 'l o .00 ' fl ' I 1 I I U 2 4 6 8 10 12 16 20 ETHANOL CONCENTRATION 66 Figure 23. Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving pCPA between two preference test sequences. Figure 24. Mean g/kg alcohol consumed as a function of ethanol concentration for animals receiving CMC vehicle between two preference test sequences. MEAN G/KG ALCOHOL CONSUMED 67 ‘ Post-pCPA 300. 2.0-l l. 0. I U I I I I 4.0 . O Pre-CMC A Post-CMC 3. 0 q 200. . 1.0 . . . 0.0 I I I I I I I 2 4 6 8 10 12 16 ETHANOL CONCENTRATION 68 These data reveal that post-pCPA ethanol selection is pp£_ decreased from baseline when there has been no association between ethanol preference testing and pCPA administration. The brains of pCPA-treated rats should still have been quite depleted of 5-HT, yet both preference and g/kg alcohol consumed showed only non—significant increases during the post-pCPA test. Even if brain serotonin level was on the rise in these animals, their brains should have been in a condition similar to those of animals shown in Figures 5 and 7 who decreased their selection of ethanol significantly in the post-pCPA period. It therefore seems rather questionable to use decreased brain serotonin level as an explanation for the greatly depressed post-pCPA ethanol selection seen when the drug is given during preference test two . Control animals showed a greater acclimation to ethanol than pCPA-treated rats as seen by their significant increase in g/kg alcohol consumed during the post-CMC test. Thus although pCPA may not have decreased ethanol selection, it may have prevented an acclimation effect. This interpretation is weakened, however, by the fact that the preference of control animals did not also rise significantly. EXPERIMENT III This experiment was intended to determine whether the changes in ethanol self-selection resulting from the use of pCPA to deplete brain serotonin were specific to ethanol solutions. The effects of pCPA treatment were assessed when preference testing procedures similar to those of Experiment 11 were employed but the choice was between either a glucose, saccharin or sodium chloride solution and tap water. Saccharin preference was tested because such solutions are preferred at lower concentrations and rejected at higher ones, as is ethanol. In addition, saccharin has no caloric value. Glucose preference was tested because glucose does have caloric value, as does ethanol, despite the fact that it is preferred at even very high concentrations. And finally, sodium chloride was employed because it also has both accepted and rejected concentration ranges, while having quite significant metabolic consequences for the maintenance of water balance, but no caloric value. Method Subjects Forty-eight adult male albino rats supplied by Holtzman Co. were used. All were 120 days old at the start of experimentation. 69 70 Housing, feeding and lighting conditions were the same as those of Experiment 11. Procedure The animals were randomly divided into six groups of eight rats each and allowed at least two weeks of adaptation to the laboratory before preference testing began. As in Experiment II, three 8-day preference test sequences were conducted. The 3-bottle, 24-hour preference method was again employed with body weights and fluid intakes recorded at approximately the same time each day. Two groups (one experimental and one control) received a choice between tap water and a glucose solution; two received a choice between tap water and a saccharin solution; and two received a choice between tap water and a sodium chloride solution. Concentrations of the solutions offered were increased on each day of a preference sequence as follows: g1ucose--15, 20, 25, 30, 35, 40, 45, and 50% saccharin--.2, .4, .6, .8, 1.2, 1.6, 2.0, and 2.4% sodium chloride--.3, .6, .9, 1.2, 1.5, 1.8, 2.1, and 2.4% Each group received the same test solution on all three preference sequences. Solutions were prepared fresh daily on a weight/weight basis. Drug preparation and administration were the same as in Experiment 1. Beginning two days before the second preference test, the 10 consecutive days of drug or vehicle treatment consisted of: BEEF. TEST #1 PREF. TEST #2 PREF. TEST #3 no drug 100 mg/kg pCPA no drug .no drug vehicle no drug 71 Four water-only days separated each preference sequence. Results and Discussion The same measures of