Interaction of Benzodiazepines with Central Nervous Glycine Receptors: Possible Mechanism of Action (Strychnine Binding/Behavioral Tests) ANNE B

Proc. Nat. Acad. Sci. USA Vol. 71, No. 6, pp. 2246-2250, June 1974

Interaction of Benzodiazepines with Central Nervous Glycine Receptors: Possible Mechanism of Action (strychnine binding/behavioral tests) ANNE B. YOUNG, STEPHEN R. ZUKIN, AND SOLOMON H. SNYDER* Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Communicaed by Cheves Walling, January 29, 1974

ABSTRACT Interaction of 21 benzodiazepines with the ously (4). The specific activity of the labeled strychnine was glycine receptor in the brainstem and spinal cord of rat have been evaluated in terms of their displacement of 13 Ci/mmol, with concentrations determined by comparison ['HIstrychnine binding. The rank order of potency of the with the ultraviolet absorption of standard solutions at 254 21 drugs in displacing specific ['H1strychnine binding cor- nm. relates (p < 0.005) with their rank order of potency in a variety of pharmacological and behavioral tests in Tissue Preparation. Male Sprague-Dawley rats (100-150 g) humans and animals that predict clinical efficacy. There were decapitated and the brainstems and spinal cords re- is a 50-fold variation in potency of the series of benzo- moved. Crude synaptic membrane fractions of these tissues diazepines with mean effective dose (ED5.) values ranging were prepared by the differential centrifugation technique from 19juM to >1000 pM. Diazepam (Valium®) and chlordiazepoxide (Librium®) have EDN's of 26 MM and 200 pM, previously reported (4). respectively, whereas the ED5. for glycine is 25 ;M. The inhibitory effects of 10 of the agents in two other central ner- Binding A8say. To measure specific binding of strychnine vous system membrane receptor assays, for the opiate to spinal cord membranes, aliquots of crude synaptic mem- receptor and the muscarinic cholinergic receptor, do not branes (0.3-1.0 mg of protein) were incubated in triplicate at correlate with any of the in vivo pharmacologic and be- 40 for 10 min in 2 ml of 0.05 M Na-K phosphate buffer (pH havioral tests. The benzodiazepines may exert their anti- 7.1) containing 200 mM NaCl and 2 nM anxiety, anticonvulsant and muscle-relaxant effects by ['H]strychnine mimicking the effects of the neurotransmitter glycine at (38,000 cpm) alone or in the presence of 1 mM glycine or its central nervous system receptor sites. various concentrations of drugs. After incubation, the reaction was terminated by centrifugation for 10 min at 48,000 X g. The most prominent clinical actions of the benzodiazepines, a The supernatant fluid was decanted, and the pellet rinsed with class of drugs including such widely used agents as diazepam 5 ml, then 10 ml of ice-cold 0.15 M NaCl. Bound radioactivity (Valium®) and chlordiazepoxide (Librium®), are relief of was extracted into 1 ml of Protosol (New England Nuclear anxiety, muscular relaxation, and amelioration of convulsive Corp.), 10 ml of toluene phosphor were added and radio- states. A variety of evidence suggests that these drugs en- activity assayed by liquid scintillation spectrometry (Packard hance polysynaptic inhibitory processes in the spinal cord, Tricarb model 3385 or 3375), at a counting efficiency of 34%. brain stem, and thalamus (1). Many inhibitory processes in Specific ['Histrychnine binding was obtained by subtract- these areas are medicated by glycine-containing interneurons ing from the total bound radioactivity the amount not dis- (2, 3). Recently we have identified specific binding of radio- placed by high concentrations (1 mM) of glycine. labeled strychnine associated with glycine receptor sites in the mammalian spinal cord, brain stem and thalamus (4). The Drug. Benzodiazepines were very kindly donated by Dr. relative abilities of amino acids to mimic the neurophysiologic W. Scott and Dr. R. Kuntzman of Hoffman-La Roche, New actions of glycine correlate with their affinities for these bind- Jersey. The structures of these drugs and their Roche ("RO5") ing sites, and the regional distribution of specific