- Email: [email protected]
Aging and benzodiazepine binding in the rat cerebral cortex
Life Sciences, Vol. 30, pp. 343-346 Printed in the U.S.A.
Pergamon Press
AGING AND BENZODIAZEPINE BINDING IN THE RAT CEREBRAL CORTEX C.C. Tsang 1, K.V. Speeg, 3r. 2, and G.R. Wilkinson l 1
D e p a r t m e n t of Pharmacology Vanderbilt University and D e p a r t m e n t of Medicine 2 Veterans Administration Medical C e n t e r Nashville, Tennessee 37232 (Received in final form December 3, 1981)
SUMMARY The specific binding of 3H-diazepam in the cerebral cortex was investigated in membrane preparations from 6 and 30 month old Fischer-344 rats. No agerelated differences in the association, equilibrium, or dissociation binding characteristics were observed. The increased sensitivity of the elderly to the central sedative effects of the benzodiazepines does not, therefore, appear to involve changes in binding to the receptor site located in the cortex. Clinical experience, studies (I-3), and epidemiological data (4, 5) indicate that aging, especially in patients older than 67 years, leads to an increase in the central nervous system's responsiveness to the benzodiazepines. For certain individual drugs this alteration may result from age-related changes in drug disposition (6, 7), however, there are indications that an alteration in central sensitivity may also occur (I, 2). Recently, high a f f i n i t y , saturable and stereospecific bindings sites for benzodiazepines in the brain have been discovered (8,9) and characterized (10-13) both in vivo and in vitro. Correlation of binding a f f i n i t y with the rank order of clinical potency for a wide variety of benzodiazepines (8-I 3) strongly suggests that these "receptors" are c r i t i c a l l y involved in the action of this class of drugs. Accordingly, we have investigated whethe~ post-maturity aging leads to a change in the in vitro specific binding characteristics of H-diazepam, using washed membranes of the rat cerebral cortex as a model system. METHODS Female Fischer-344 rats aged 6-7 months and 29-30 months were obtained from Charles River Breeding Laboratories (Wilmington, MA) under contract to the National Institute on Aging, and stabilized for 3-14 days with food and water ad libitum. Following decapitation, the weighed cerebral cortexes from two rats in each age group (n=20) were pooled and homogenized in 25 volumes of ice-cold Krebs-Ringer-Tris HCI 15mM buffer, pH 7.4 at 4°C, using a Brinkman Polytron at the maximum setting for l0 seconds. The homogenate was centrifuged at 48,000 x g for 10 minutes at 4°C and the resulting pellet was resuspended and washed three times with the ice-cold buffer. The final 1:~5 w/v homogenate was then immediately used to investigate the binding characteristics of H-diazepam. Binding was studied in triplicate at 4°C using an aliquot of homogenate equivalent to about 0.5 mg protein, H-diazepam (94 Ci/mmole, Amersham Corp., Arlington Heights, IL) and Krebs-Ringer-Tris HCI 15raM buffer, pH 7.4 to a final volume of 2 ml, in the presence or absence of IteM diazepam. A f t e r incubation, the samples were rapidly filtered through 0024-3205/82/040343-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
344
Aging and Benzodiazepine
Receptors
Vol. 30, No. 4, ]982
Whatman GF/C glass fiber filters and i m m e d i a t e l y washed twice with 5ml of ice-cold buffer. The filters were then placed in glass scintillation vials, 10ml Aquasol (New England Nuclear Corp., Boston, MA) was added and the r a d i o a c t i v i t y determined by liquid scintillation spectrometry using the external standards ratio method for quench correction. Specific binding was defined as the difference between total binding in the presence and absence of excess diazepam. Protein was determined by the method of Lowry et al. (14) using bovine serum albumin as a standard. The rate of association of ligand ~nd binding site(s) was studied by incubating the samples for 0.75 to 20 minutes with 2nM H - d i a z e p ~ . . Dissociation of the binding complex was also investigated by incubating with 2nM H-diazepam but [or 30 minutes followed by serial sampling over lg minutes after the addition of 2p.M diazepam. Logarithmic transformation of the time courses of association and dissociation were used to estimate the rate constants of these processes(IS). Equilibri%m binding parameters, K_ and Bmax, were determined by incubating with 0.5 to 10nM H-dlazepam for 30 minutes prior to f i l t r a t i o n . The resulting data was then analyzed according to Scatchard (16). A t w o - t a i l e d Student's t-test was used to compare the mean parameter values obtained from the two groups of rats with p < 0.05 being taken as the l i m i t of significance. _
.