self—selection and the same statistical procedures were employed in Experiment III as had been used in Experiment 11. Table 4 provides the drug treatments, mean daily preference ratios and mean daily g/kg alcohol consumed for groups in this experiment. Statistical analyses are based upon these data. Saccharin Figures 26 and 27 show the mean prOportion saccharin solution consumed by animals receiving pCPA or CMC vehicle, respectively, during the second preference sequence. Significant differences were found among the tests for the pCPA group only (F = 4.12, df 2/14, p <.05). Treatment with pCPA significantly decreased preference from baseline only during the drug period itself (p <.05) and, as Figure 25 reveals, the effect was primarily at the lower concentrations. Mean g/kg saccharin consumed for the pCPA group can be seen in Figure 27, while the same measure is plotted for CMC vehicle controls in Figure 28. As with preference, only the pCPA treated group displayed significant differences among the three tests (F = 4.20, df 2/14, p <.05). Mean g/kg saccharin ingested during the post-pCPA period was significantly less than baseline (p <.05). A similarity, although slightly obscure, can be seen between these saccharin solution results and those obtained with ethanol under the same test procedures (see Figures 5 and 7). Preference for each solution, but not g/kg consumed, was significantly decreased during PCPA treatment. Both measures of selection were significantly reduced 72 Table 4.--Drug treatments, mean daily preference ratios (:_S.E.) and mean daily g/kg alcohol consumed (:_S.E.) for each group of Experiment III. Drug Mean Daily Mean Daily g/kg Treatment Pref. Ratio Alc. Consumed SACCHARIN Test #1 no drug 45 i 0.34 i .02 Test #2 pCPA 32 i 0.30 : Test #3 no drug 36 i 0.24 i .03 Test #1 no drug 39 i 0.32 i 0.06 Test #2 CMC 42 i 0.31 i 0.06 Test #3 no drug 38 i 0.27 i 0.05 GLUCOSE Test #1 no drug 63 i 22 i .5 Test #2 pCPA 52 i 28 i Test #3 no drug 63 i 18 i .8 Test #1 no drug 78 i 30 i .9 Test #2 CMC 73 i 25 i Test #3 no drug 76 i 26 i .l SODIUM CHLORIDE Test #1 no drug 30 i 0.39 i 0.06 Test #2 pCPA 36 i 0.76 i 0.20 Test #3 no drug 15 i 0.20 i 0.08 Test #1 no drug 27 i 0.31 i 0.05 Test #2 CMC 27 i 0.31 i 0.06 Test #3 no drug 22 i 0.23 i 0.04 73 Figure 25. Mean prOportion saccharin solution to total fluids consumed as a function of saccharin concentration for animals receiving pCPA during the second of three preference test sequences. Figure 26. Mean proportion saccharin solution to total fluids consumed as a function of saccharin concentration for animals receiving CMC vehicle during the second of three preference test sequences. MEAN PROPORTION SACCHARIN TO TOTAL FLUIDS CONSUMED 74 l ' 00' . Baseline I pCPA A Post-drug .75. . 50.4 . 25. \— I I I I T I I ‘— 1.004 0 Baseline D CMC A Post-drug .751 .50. . 25 . . 00 t .2 .4 .6 .8 1.2 1.6 2.0 2.4 SACCHARIN CONCENTRAT ION 75 Figure 27. Mean g/kg saccharin consumed as a function of saccharin concentration for animals receiving pCPA during the second of three preference test sequences. Figure 28. Mean g/kg saccharin consumed as a function of saccharin concentration for animals receiving CMC vehicle during the second of three preference test sequences. MEAN G/KG SACCHARIN CONSUMED 76 l ' 00! 0 Baseline I pCPA A Post-drug . 75. .50. 1.00. 0 Baseline D CMC A Post-drug .75- SACCHARIN CONCENTRATION 77 from baseline during the post-pCPA test sequence for ethanol; only g/kg showed a significant post-pCPA drop when saccharin was tested. Mean post-drug preference for saccharin, although not different from baseline preference, was not significantly above the reduced preference during pCPA treatment. To summarize, the results for saccharin and ethanol were quite similar except that preference for saccharin was not as depressed post-drug. Control animals intubated with CMC and tested with either ethanol (Figures 6 and 8) or saccharin (Figures 26 and 28) showed no significant changes whatsoever during any of the three preference tests. The findings for ethanol and saccharin do not correlate perfectly, but they do suggest that pCPA is similarly affecting the self-selection of each. It can be concluded then that pCPA's effects are definitely not specific to ethanol. Unfortunately, there are no other published studies with which to compare the saccharin results. A conflict is