strychnine designations are shown in Figs. 1 and 2. binding in the central nervous system closely parallels the dis- Carisoprodol, meprobamate, methaqualone, and tybamate tribution of glycine as well as neurophysiological sensitivity to were kindly donated by Carter-Wallace Laboratories of New glycine (3, 5). In the present study we have examined the Jersey; methocarbamol by Robins Research Laboratories; relative affinities of a series of benzodiazepines for specific and chlorzoxazone from McNeil Laboratories, Fort Washing- strychnine-binding sites in the brain stem, spinal cord, and ton, Pa. Mephenesin was kindly donated by Dorsey Labora- thalamus of the rat and have correlated this data with the tories, Lincoln, Neb. pharmacological actions of the benzodiazepines. Specific binding of ['H]dihydromorphine was assayed in brain homogenates as described separately (9). MATERIALS AND METHODS Binding of ['H ]quinuclidinyl benzilate (QNB) to muscarinic Strychnine was labeled by catalytic tritium exchange at cholinergic receptors was assayed as previously described New England Nuclear Corp. and purified as described prey- (10). RESULTS AND DISCUSSION Abbreviation: ED5o, mean effective dose. * To whom reprint requests should be sent at the Department of A variety of benzodiazepines (Figs. 1 and 2) are effective in Pharmacology. displacing specific [3H]strychnine binding (Table 1). The 2246 Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Benzodiazepines and Glycine Receptors 2247 RO 5-6789 (OXAZEPAM) H° 4 N cK%>

p RO 5-3785 RO 5-2181 H ,0 NHCH DRUG RI Rp x H RO 5-2904 - CFN - -H OH CI NH RO 5-3059 _ _-Hu (NITRAZEPAM) RO 5-3027 -Cl e- CI -H

RO 5-4023 - NO2 O-C I -H (CLONAZ EPAM) c-CF3 -H RO 5-3590 -NO2 RO 5-4933 H RO 5-3636 CHI RO 5-3350 -Br -H (BROMAZEPAM) O-P HCI RO 5-4528 -CN -CH3 cl\CHI DIAZE PAM -Cl -CH3 RO 5-4200 o-CF -CH3 (FLUNITRAZEPAM) -NOg RO 5-4864 -Cl p..CI -CH3 RO 5-4556 CH3 RO 5-4964 Cr3 RO 5-5807 -Cl -CHgCONHCH3 (MEDAZEPAM) RO 5-6901 -Cl O-F -CH (FLURAZEPAM) H2N(CgH5)g *HCI *2 HCI o-F RO 5-6227 -Cl -(CH9)3N (C"2)3 CIl~ HZNXN FIG. 1. Structures of a series of 1,3-dihydro-5-phenyl-2H-1,4- benzodiazepine-2-one derivatives. FIG. 2. Structures of benzodiazepine derivatives in which ring series of 21 drugs exhibits a 50-fold variation in potency with B differs from that of the drugs depicted in Fig. 1. EDso values ranging from 19MuM to >1000 MM. By comparison, glycine has an ED50 of 25 ,M. Diazepam (Valium®) and elicited by minimal or maximal electroshock; these tests are chlordiazepoxide (Librium®), the two most frequently pre- also less effective predictors of benzodiazepine action in scribed drugs in the United States, have ED50 values of 26 humans (6). MAM and 200 MM, respectively. Nitrazepam (Mogadon®) and Thirteen of the benzodiazepines (Fig. 1) possess a common oxazepam (Serax®), also used in this country as anti-anxiety B-ring structure, except for variations in the X-substituent. agents, have ED50 values of 20 ,M and 90 MuM, while fluraze- Dreiding molecular models of glycine can be readily super- pam (Dalmane®), used as a hypnotic, has an ED5o of 28 MM. imposed upon a portion of the B-ring of these 13 agents. By The most potent drug in our series was flunitrazepam, which contrast, models of glycine cannot be as effectively super- is used clinically in Europe. Clonazepam has an ED60 of 32.5 imposed upon the B-rings of the drugs shown in Fig. 2. Inter- MAM and medazepam, used in Europe, is much less effective, estingly, the drugs having the B-ring structure shown in Fig. 1 with an ED5o of 340MAM. tend to be more potent than those having the variant struc- To ascertain whether the interactions of benzodiazepines tures shown in Fig. 2, as indicated in Fig. 3, where the latter with specific strychnine-binding sites are related to their are italicized. The mean ED50 for the drugs in Fig. 1 is signifi- clinical activities, we compared the clinical potency and ac- cantly lower than that for the drugs in Fig. 2 by a Mann- tivity in pharmacological tests that predict clinical activity Whitney U-test (p < 0.01). Despite this implication that (6) with