.
l )
.
RESULTS The rate of formation of the specific 3H-diazepam-binding site complex was rapid with equilibrium being reached within 10-I5 minutes. Logarithmic transformation of the data indicated thatlthe a~sociation was monophasic with a rate constant (k 1) in the young rats of about 0.l n M - ' m i n - " (Table l). Dissociation of the specific binding complex followed firstorder kinetics (k l ) over the period of study with a h a l f - l i f e of 4-5 minutes (Table 1). No differences were-observed in the kinetics of the binding process to homogenates obtained from the young and old rats. Incubation with various concentrations of 3H-diazepam under equilibrium conditions showed that the specific binding was saturable. Scatchard analysis indicated that over the concentration range 0.5 to 10.0 nM only a single class of binding sites was present with a dissociation constant (Kr~) of about 3 nM and a maximal binding capacity (Bmax) of about 900 fmoles/mg protein.'" Again there were no statistical differences in the cortical synaptosomal binding characteristics in young and old rats (Table I). TABLE l Specific Binding Parameters (Mean -+ S.D.) of 3H-13iazepam to Cerebral Cortex Homogenate of Young (6 Month) and Old (30 Month) Fischer-34~ Rats Young n--10
Old n=10
k l , n M - l . m i n -1
0.094 _+ 0.042
0.I00 _+ 0.039
k_l, min-1
0.156 +- 0.036
0.144 +_ 0.029
k_l/kL, nM
2.44 +_ 2.20
1.62 +- 0.56
KD, nM
3.I0 + 0.56
3.02 -+ 0.75
[~max, f m o l e / r n g p r o t e i n
973 +- 165
934 _+ 94
No significant differences exist between the mean values for the young and old rats.
Vol. 30, No. 4, 1982
Aging and Benzodiazepine Receptors
345
DISCUSSION Aging is often associated with changing drug responsiveness and, in the case of centrallyacting agents, it often leads to an enhanced pharmacological effect. With benzodiazepines such an increase in sensitivity is well-recognized clinically (l-5), and a similar phenomenon also appears to exist in the Fischer-344 rat (17). The relative roles and importance of changes in either drug disposition and/or pharmacodynamic [actors in such altered responsiveness is not well-defined, however, it is frequently speculated that an age-related change in receptor characteristics is probably contributory. The absence of any major age-related changes in the pharmacokinetics of diazepam in the Fischer-3~4 rat (l g) despite alterations in its effect on learning/memory (]7) supports this hypothesis. Howegver, the present findings indicate that receptor function, as assessed by specific binding of "H-diazepam to a membrane preparation from rat cortex, is not different in old animals compared to young but mature rats. Subsequent to completion of the present study, similar results were recently reported for the cortex and several other brain regions with the exception of the ~ippocampus by Memo et al. (19), and in a preliminary report (20) the equilibrium binding of H-flunitrazepam was found not to be significantly different in the cortex and cerebellum of young and senescent rats. The benzodiazepine specific binding site in the brain is now known to be part of a functionally coupled GABA-benzodiazepine-ionophorewhich may be modulated by a variety of [actors (10-13). It is, therefore, conceivable that the couplingof this receptor complex might be affected by aging in a fashion resulting in increased responsiveness not detectable by in vitro binding determinations using an essentially GABA-free membrane preparation. Possible causes for such a change might include age-related changes in the brain levels of GABA (21), anions such as chloride, or an endogenous inhibitor(s) of binding (10-13). Additionally, it is clear that specific binding is only an initial recognition step that leads to a sequence of events terminating in a v a r i e t y of pharmacological effects. L i t t l e is known or understood of these subsequent steps and it is possible that aging leads to changes at this level of the benzodiazepine's mechanism of action. Benzodiazepine receptors are unevenly distributed throughout the brain, with the highest concentration found in the cerebral cortex (g, 9). Moreover, it is becoming increasingly clear that at least two types of receptors are present with differential localization and with distinctive pharmacological, biochemical, and possibly functional characteristics (22-24). The present study did not distinguish between the receptor sub-populations, however, Pedigo et al. have reported no differences in the effects of aging on the two types of receptors in the rat cortex and cerebellum (20). It has been suggested (22-2t~) that the Type I site is involved in the anxiolytic properties of the benzodiazepines, however, the roles of the different receptors and more particularly the precise brain region important in any particular effect are not well-defined. In this regard it should be noted that the reported increases in responsiveness of elderly patients (I-5) are to the sedative effects of the benzodiazepines. It may well be that age-related changes in specific binding to a subpopulation of receptors in brain regions other than the cortex might occur (19) and be pharmacologically relevant to the enhanced drug effects observed in the elderly. ACKNOWLEDGEMENTS The technical assistance of Sherry Wang is greatly appreciated, and the research was supported by grant AG 01395 from the United States Public Health Service.