apparent with the statement made by Cicero and Hill in 1970. They mentioned unpublished observations of increased saccharin intake due to pCPA. Glucose Mean proportion glucose consumed during pCPA or CMC vehicle treatment is plotted in Figures 29 and 30, respectively. Analysis revealed significant differences among the three tests for the pCPA group (F = 5.31, df 2/14, p <.025), while no reliable differences were found for vehicle controls. During pCPA treatment, preference for glucose was significantly below that during the baseline and post-drug Sequences (p <.05 each time). This effect was evident for nearly all 78 Figure 29. Mean proportion glucose solution to total fluids consumed as a function of glucose concentration for animals receiving pCPA during the second of three preference test sequences. Figure 30. Mean prOportion glucose solution to total fluids consumed as a function of glucose concentration for animals receiving CMC vehicle during the second of three preference test sequences. MEAN PROPORTION GLUCOSE TO TOTAL FLUIDS CONSUMED 1.00‘ “75$ .50. .254 79 0 Baseline I pCPA A Post-drug 1.00- I r I I u CMC A Post-drug j 0 Baseline .75d .50-‘ .25" .00 w I w I w r I I 15 20 25 30 35 40 45 50 GLUCOSE CONCENTRATION 80 concentrations. Figure 31 shows mean g/kg glucose consumed for the pCPA group. It presents quite a different picture than was seen with preference. Significant differences were found among the tests (F = 8.08, df 2/14, p <.005), but rather than being suppressed, g/kg glucose ingested was significantly higher during pCPA treatment than during baseline (p <.05) or the post-drug period (p <.005). Regarding the control animals receiving CMC (Figure 32), significant differences were also found in g/kg consumed among the three sequences (F = 9.57, df 2/14, p <.005). Their glucose ingestion, however, was significantly reduced from baseline during CMC treatment (p <.005) and remained depressed during test number three (p <.005). The elevated consumption of glucose (in g/kg) during pCPA treatment is especially noteworthy when one considers that vehicle intubation actually reduced glucose intake. This finding agrees with an experiment by Nancy and Kilby (1973) which showed sucrose ingestion to be increased by daily 100 mg/kg i.p. injections of pCPA. Four or 12-hour preference tests were employed by them with random presentation of sucrose solutions ranging from 0.5 to 16%. They found preference to be elevated also, while such was not the case here. The discrepancy may be due in part to the longer 24-hour testing procedure employed in this study which allowed more time for post-ingestional factors influencing water intake to come into play. Brody (1970) also reported increases in glucose consumption in unpublished experiments. Nance and Kilby, however, are the only investigators who have reported a successful attempt to reverse pCPA's effects on self-selection by giving 5-HTP. 81 Figure 31. Mean g/kg glucose consumed as a function of glucose concentration for animals receiving pCPA during the second of three preference test sequences. Figure 32. Mean g/kg glucose consumed as a function of glucose concentration for animals receiving CMC vehicle during the second of three preference test sequences. MEAN G/KG GLUCOSE CONSUMED 82 40! . Baseline I pCPA A Post-drug 30* 20- 10-1 I I U i ' T j w‘ 40. ' 0 Baseline D CMC A Post-drug 3o, ’ ° - I 20 . 10 .. O U I I I I T I I 15 20 25 30 35 40 45 50 GLUCOSE CONCENTRATION 83 It may well be that 5-HT neurons are involved in the regulation of carbohydrate intake as Nance and Kilby (1973) suggest. Caloric value of the solution alone apparently does not determine whether pCPA will enhance or reduce its consumption. Both ethanol and glucose contain calories, but only glucose consumption was enhanced. It may be that glucose, an easily digestable source of calories, is less irritating to the digestive mucosa than either dry diet or ethanol. Sweetness of the solution, per se, cannot be the sole determinant of pCPA's effects either. Saccharin, at least at lower concentrations, _ is sweet, as is glucose. Yet saccharin intake was depressed while glucose consumption rose with pCPA treatment. Sodium Chloride Mean pr0portion sodium chloride solution consumed is shown in Figures 33 (pCPA group) and 34 (CMC controls). Analysis revealed significant differences in preference among