potency in displacing [3H]strychnine binding (Fig. 3, potency correlates with structural similarity to glycine, recent Table 2). Potencies in displacing [8H]strychnine binding cor- studies indicate that strychnine and glycine interact at recep- relate very closely with potencies in a "human bioassay" tor sites in an allosteric fashion (7). Thus, some agents such as based on the minimal dose at which 50% of subjects experi- diazonium tetrazole and acetic anhydride strongly inhibit the ence subjective effects. Similar close correlations occur ability of glycine to displace [3H]strychnine binding but only between displacement of [3H strychnine binding and the slightly affect the ability of nonradioactive strychnine to dis- potencies of benzodiazepines in several animal tests which place [3H]strychnine. Moreover, the Hill coefficient, n, for have been found to be effective predictors of drug potency in displacement of bound [3Hjstrychnine by nonradioactive humans; thus, the potencies in the mouse antifighting, mon- strychnine is 1.0, while the coefficient for glycine displacement key taming, mouse and cat muscle relaxation, antipentylene- of ['H]strychnine is 1.7, indicating a cooperative interaction tetrazole seizure and rat continuous avoidance tests correlate of glycine with strychnine binding (Fig. 4). Displacement of with the strychnine displacement potencies in highly signifi- [1H]strychnine by benzodiazepines has a Hill coefficient of cant fashion. Considerably lower correlation is obtained with 1.0, the same as for strychnine (Fig. 4). This suggests that discrete-trial conditioning experiments, failure of test animals benzodiazepines interact with the strychnine rather than the to escape from electric shock, and prevention of convulsions glycine site of the receptor. Downloaded by guest on September 25, 2021 2248 Physiology: Young et al. Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 1. Interactions of various psychotropic agents TABLE 2. Benzodiazepines: Correlation of behavioral with central nervous glycine, opiate, and muscarinic effects with displacement of [3H]strychnine binding and cholinergic receptors not with opiate or muscarinic cholinergic receptor binding Inhibition of Inhibition of [3H ]strychnine [3H],strychnine A. Benzodiazepines: Correlation of [3H]strychnine binding binding binding displacement with behavioral effects Drug (EDso, jM) Drug (ED60, pM) No. of Statistical Spearman RO 5-4200 RO 5-4528 67.0 drugs signifi- correlation (flunitrazepam) 19.0 RO 5-3590 70.0 Behavioral test tested cance, p coefficient RO 5-3350 RO 5-4933 74.0 (bromazepam) 19.5 RO 5-3785 80.0 Human bioassay 20 <0.001 0.74 RO 5-3059 RO 5-6789 (nitrazepam) 20.0 (oxazepam) 90.0 Fighting mouse test 21 <0. 001 0.71 RO 5-2904 24.0 RO 5-4864 100.0 Antipentylene tetrazole test, mice 20 <0. 004 0.63 Diazepam 26.0 RO 5-5807 104.0 Continuous avoidance, shock RO 5-6901 Chlordiazepoxide 200.0 rate increase, rat 20 <0.004 0.63 (flurazepam) 28.0 RO 5-4556 RO 5-4023 (medazepam) 340.0 Cat muscle relaxation 19 <0. 005 0.67 (clonazepam) 32.5 RO 5-2181 900.0 Monkey taming 19 <0.005 0.65 RO 5-3027 56.0 RO 5-3636 1000.0 Mouse muscle relaxation 20 <0.005 0.61 RO 5-6227 65.0 RO 5-4964 1000.0 Antimaximal electroshock, mice 19 <0. 05 0.46 Discrete trials "trace" avoidance, Inhibition of noise response failure, rat 14 <0. 10 0.51 specific [3H]- Inhibition of dihydromorphine l'H]QNB Antiminimal electroshock, mice 19 <0. 20 0.33 binding binding Continuous avoidance, escape Drug (100 pAM) (% inhibition) Drug (250 MM) (% inhibition) failure, rat 19 <0. 