REFEPENCES 1. 2.
C.M. CASTLEDEN, C.F. GEORGE, D. MARCER and C. HALLETT, Brit. Med. 3. I_ 1012, (1977). M.M. REIDENBERG, M. LEVY, 1-1. WARNER, C.B. COUTINHO, M.A. SCHWARTZ, G.
346
3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24.
Aging and Benzodiazepine Receptors
Vol. 30, No. 4, 1982
YU and 3. CHERIPKO, Clin. Pharmacol. Ther. 23 371-374 (1978) H.G. GILES, S.M. MACLEOD, 3.R. WRIGHT and E.M. SELLERS, Can. Med. Assoc. 3. II8 513-514(1978). BOSTON COLLABORATIVE DRUG SURVEILLANCE PROGRAM, New Engl. 3. Med. 288 277-280 (1973). D.3. GREENBLATT, M.D. ALLEN and R.I. SHADER, Clin. Pharmacol. Ther. 21 355361 (1977). G.R. WILKINSON, Acta. Psychiat. Scand. Suppl 274 56-74 (1978). D.J. G R E E N B L A T T a n d R . I . SHADER, Arzneim.Forsch. 30 886-890 (1980). R.F. SQUIRES and C. BRAESTRUP, Nature 266 732-734-C1977). H. M O H L E R a n d T . OKADA, Science 198 849-871 (1977). E. COSTA, G. DI CHIARA and G.L. GESSA, GABA and the Benzodiazepine Receptors (Advances in Biochemical Psychopharmacology Vol. 26), Raven Press, New York ( 1980). R.C. SPETH, R.W. 3OHNSON, 3. REGAN, T. REISINE, R.M. KOBAYASHI, N. BRESOLIN, W.R. ROESKEand H.I. YAMAMURA, Fed. Proc. 39 3032-3038(1980). C. BRAESTRUP and M. NIELSEN, Arzneim.Forsch. 30 852-857--(1980). S.M. PAUL, P.3. MARANGOS and P. SKOLNICK, Biol-- Psychiat. 16 213-229 (19gl). O.H. LOWRY, N.3. ROSEBOROUGH, A.L. FARR and R.3. RANDALL, 3. Biol. Chem. 193 267-275 (1971). R.---C.SPETH, G.3. WASTEK, P.C. 3OHNSON and H.I. YAMAMURA, Life Sci. 22 g59866 (1978). G. SCATCHARD, Ann. N.Y. Acad. Sci. 7.__11660-672 (1949). H.L. KOMISKEY, T.bl. COOK, C.-F. LIN and W.L. HAYTON, Psychopharmacol. 73 30t~-305 (19gl). C . F . C . TSANG and G.R. WILKINSON, Pharmacologist 22 230 (1980). M. MEMO, P.F. SPANO and M. TRABUCCHI, 3. Pharm. Pharmacol. __3364 (1981). N.W. PEDIGO, 3.N. McDOUGAL, T.F. BURKS and H.I. YAMAMURA, Fed. Proc. 40 311 (1981). P.L. McGEER and E.G. McGEER, 3. Neurochem. 26 65-76 (1976). A.S. LIPPA, D. CRITCHETT, M.C. SANO, C.A. KLEPNER, E.N. GREENBLATT, 3. COUPET, and B. BEER, Pharmac. Biochem. Behav. __10 831-843 (1979). C.A. KLEPNER, A.S. LIPPA, 13.I. BENSON, M.C. SANO and B. BEER, Pharmacol. Biochem. Behav. 11 477-462 (1979). E.F. WILLIAMS, K.--C. RICE, S.M. PAUL and P. SKOLNICK, 3. Neurochem. 35 591-597 (1980).