the three sequences for the pCPA group (F = 9.30, df 2/14, p <.005) but no differences for controls. Treatment with pCPA caused sodium chloride preference to decrease significantly from baseline (p <.01) and drug sequence (p <.005) during the post-pCPA test. To statistically test for concentration effects during pCPA treatment, (Figure 33) a mean daily preference ratio was calculated for each animal for each test at concentrations 539% NaCl and at concentrations :1.2% NaCl. Related measures t-tests (one-tailed) were then conducted. These revealed that for concentrations 339% NaCl, post—drug preference was significantly different from both drug preference (t = 5.66, df 7, p <.001) and baseline preference (t = 7.00, df 7, p <.001). At concentrations 31.2% NaCl, drug preference was not significantly different than either baseline or post—drug preference. 84 Figure 33. Mean proportion NaCl solution to total fluids consumed as a function of NaCl concentration for animals receiving pCPA during the second of three preference test sequences. Figure 34. Mean proportion NaCl solution to total fluids consumed as a function of NaCl concentration for animals receiving CMC vehicle during the second of three preference test sequences. MEAN PROPORTION SODIUM CHLORIDE TO TOTAL FLUIDS CONSUMED 85 1.00‘ 0 Baseline I pCPA APost-drug .75. .50q .25. ‘~.--> t“ 17 I I r I I I— 1.00. OBaseline DCMC APost-drug .75 d .50 a .25 4 .00 SODIUM CHLORIDE CONCENTRATION 86 Figures 35 and 36 show mean g/kg NaCl consumed for the same two groups. Animals given pCPA (Figure 35) showed significant differences in NaCl consumption among the three tests (F = 7.65, df 2/14, p <.01). Mean g/kg consumed was significantly higher during pCPA treatment than it was during either baseline (p <.05) or the post-drug sequence (p <.005). There were no significant differences among the tests for the animals receiving CMC. Concentration effects during pCPA treatment (Figure 35) were again statistically analyzed using one-tailed t-test for related measures. Mean g/kg alcohol consumed were calculated for each animal for each test at concentrations 539% NaCl and :l.2% NaCl. For the lower concentrations post-drug intake was significantly below baseline (t = 9.0, df 7, p <.001) and drug treatment intakes (t = 4.9, df 7, p <.005). Alcohol intake in g/kg during drug treatment concentrations :l.2% NaCl was significantly below baseline (t = 2.17, df 7, p <.05) and post-drug intakes (t = 2.30, df 7, p <.05). These results provide the only example of an increased con- sumption of the little preferred concentrations of a solution near the end of pCPA treatment. Unfortunately, duration of pCPA adminis- tration is confounded with concentration. This makes it impossible to determine from these data whether animals increased their intake of NaCl at high concentrations only or whether they would also have increased at lower concentrations if they had come later in treatment. Preliminary data from three additional animals given a choice between 1.8% NaCl and tap water while receiving daily doses of pCPA indicated that the duration of drug administration may be the important factor. Further experiments are needed to make a precise determination. Ilandom presentation of a range of NaCl solutions during pCPA treatment 87 Figure 35. Mean g/kg NaCl consumed as a function of NaCl concentration for animals receiving pCPA during the second of three preference test sequences. Figure 36. Mean g/kg NaCl consumed as a function of NaCl concentration for animals receiving CMC vehicle during the second of three preference test sequences. MEAN G/KG SODIUM CHLORIDE CONSUMED 88 l ' 00.1 . Baseline I pCPA ‘ Post-drug 075‘ ‘ . 504 . 25¢ w 1. oo .. ' ' ‘ oBaseline DCMC APost—drug .75 . o 50 I . O 25 d I. .ooA:1 I I I I I I I .6 .9 1.2 1.5 1.8 2.1 2.4 SODIUM CHLORIDE CONCENTRATION 89 would be enlightening. There are no data in the literature concerning pCPA's effects on NaCl consumption with which to compare these results. If an increased need for sodium chloride does result from brain serotonin depletion, this may indicate that serotonergic neurons may modulate the gustatory, olfactory or other areas within the brain which are involed in sodium appetite. On the other hand, if the enhanced NaCl intake merely reflects drug toxicity, it may mean that aldosterone secretion is inhibited or kidney reabsorption