45 0.20 RO 5-3027 41 RO 5-4964 75 B. Benzodiazepines: Affinity for muscarinic cholinergic Diazepam 32 Diazepam 32 receptor fails to correlate with behavioral effects RO 5-4200 RO 5-3027 24 (flunitrazepam) 29 RO 5-3636 22 Human bioassay 9 -0.50 RO 5-4964 26 RO 5-4200 Fighting mouse test 10 -0.03 RO 5-3636 23 (flunitrazepam) 16 Antipentylene tetrazole test, mice 10 -0.36 RO 5-3590 22 Chlordiazepoxide 13 rate RO 5-2904 17 RO 5-3059 Continuous avoidance, shock RO 5-3059 (nitrazepam) 12 increase, rat 10 -0.29 (nitrazepam) 13 RO 5-2904 10 Cat muscle relaxation s 9 -0.44 RO 5-6789 RO 5-3590 6 Monkey taming 9 -0.14 (oxazepam) 11 RO 5-6789 10 -0.22 0.2 5 Mouse muscle relaxation Chlordiazepoxide (oxazepam) Antimaximal electroshock, Mims 10 -0.06 10 -0.22 Central muscle relaxants that had no effect on Antiminimal electroshock, mix,. [3H]strychnine binding at 10 mM Continuous avoidance, escape., failure, rat 10 +0.36 Carisoprodol Meprobamate Mephenesin Tybamate Chloroxazone Methocarbamol Methaqualone C. Benzodiazepines: Affinity for opiate receptor fails to correlate with behavioral effects Drugs having no effect on [1H]strychnine binding at 100 pM Human bioassay 9 0.13 Tetracycline Methysergide Haloperidol Fighting mouse test 10 0.44 Aminosalicylic acid Hippuric Acid Hydroxyzine Antipentylene tetrazole test, mice 10 0.17 Nicotinamide Melatonin Diethyl carbamazine Continuous avoidance, shock rate Pyridoxal * HC1 Tolbutamide Lidocaine- HCO rat 10 -0.10 Dilantin Alloxan Dichlorphenamide increase, a-Naphthylthiourea Ketamine Chlorphenesin Cat muscle relaxation 9 0.18 Urea Lithium Carbonate Ethosuximide Monkey taming 9 0.12 Acetazolamide Ethanol Phenacemide Mouse muscle relaxation 10 0.09 Ethoxzolamide N0050 Carbamazepine Antimaximal electroshock, mice 10 0.22 Methyl-DOPA Reserpine Acetophenetidine 10 Ergocristine Benactyzine Antiminimal electroshock, mice 0.07 Continuous avoidance, escape 10 0.11 Assay of specific [3H]strychnine binding was performed as described in failure, rat Materials and Methods. [3H]Strychnine binding was assayed in triplicate in the presence of six different concentrations of drug and the mean effective of was as described in text. dose (ED60) determined by log-probit analysis., The ED60 values represent Assay [3H]strychnine binding the mean from three separate experiments which varied less than 10%. Assays of [3H]dihydromorphine (9) and [3H]quinuclidinyl Assay of [3Hldihydromorphine (9) and [3H]quinuelidinyl benzilate (10) bind- benzilate (10) binding were performed as previously described. ing were performed as previously described. Values represent the mean of The rank order of potency of the benzodiazepines in inhibiting triplicate determinations and the experiments wvere replicated twice. receptor binding was correlated with the rank order of potency of these drugs in a variety of pharmacological and behavioral rank correlation The first of tests (6) using the Spearman analysis. The close correlation between pharmacological activity eight tests have been shown to correlate well with clinical efficacy the benzodiazepines and displacement of [5H]strychnine bind- of the benzodiazepines, whereas the last three tests have been ing strongly suggests that these drugs exert their pharma- shown to be poor predictors of clinical potency (6). cological activities by interacting with the glycine receptor. Conceivably, relative pharmacological potencies of these might also determine the access of the drugs to strychnine- drugs in vivo are related simply to their ability to reach brain binding sites in our synaptic membrane preparations. If such receptors becai4se of factors, such as lipid solubility, which were the case, the same factors would be expected to govern Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Benzodiazepines and Glycine Receptors 2249

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z 35R 0 3340yWAZ. 3%90 Ia: 4 30027 O< ST0 ~4'of. *m p <0.O 8NHBIIONO*Z. TYHIE IDN 4200 3027 0 .a . a C -. 4 8 12 16 - 20 4 8 12 16 20 4 8 12 16 20 INHIBITION OF [HSTRYCHNINE BINDING INHIBITION OF [3H) STRYCHNINE BINDING INHIBITION OF [3H]STRYCHNINE BINDING RANK RANK RAN K FIG. 3. Plots of the rank potency of a series of benzodiazepines in displacing bound [3H]strychnine versus the rank potency of these drugs in a variety of pharmacological and behavioral tests (6). Rank correlation coefficients (p) and their significance (p) were determined by the Spearman rank correlation analysis. All tests shown except the continuous avoidance, escape failure in the rat have been shown to correlate closely with the clinical efficacy of the drugs (6). CHLOR. is chlordiazepoxide, DIAZ. is diazepam.