Pergamon Press
AGING AND BENZODIAZEPINE BINDING IN THE RAT CEREBRAL CORTEX C.C. Tsang 1, K.V. Speeg, 3r. 2, and G.R. Wilkinson l 1
D e p a r t m e n t of Pharmacology Vanderbilt University and D e p a r t m e n t of Medicine 2 Veterans Administration Medical C e n t e r Nashville, Tennessee 37232 (Received in final form December 3, 1981)
SUMMARY The specific binding of 3H-diazepam in the cerebral cortex was investigated in membrane preparations from 6 and 30 month old Fischer-344 rats. No agerelated differences in the association, equilibrium, or dissociation binding characteristics were observed. The increased sensitivity of the elderly to the central sedative effects of the benzodiazepines does not, therefore, appear to involve changes in binding to the receptor site located in the cortex. Clinical experience, studies (I-3), and epidemiological data (4, 5) indicate that aging, especially in patients older than 67 years, leads to an increase in the central nervous system's responsiveness to the benzodiazepines. For certain individual drugs this alteration may result from age-related changes in drug disposition (6, 7), however, there are indications that an alteration in central sensitivity may also occur (I, 2). Recently, high a f f i n i t y , saturable and stereospecific bindings sites for benzodiazepines in the brain have been discovered (8,9) and characterized (10-13) both in vivo and in vitro. Correlation of binding a f f i n i t y with the rank order of clinical potency for a wide variety of benzodiazepines (8-I 3) strongly suggests that these "receptors" are c r i t i c a l l y involved in the action of this class of drugs. Accordingly, we have investigated whethe~ post-maturity aging leads to a change in the in vitro specific binding characteristics of H-diazepam, using washed membranes of the rat cerebral cortex as a model system. METHODS Female Fischer-344 rats aged 6-7 months and 29-30 months were obtained from Charles River Breeding Laboratories (Wilmington, MA) under contract to the National Institute on Aging, and stabilized for 3-14 days with food and water ad libitum. Following decapitation, the weighed cerebral cortexes from two rats in each age group (n=20) were pooled and homogenized in 25 volumes of ice-cold Krebs-Ringer-Tris HCI 15mM buffer, pH 7.4 at 4°C, using a Brinkman Polytron at the maximum setting for l0 seconds. The homogenate was centrifuged at 48,000 x g for 10 minutes at 4°C and the resulting pellet was resuspended and washed three times with the ice-cold buffer. The final 1:~5 w/v homogenate was then immediately used to investigate the binding characteristics of H-diazepam. Binding was studied in triplicate at 4°C using an aliquot of homogenate equivalent to about 0.5 mg protein, H-diazepam (94 Ci/mmole, Amersham Corp., Arlington Heights, IL) and Krebs-Ringer-Tris HCI 15raM buffer, pH 7.4 to a final volume of 2 ml, in the presence or absence of IteM diazepam. A f t e r incubation, the samples were rapidly filtered through 0024-3205/82/040343-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
344
Aging and Benzodiazepine
Receptors
Vol. 30, No. 4, ]982
Whatman GF/C glass fiber filters and i m m e d i a t e l y washed twice with 5ml of ice-cold buffer. The filters were then placed in glass scintillation vials, 10ml Aquasol (New England Nuclear Corp., Boston, MA) was added and the r a d i o a c t i v i t y determined by liquid scintillation spectrometry using the external standards ratio method for quench correction. Specific binding was defined as the difference between total binding in the presence and absence of excess diazepam. Protein was determined by the method of Lowry et al. (14) using bovine serum albumin as a standard. The rate of association of ligand ~nd binding site(s) was studied by incubating the samples for 0.75 to 20 minutes with 2nM H - d i a z e p ~ . . Dissociation of the binding complex was also investigated by incubating with 2nM H-diazepam but [or 30 minutes followed by serial sampling over lg minutes after the addition of 2p.M diazepam. Logarithmic transformation of the time courses of association and dissociation were used to estimate the rate constants of these processes(IS). Equilibri%m binding parameters, K_ and Bmax, were determined by incubating with 0.5 to 10nM H-dlazepam for 30 minutes prior to f i l t r a t i o n . The resulting data was then analyzed according to Scatchard (16). A t w o - t a i l e d Student's t-test was used to compare the mean parameter values obtained from the two groups of rats with p < 0.05 being taken as the l i m i t of significance. _
.