of sodium is disturbed. However, the metabolism data of Experiment I did not reveal elevated urine sodium concentrations toward the end of drug treatment. It would be interesting to give rats a shorter preference test (one, four, or 12 hours) with a choice between tap water and a normally non- preferred NaCl solution after one 316 mg/kg dose of pCPA. This and other research along these lines is definitely called for. Why there should have been a significantly reduced NaCl intake at the start of preference test three remains unclear. One possibility is that a sequestering of sodium occurred during late drug treatment. Such a sodium accumulation could then have been reduced by an avoidance of the NaCl solution in the post-drug test. Another possibility is that the reduction represents a transitory aversive conditioning factor. A review of the data for each case where a drug was adminis- tered on the second of three preference sequences reveals that, regardless of the solution being offered, either preference and/or g/kg alcohol consumed was suppressed at the start of the post-drug test. GENERAL DISCUSSION The primary thrust of these experiments was to further investi- gate the hypothesized relationship between brain serotonin and ethanol self-selection through the use of pharmacological techniques. The results of several previous experiments were confirmed concerning the effects of pCPA (Myers 6 Veale, 1968; Myers 8 Cicero, 1969; Veale G Myers, 1970; Frey, Magnussen, G Nielsen, 1970; Myers 5 Tytell, 1972; Myers 6 Martin, 1973) and S-HTP (Myers, Evans, G Yaksh, 1972; Geller, 1973) on ethanol self-selection. Both drugs suppressed ethanol intake, each having a more pronounced effect after drug treatment had ended. The pCPA results of Geller (1973) indicating that pCPA enhances ethanol selection were definitely not confirmed. Several lines of evidence converge to support the position taken by Nachman, Lester, and Le Magnen (1970) and Holman, Hoyland, and Shillito (1974) that the reduced ethanol consumption may well represent a conditioned aversion resulting from the pairing of noxious drug effects with the distinct taste of ethanol. The evidence is as follows: (1) chronic administration of both pCPA and HMD + S-HTP caused a reduction in body weight indicating noxious drug effects, (2) pCPA and HMD + S-HTP treatments each reduced the selection of ethanol, although they should have had Opposite effects on brain 90 91 serotonin, (3) HMD + S-HTP (at the one dose employed) failed to reverse the effects of pCPA and restore ethanol selection to baseline levels, (4) pCPA had little effect on ethanol selection when its administration did not coincide with ethanol drinking, and (S) pCPA similarly affected the selection of ethanol and saccharin solutions, indicating that its effects are not specific to ethanol. It is difficult to explain why pCPA affected the consumption of saccharin in a manner rather similar to ethanol, while the intakes of glucose and sodium chloride solutions were affected in quite different ways. The reason may be that glucose, being easily digestable, and sodium chloride, having consequences for water balance, helped the animals to counteract some aspects of drug toxicity. The animals may also have been deconditioned to these substances since they undoubtedly had previous experience with both sodium chloride and carbohydrates. Ethanol, on the other hand, being irritating to the gastrointestinal tract, and saccharin, having no caloric value were probably more likely candidates for conditioned aversion formation. In addition, these substances were novel to the animals. This is an explanation based primarily on post-ingestional factors, rather than on changes in the sensitivity of the chemical senses, taste and small. Sensitivity changes were not assessed in the present experiments. Stronger conclusions concerning brain serotonin's role in fluid self-selection might result from the use of these pharmacological agents if much shorter (i.e., one or four hour) preference tests were used and at least a day intervened between drug administration and preference testing. Also, random or counterbalanced presentation of test concentrations would help to eliminate the confounding of two 92 variables, namely, duration of drug treatment and concentration. These methods should much more reliably determine the