interactions of benzodiazepines with other central nervous indicates that the displacing actions of the benzodiazepines system receptors in vitro. To examine this possibility, we represent unique effects. measured the abilities of 10 of the benzodiazepines, including Since anxiolytic properties of the benzodiazepines are the most and least potent, to interact with opiate and mus- closely correlated with the muscle relaxant effects of these carinic cholinergic brain receptors. Displacement of ['H]dihydromorphine binding to opiate receptor sites, or displacement of binding of [3H]quinuclidinyl benzilate to muscarinic cholinergic receptor sites in membrane preparations of rat brain requires concentrations of benzodiazepines 10-50 times higher than are needed for [8H]strychnine displacement (Table 1). Inhibition of opiate and muscarinic receptor binding by benzodiazepines fails to correlate with any of the pharmacological tests (Table 2B, C). Thus, it is highly unlikely ;' n[ = 1 | n = 1.0 n= n 1.O GLYCINE DIAZEPAM RO 5-4200 \ that the correlation of pharmacological activity of benzodiaze- } . ~~~~STRYCHNE

pines with their displacement of [3H]strychnine binding can I be explained simply by nonspecific membrane actions. _1 -5 -4-5 -4 -5 -4 -9.4 -8. Drugs other than the benzodiazepines, most notably pro- LOG [DISPLACER] panediols, exert muscle relaxant effects. These agents fail to FIG. 4. Displacement of [3H]strychnine binding by non- displace [3H]strychnine binding even at concentrations as radioactive glycine, strychnine, diazepam, and RO 5-4200 high as 10 mM. The bearing of their displacing abilities upon (flunitrazepam). Synaptic membrane suspensions (0.6 mg of their mechanism of action must be viewed with caution, since protein per tube) were incubated with 2 nM [3H]strychnine (38,000 cpm) and increasing amounts of displacer at 40 for 10 min. these agents are extremely weak in vivo compared to the Nonspecific binding obtained in the presence of 0.1 mM strych- benzodiazepines. For example, in clinical usage meprobamate nine has been subtracted from all experimental values. The data (Miltown®) is less than 1% as potent as diazepam. The failure are plotted according to the Hill equation. Values are the means of a wide range of psychotropic drugs lacking prominent anti- of triplicate determinations which varied less than 10%. The anxiety actions to displace [3H]strychnine binding (Table 1) experiment has been replicated at least twice. Downloaded by guest on September 25, 2021 0050IQIQ Physiology: Young et al. Proc. Nat. Acad. Sci. USA 71 (1974)

drugs, muscle relaxing actions might be responsible for the F. H. (1970) J. Neurol. Sci. 13, 189-195; Stratten, W. P. & amelioration of anxiety. Alternatively, antianxiety effects, Barnes, C. D. (1971) Neuropharmacol. 10, 68.5-696; Tseng, T. -C. & Wang, S. C. (1971) J. Pharmacol. Eip. Ther. 178, anticonvulsant. effects, and muscle relaxant effects might be 350-360. exerted at different parts of the central nervous system but 2. Aprison, M. H. & Werman, R. (1965) Life Sci. 4, 207.5- utilize the same neuronal mechanism. Glycine neurons and 2083; Graham, L. T., Jr., Shank, R. P. Werman, R. & Apri- receptors apparently exist in both spinal and supraspinal areas son, M. H. (1967) J. Neurochem. 14, 46.5-472; Davidoff, (2-5). It is conceivable that muscle relaxant actions involve an R. A., Graham, L. T., Jr., Shank, R. P., Werman, R. & Aprison, M. H. (1967) J. Neurochem. 