.
l )
.
RESULTS The rate of formation of the specific 3H-diazepam-binding site complex was rapid with equilibrium being reached within 10-I5 minutes. Logarithmic transformation of the data indicated thatlthe a~sociation was monophasic with a rate constant (k 1) in the young rats of about 0.l n M - ' m i n - " (Table l). Dissociation of the specific binding complex followed firstorder kinetics (k l ) over the period of study with a h a l f - l i f e of 4-5 minutes (Table 1). No differences were-observed in the kinetics of the binding process to homogenates obtained from the young and old rats. Incubation with various concentrations of 3H-diazepam under equilibrium conditions showed that the specific binding was saturable. Scatchard analysis indicated that over the concentration range 0.5 to 10.0 nM only a single class of binding sites was present with a dissociation constant (Kr~) of about 3 nM and a maximal binding capacity (Bmax) of about 900 fmoles/mg protein.'" Again there were no statistical differences in the cortical synaptosomal binding characteristics in young and old rats (Table I). TABLE l Specific Binding Parameters (Mean -+ S.D.) of 3H-13iazepam to Cerebral Cortex Homogenate of Young (6 Month) and Old (30 Month) Fischer-34~ Rats Young n--10
Old n=10
k l , n M - l . m i n -1
0.094 _+ 0.042
0.I00 _+ 0.039
k_l, min-1
0.156 +- 0.036
0.144 +_ 0.029
k_l/kL, nM
2.44 +_ 2.20
1.62 +- 0.56
KD, nM
3.I0 + 0.56
3.02 -+ 0.75
[~max, f m o l e / r n g p r o t e i n
973 +- 165
934 _+ 94
No significant differences exist between the mean values for the young and old rats.
Vol. 30, No. 4, 1982
Aging and Benzodiazepine Receptors
345
DISCUSSION Aging is often associated with changing drug responsiveness and, in the case of centrallyacting agents, it often leads to an enhanced pharmacological effect. With benzodiazepines such an increase in sensitivity is well-recognized clinically (l-5), and a similar phenomenon also appears to exist in the Fischer-344 rat (17). The relative roles and importance of changes in either drug disposition and/or pharmacodynamic [actors in such altered responsiveness is not well-defined, however, it is frequently speculated that an age-related change in receptor characteristics is probably contributory. The absence of any major age-related changes in the pharmacokinetics of diazepam in the Fischer-3~4 rat (l g) despite alterations in its effect on learning/memory (]7) supports this hypothesis. Howegver, the present findings indicate that receptor function, as assessed by specific binding of "H-diazepam to a membrane preparation from rat cortex, is not different in old animals compared to young but mature rats. Subsequent to completion of the present study, similar results were recently reported for the cortex and several other brain regions with the exception of the ~ippocampus by Memo et al. (19), and in a preliminary report (20) the equilibrium binding of H-flunitrazepam was found not to be significantly different in the cortex and cerebellum of young and senescent rats. The benzodiazepine specific binding site in the brain is now known to be part of a functionally coupled GABA-benzodiazepine-ionophorewhich may be modulated by a variety of [actors (10-13). It is, therefore, conceivable that the couplingof this receptor complex might be affected by aging in a fashion resulting in increased responsiveness not detectable by in vitro binding determinations using an essentially GABA-free membrane preparation. Possible causes for such a change might include age-related changes in the brain levels of GABA (21), anions such as chloride, or an endogenous inhibitor(s) of binding (10-13). Additionally, it is clear that specific binding is only an initial recognition step that leads to a sequence of events terminating in a v a r i e t y of pharmacological effects. L i t t l e is known or understood of these subsequent steps and it is possible that aging leads to changes at this level of the benzodiazepine's mechanism of action. Benzodiazepine receptors are unevenly distributed throughout the brain, with the highest concentration found in the cerebral cortex (g, 9). Moreover, it is becoming increasingly clear that at least two types of receptors are present with differential localization and with distinctive pharmacological, biochemical, and possibly functional characteristics (22-24). The present study did not distinguish between the receptor sub-populations, however, Pedigo et al. have reported no differences in the effects of aging on the two types of receptors in the rat cortex and cerebellum (20). It has been suggested (22-2t~) that the Type I site is involved in the anxiolytic properties of the benzodiazepines, however, the roles of the different receptors and more particularly the precise brain region important in any particular effect are not well-defined. In this regard it should be noted that the reported increases in responsiveness of elderly patients (I-5) are to the sedative effects of the benzodiazepines. It may well be that age-related changes in specific binding to a subpopulation of receptors in brain regions other than the cortex might occur (19) and be pharmacologically relevant to the enhanced drug effects observed in the elderly. ACKNOWLEDGEMENTS The technical assistance of Sherry Wang is greatly appreciated, and the research was supported by grant AG 01395 from the United States Public Health Service.