significance of gustatory and/or olfactory influences, in particular. Undoubtedly, the greatest liability limiting the interpretation of the present fluid selection experiments, and many other experiments in this area, is the lack of closely corresponding brain S-HT assay data. In those instances where assays have been carried out by others, usually only one or a few S-HT determinations have been made, often coming at the end of drug treatment. No data are available which provide a function for the recovery of brain serotonin level after chronic pCPA or S-HTP adminiatration. This is quite disturbing since many of the effects of these drugs on fluid selection occur in the. post-drug period. Veale and Myers (1970) allude to preliminary assays revealing an overshoot of brain S-HT level after chronic pCPA adminis- tration. No details of these assays have appeared in print. Holman, Hoyland, and Shillito (1974), who used a preference testing methodology very similar to that of Experiments II and III, did made several S-HT determinations following pCPA treatment. They discovered that whole-brain S-HT was reduced during drug administra- tion (sequence two) but not different from control at the end of post-drug sequence three. Their dose regimen (316 mg/kg pCPA two days before sequence two and 100 mg/kg pCPA four days later) was not really chronic, however, and the drug was given intraperitoneally. Despite reduced brain serotonin, the selection of ethanol was not affected by pCPA treatment. The pharmacological manipulations used in these experiments each have their own inherent limitations. When using pCPA one can 93 never reduce brain serotonin level to zero, and the significance of remaining S-HT stores cannot be determined. Peripheral S-HT is depleted by pCPA also. With S-HTP there is no true physiological distribution within the brain and its effects are relatively short- lived. These complications restrict the interpretations which can be drawn from any study using these two drugs to alter brain serotonin level. In addition, manipulation of whole—brain serotonin provides absolutely no insight whatsoever into precisely where in the brain the crucial serotonergic terminals might be located. The use of pCPA and HMD + S-HTP in a relatively long-term preference situation poses other problems due to their influences on water intake as shown in Experiment I. In addition, over a period of days both must be relatively noxious (and probably toxic) to the animals, since each causes a significant reduction in body weight. Despite the methodological limitations, the present experiments have several major procedural advantages over all previous investi- gations of serotonin's involvement in self-selection behavior. First, the possibility of attributing conflicting or unexpected results to strain or supplier differences (Tilson G Rech, 1974) has been eliminated. Second, standardized procedures were employed throughout the experiments and all animals were of very similar ages and body weights. Third, this is the only study in which the effects of pCPA on a variety of test solutions has been determined under such exacting experimental conditions. That there may yet be some relationship between brain serotonin level and ethanol consumption has been indicated by the recent investigations of Ho, Tasi, Chen, Begleiter, and Kissin (1974) and 94 Myers and Melchior (1975). Each group injected 5,6edihydroxytryptamine, either intracisternally or intraventricularly, and found it to enhance ethanol selection. Unfortunately, 5,6-DHT may not be as specific a toxin for S-HT neurons as some would hope (Bjorklund, Nobin, G Stenevi, 1973; Longo, Scotti de Carolis, Liuzzi, G Massotti, 1974; Nygren, Fuxe, Jonsson, 6 Olson, 1974). Two methods of altering central serotonergic processes which no one has utilized in the study of ethanol choice behavior include lesioning and electrical stimulation of the dorsal and/or median raphe nuclei. Application of these tech- niques should prove enlightening, despite their drawbacks. 