14, 1025-1031; enhancement of glycine-mediated synaptic inhibition in the Matus, A. I. & Dennison, M. E. (1971) Brain Res. 32, 195- spinal cord, while antianxiety effects result from enhanced 197; Hbkfelt, T. & Ljungdahl, A. (1971) Brain Res. 32, synaptic inhibition in the brain stem or higher centers. In- 189-194. fluences of benzodiazepines on limbic system activity (6) 3. Curtis, D. R., Hbsli, L. & Johnstori, G. A. R. (1968) Exp. Brain Res. 6, 1-18; Kelly, J. S. & Krnjevic, K. (1969) Exp. might involve glycine receptors in limbic structures (4) or Brain Res. 9, 1.55-163; Galindo, A., Krnjevic, K. & Schwartz, might result secondarily from effects on brainstem glycine S. (1967) J. Physiol. (London) 192, 3.59-377; Werman, R., receptors. The effects of benzodiazepines on seizure activity, Davidoff, R. A. & Aprison, M. H. (1968) J. Neurophysiol. both cortically and subcortically induced, may result from 31, 81-95. potentiation of inhibitory pathways from the brainstem 4. Young, A. B. & Snyder, S. H. (1973) Proc. Nat. Acad. Sci. USA 70, 2832-2836; Snyder, S. H., Young, A.-B., Bennett, reticular activating system. J. P. & Mulder, A. H. (1973) Fed. Proc. 32, 2039-2047. The concentration of benzodiazepine in the blood and cen- 5. Aprison, M. H., Shank, R. P., Davidoff, R. A. & Werman, tral nervous system at pharmacologically active doses (8) is R. (1968) Life Sci. 7, 583-590; Aprison, M. H., Shank, similar to the concentration required to displace [3H ]strychnine R. P. & Davidoff, R. A. (1969) Comp. Biochem. Physiol. 28, 1345-135a5; Arregui, A., Logan, W. J., Bennett, J. P. & binding. Because of the impressive correlation between the Snyder, S. H. (1972) Proc. Nat. Acad. Sci. USA 69, 3485- ability of the benkodiazepines to displace specific strychnine 3489. binding associated with glycine receptor sites and their phar- 6. Zbinden, G. & Randall, L. 0. (1967) Advan. Pharmacol. 5, macological actions, wb propose that the benzodiazepine drugs 213-291. produce their antianxiety, anticonvulsant and muscle-relaxant 7. Young, A. B. & Snyder, S. H., in preparation. 8. Morselli, P. L., Cassano, G. B., Placidi, G. F., Muscettola, effects by mimicking the effects of glycine at its receptor sites G. B. & Rizzo, M. (1973) in The Benzodiazepines, eds. by in the central nervous system. Garattini, S., Mussini, E. & Randall, L. 0. (Raven Press, New York), pp. 129-143. A.B.Y. is a graduate student and recipient of a fellowship from 9. Pert, C. B. & Snyder, S. H. (1973) Science 179, 1011-1014; the Scottish Rite Foundation. S.R.Z. is a year V medical Pert, C. B. & Snyder, S. H. (1973) Proc. Nat. Acad. Sci. student. This research was supported by USPHS Grant MH- USA 70, 2243-2247; Kuhar, M. J., Pert, C. B. & Snyder, 18501, RSDA Award MH-33128 to S.H.S. and grants of the Johh S. H. (1973) Nature 245, 447-450; Pert, C. B., Pasternak, A. Hartford Foundation and Nelson Research and Development, G. & Snyder, S. H. (1973) Science 182, 13.59-1361. Inc. 10. Yamamura, H. I. & Snyder, S. H. (1974) Proc. Nat. Acad. Sci. USA, 71, 1725-1729; Yamamura, H. I., Kuhar, M. J., 1. Schmidt, R. F., Vogel, M. E. & Zimmermann, M. (1967) Greenberg, D. & Snyder, S. H. (1974) Brain Res. 66, 541- Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol. 258, 546; Snyder, S. H., Banerjee, S. P., Yamamura, H. I. & 69-42; Przybyla, A. C. & Wang, S. C. (1968) J. Pharmacol. Greenberg, D. M. (1974) Science, in press; Snyder, S. H., Exp. Ther. 163, 439-447; Hudson, R. D. & Wolpert, M. K. Greenberg, D. M. & Yamamura, H. I. (1974) Arch. Gen. (1970) Neuropharmacol. 9, 481-488; Takanishi, T. & Norris, Psychiat., in press. Downloaded by guest on September 25, 2021