REFEPENCES 1. 2.
C.M. CASTLEDEN, C.F. GEORGE, D. MARCER and C. HALLETT, Brit. Med. 3. I_ 1012, (1977). M.M. REIDENBERG, M. LEVY, 1-1. WARNER, C.B. COUTINHO, M.A. SCHWARTZ, G.
346
3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24.
Aging and Benzodiazepine Receptors
Vol. 30, No. 4, 1982
YU and 3. CHERIPKO, Clin. Pharmacol. Ther. 23 371-374 (1978) H.G. GILES, S.M. MACLEOD, 3.R. WRIGHT and E.M. SELLERS, Can. Med. Assoc. 3. II8 513-514(1978). BOSTON COLLABORATIVE DRUG SURVEILLANCE PROGRAM, New Engl. 3. Med. 288 277-280 (1973). D.3. GREENBLATT, M.D. ALLEN and R.I. SHADER, Clin. Pharmacol. Ther. 21 355361 (1977). G.R. WILKINSON, Acta. Psychiat. Scand. Suppl 274 56-74 (1978). D.J. G R E E N B L A T T a n d R . I . SHADER, Arzneim.Forsch. 30 886-890 (1980). R.F. SQUIRES and C. BRAESTRUP, Nature 266 732-734-C1977). H. M O H L E R a n d T . OKADA, Science 198 849-871 (1977). E. COSTA, G. DI CHIARA and G.L. GESSA, GABA and the Benzodiazepine Receptors (Advances in Biochemical Psychopharmacology Vol. 26), Raven Press, New York ( 1980). R.C. SPETH, R.W. 3OHNSON, 3. REGAN, T. REISINE, R.M. KOBAYASHI, N. BRESOLIN, W.R. ROESKEand H.I. YAMAMURA, Fed. Proc. 39 3032-3038(1980). C. BRAESTRUP and M. NIELSEN, Arzneim.Forsch. 30 852-857--(1980). S.M. PAUL, P.3. MARANGOS and P. SKOLNICK, Biol-- Psychiat. 16 213-229 (19gl). O.H. LOWRY, N.3. ROSEBOROUGH, A.L. FARR and R.3. RANDALL, 3. Biol. Chem. 193 267-275 (1971). R.---C.SPETH, G.3. WASTEK, P.C. 3OHNSON and H.I. YAMAMURA, Life Sci. 22 g59866 (1978). G. SCATCHARD, Ann. N.Y. Acad. Sci. 7.__11660-672 (1949). H.L. KOMISKEY, T.bl. COOK, C.-F. LIN and W.L. HAYTON, Psychopharmacol. 73 30t~-305 (19gl). C . F . C . TSANG and G.R. WILKINSON, Pharmacologist 22 230 (1980). M. MEMO, P.F. SPANO and M. TRABUCCHI, 3. Pharm. Pharmacol. __3364 (1981). N.W. PEDIGO, 3.N. McDOUGAL, T.F. BURKS and H.I. YAMAMURA, Fed. Proc. 40 311 (1981). P.L. McGEER and E.G. McGEER, 3. Neurochem. 26 65-76 (1976). A.S. LIPPA, D. CRITCHETT, M.C. SANO, C.A. KLEPNER, E.N. GREENBLATT, 3. COUPET, and B. BEER, Pharmac. Biochem. Behav. __10 831-843 (1979). C.A. KLEPNER, A.S. LIPPA, 13.I. BENSON, M.C. SANO and B. BEER, Pharmacol. Biochem. Behav. 11 477-462 (1979). E.F. WILLIAMS, K.--C. RICE, S.M. PAUL and P. SKOLNICK, 3. Neurochem. 35 591-597 (1980).