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The effects of p-chlorophenylalanine on morphine analgesia, tolerance and dependence development in two strains of rats. Psychopharmacologia, 1974, 35, 45-60. Veale, W. L., G R. D. Myers. Decrease in ethanol intake in rats following administration of p-chlorophenylalanine. Neuro- pharmacology, 1970, 9, 317-326. Weissman, A. Behavioral pharmacology of p-chlorophenylalanine (p-CPA). In: Serotonin and Behavior. J. Barchas G E. Usdin (Eds.). New York: Academic Press, 1973, pp. 246-247. Winer, B. J. Statistical Principles in Experimental Desigg, New York: McGraw-Hill, 1962, 672 pp. APPENDICES APPENDIX A DRUGS, SUPPLIES, AND EQUIPMENT APPENDIX A DRUGS, SUPPLIES, AND EQUIPMENT m l. DL-p-chlorophenylalanine (courtesy of Pfizer Inc.) 2. L-a-hydrazino-a-methyl-8-3,4-dihydroxyphenyl propionic acid (courtesy Merck, Sharp G Dohme Co.) 3. DL-S-hydroxytryptophan (Sigma Chemical Co.) Supplies 1. Carboxymethylcellulose (Sigma Chemical Co.) 2. Sodium saccharin (Sigma Chemical Co.) 3. Sodium chloride (M.S.U. General Stores) 4. Glucose (M.S.U. General Stores) 5. Stainless steel drinking spouts (M.S.U. Center for Laboratory Animal Resources) 6. 125 m1. glass bottles (M.S.U. General Stores) 7. #0 rubber stoppers (M.S.U. General Stores) 8. Sec plastic syringes (VWR Scientific) Eguipment 1. Metabolism cages with bases (Acme Metal Products Co.) 2. Dial-o-gram balance (Ohaus) 3. Autogram balance (Ohaus) 4. Magnetic stirrer (Corning Glass Works) 100 101 S. Refractometer (American Optical) 6. Flame photometer model 143 (American Instrumentation Laboratories) APPENDIX B SUMMARY DATA--EXPERIMENT I APPENDIX B SUMMARY DATA--EXPERIMENT I Mean Water Intakes for Rats in Standard Cages pmm 54nP Day Vehicle pCPA-SO pCPA-100 pCPA-200 Vehicle S-HTP-ZS 1 35 39 48 45 4o 47 2 40 52 50 41 43 45 3 43 45 43 40 41 44 4 4o 46 44 43 39 44 5 45 44 46 45 4o 44 6 38 41 41 4o 40 42 7 43 47 44 44 44 41 8 39 46 44 41 39 40 9 32 4o 44 50 37 34 10 36 55 70 107 39 37 11 33 55 86 88 33 41 12 35 47 68 72 32 40 13 38 46 63 66 33 47 14 32 43 55 63 33 43 15 36 42 58 58 35 42 16 33 44 57 52 34 43 17 32 52 62 53 34 4o 18 33 56 58 57 34 42 19 41 44 54 48 4o 56 20 4o 44 56 47 41 55 21 4o 40 44 41 4o 49 22 39 42 34 36 39 46 102 103 fimA 54nP Day Vehicle pCPA-50 pCPA-100 pCPA-200 Vehicle S-HTP-ZS 23 38 38 37 35 39 45 24 41 43 37 34 43 49 25 43 47 38 37 43 46 26 4o 45 42 40 4o 47 27 41 46 44 41 39 43 28 43 47 42 42 45 48 29 42 55 45 41 42 47 3o 40 45 4o 41 38 45 31 42 43 41 42 38 43 32 42 53 51 51 44 48 33 41 48 45 , 43 41 44 34 39 45 45 47 42 44 35 49 57 52 46 42 53 36 42 53 51 43 41 51 37 41 S3 45 44 36 4S 104 Mean Body Weights for Rats in Standard Cages pCPA , S-HTP Day Vehicle pCPA-50 pCPA-100 pCPA-200 Vehicle S-HTP-ZS l 486 497 490 509 498 495 2 487 505 494 512 503 498 3 491 507 495 517 505 503 4 494 509 497 517 506 505 5 497 507 502 519 509 508 6 496 512 501 522 510 508 7 499 515 506 524 514 513 8 498 515 506 526 514 515 9 498 513 501 521 514 514 10 499 508 495 515 513 504 11 498 500: 483 494 509 499 12 495 494 474 479 505 490 13 496 489 468 470 503 488 14 494 483 460 464 501 484 15 494 480 457 460 499 481 16 493 473 449 454 497 479 17 493 469 448 451 499 478 18 491 464 445 446 498 478 19 493 467 449 450 502 486 20 496 473 459 459 502 493 21 502 478 464 465 509 497 22 502 482 466 468 510 500 23 505 482 471 471 511 501 24 506 485 472 474 515 504 25 509 493 475 479 515 510 26 510 492 480 485 518 511 27 512 497 483 489 518 519 28 515 501 485 492 522 518 29 516 504 488 495 525 520 30 514 507 488 498 527 520 31 519 510 493 504 528 522 105 pCPA S-HTP Day Vehicle pCPA-50 pCPA-100 pCPA-200 Vehicle S-HTP-ZS 32 519 515 501 511 529 525 33 521 518 504 514 533 526 34 522 522 505 519 532 528 35 524 524 510 521 536 530 36 526 529 514 525 539 533 37 528 533 518 533 540 535 APPENDIX C SUMMARY DATA-~EXPERIMENT II APPENDIX C SUMMARY DATA--EXPERIMENT II Mean Daily Preference Ratios for Each Animal pCPA (Test #2) Test #1 50 31 49 20 30 63 27 19 (x = 36 s 5.6) Test #2 10 6 29 6 27 36 33 6 (x = 19 s 4.7) Test #3 28 2 l 15 4 28 19 3 (x = 12 i 4.1) pCPA Vehicle (Test #2) Test #1 17 60 35 29 40 26 32 46 (3 = 36 i 4.7) Test #2 2 68 36 49 58 45 43 14 (x = 39 t 7.7) Test #3 0 67 42 so 54 3o 39 39 (X = 40 i 7.0) pCPA (Test #2) + S-HTP (Test #3) Test #1 37 24 34 22 47 57 47 43 (X = 39 t 4.2) Test #2 13 1 14 o 19 36 9 26 (X = 15 i 4.3) Test #3 5 0 11 o 2 55 29 39 (x = 18 s 7.4) pCPA (Test #2) + Vehicle (Test #3) Test #1 28 33 41 33 26 25 36 60 (3 = 35 i 4.0) Test #2 4 so 17 28 21 6 1 16 (x = 18 t 5.6) Test #3 27 40 l 7 11 20 0 39 (x = 18 i 5.6) 5-HTP (Test #2) Test #1 23 52 39 27 41 46 39 50 (8 = 40 i 3.6) Test #2 13 ll 3 38 24 28 27 11 (x = 19 t 4.1) Test #3 8 2o 1 27 40 28 ‘6 34 (x = 20 i 5.0) S-HTP Vehicle (Test #2) Test #1 46 30 51 51 33 46 71 26 (x = 44 i 5.1) Test #2 54 36 24 62 29 57 49 2 (x = 39 i 7.2) Test #3 54 52 54 50 3o 62 56 1 (x = 45 i 7.1) 106 107 pCPA (Test #1) Test #1 10 3 19 2 2 31 11 (x = 37 s 7.6) Test #2 18 41 50 2 3 14 17 (X = 21 i 6.9) Test #3 33 64 56 23 7 49 30 (X = 37 t 7.6) pCPA Vehicle (Test #1) Test #1 17 19 18 23 29 3 18 25 (g = 19 i 2.7) Test #2 24 8 27 43 53 29 11 12 (X = 26 i 5.6) Test #3 44 2 5 37 SS 39 21 46 (X = 31 t 6.9) pCPA (Between Tests) Test #1 21 32 20 28 42 31 19 27 (X = 28 i 2.7) Test #2 16 41 2 48 78 41 34 15 (X = 34 i 8.4) pCPA Vehicle (Between Tests) Test #1 21 27 46 44 37 26 19 13 (g = 29 s 4.2) Test #2 63 54 58 58 50 29 3 22 (X = 42 i 7.6) Mean Daily g/kg Alcohol Consumed by Bach Animal pCPA (Test #2) Test #1 2.18 3.86 1.45 0.82 2.58 1.06 3.56 0.88 (X=2.05:0.42) Test #2 2.34 3.13 2.26 0.59 0.37 0.35 2.45 0.61 (X=l.51i0.40) Test #3 0.19 1.58 1.22 0.19 1.55 0.18 0.19 0.71 (X=0.7310.22) pCPA Vehicle (Test #2) Test #1 0.63 3.61 1.52 1.92 1.95 2.05 1.64 1.58 (X=l.86tO.29) Test #2 0.21 3.16 1.56 2.66 3.41 3.32 2.22 0.65 (X=2.15:0.44) Test #3 0.00 3.72 2.14 3.80 3.25 2.25 2.51 1.38 (X=2.38:0.45) pCPA (Test #2) + 5-HTP (Test #3) Test #1 1.21 0.78 1.56 0.75 3.36 3.01 2.24 2.36 (X=l.91t0.35) Test #2 1.06 0.09 0.83 0.00 1.49 3.57 1.03 2.71 (X=1.35:0.44) Test #3 0.09 0.03 0.11 0.01 0.08 3.85 1.99 1.93 (X=1.01:0.51) pCPA (Test #2) + Vehicle (Test #3) Test #1 1.46 1.76 1.60 2.02 1.64 1.08 1.70 3.12 (X=1.80:0.21) Test #2 0.22 3.77 1.52 2.39 1.66 0.26 0.12 1.21 (X=1.39:0.44) Test #3 0.94 1.94 0.02 0.32 1.09 0.58 0.00 1.26 (X=0.77iO.24) S-HTP (Test #2) Test #1 0.70 2.00 1.50 2.23 2.28 1.70 1.58 2.02 (X=1.7510.18) Test #2 0.53 0.69 0.35 1.59 1.60 1.60 2.19 0.86 (x=1.18:0.23) Test #3 0.27 0.57 0.02 1.05 3.01 1.21 0.07 1.30 (X=0.94i0.34) 108 S-HTP Vehicle (Test #2) Test #1 2.42 0.79 2.10 2.13 1.39 Test #2 3.48 1.07 2.31 2.79 1.87 Test #3 4.36 2.01 2.52 2.42 1.32 pCPA (Test #1) Test #1 1.04 0.21 0.91 0.11 0.14 Test #2 0.79 3.38 3.33 0.08 0.08 Test #3 1.73 4.88 4.77 1.29 1.03 pCPA Vehicle (Test #1) Test #1 0.73 1.16 0.63 0.65 0.94 Test #2 1.32 1.26 1.23 0.15 1.13 Test #3 2.23 3.52 2.73 0.09 0.33 pCPA (Between Tests) Test #1 1.01 1.12 0.67 1.73 2.49 Test #2 0.80 1.76 0.16 3.16 4.61 pCPA Vehicle (Between Tests) Test #1 0.80 1.51 2.89 2.97 .87 Test #2 3.98 3.92 3.94 4.24 2.86 H NCO (NbN NH .16 .38 .63 .89 .84 .91 .23 .69 .74 .29 .22 .85 .60 r—o N044?- N040 .80 .51 .96 .03 .02 .96 .98 .40 .80 .78 .92 .61 .24 mars ><1 II II II NNO Ham NH0 00 .08:0 .44i0 .42:0 .62:0. .3620 .80:0. .81:0. .65:0 .18i0 45) 50) 46) 17) .53) 59) 12) .36) .45) .21) _ .53) .35) _ .57) APPENDIX D SUMMARY DATA--EXPERIMENT III APPENDIX D SUMMARY DATA—-EXPERIMENT III Mean Daily Preference Ratios for Each Animal Saccharin (pCPA) Test #1 52 38 51 47 51 33 33 58 (x = 45 Test #2 26 44 30 24 40 29 26 39 (x = 32 Test #3 47 18 35 26 29 35 46 52 (X = 36 Saccharin (CMC) Test #1 28 51 50 45 43 30 15 49 (g = 39 Test #2 43 37 60 50 30 32 34 48 (x = 42 Test #3 23 28 54 49 18 42 41 46 (X = 38 Glucose (pCPA) Test #1 40 79 51 64 60 65 77 69 (x = 63 Test #2 24 68 50 69 50 51 64 40 (x = 52 Test #3 60 68 58 73 61 60 64 63 (x = 63 Glucose (CMC)_ Test #1 68 76 80 80 84 87 87 59 (x = 78 Test #2 70 66 78 74 77 83 81 54 (x = 73 Test #3 73 71 80 75 80 83 82 62 (x = 76 Sodium Chloride (pCPA) Test #1 30 24 29 37 15 35 48 24 (x = 30 Test #2 24 65 12 37 25 47 52 26 (x = 36 Test #3 10 16 6 12 7 6 42 18 (x = 15 Sodium Chloride (CMC) Test #1 24 32 30 30 25 25 26 27 (g = 27 Test #2 30 28 23 24 39 21 14 36 (x = 27 Test #3 26 18 14 22 25 18 23 32 (x = 22 109 l+l+l+ l+l+l+ I+I+l+ l+|+l+ l+l+l+ naoaoa .blucn u+u+ H MN .4) .7) .1) .6) .7) .6) .6) .4) .8) .4) .3) .5) .5) .2) .2) .0) L'. -h l J Mean Daily Saccharin (pCPA) Test #1 0.42 0.25 Test #2 0.23 0.27 Test #3 0.30 0.08 Saccharin (pCPA) Test #1 Test #2 Test #3 Glucose 0.21 0.30 0.14 (pCPA) Test #1 Test #2 Test #3 Glucose (CMC) Test #1 Test #2 Test #3 23.6 20.6 23.3 0.38 0.20 0.11 0.25 0.44 0.61 0.43 Sodium Chloride (pCPA) 16.6 26.1 15.8 29. 24. 23. CNN 110 g/kg Alcohol Consumed 0.26 0.14 0.15 0.24 by Each 0 N 00 000 29. 22. 17.7 17. 28. 22.8 17. 18. Animal .36 .35 .33 .59 .49 .48 Menu: #mO‘ .02) _ .04) _ .03) .06) .06) .05) .5) .7) .9) .3) .1) Test #1 0.32 0.28 0.39 Test #2 0.34 1.73 0.16 Test #3 0.10 0.21 0.07 0.47 0.71 .50 .69 .06) .20) .08) O O) :5 AAA ><1><1 H I H H"