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Displacement of serotonin from binding sites in rat cortex: The effects of biogenic “trace” amines
copyright
DISPLACEMENT
ROBERT A.
0
13278-5846~84 $0.00 + so 1984 ~erymor. Press Ltd.
OF SEROTONIN FROM BINDING SITES IN RAT CORTEX: EFFECTS OF BIOGENIC “TRACE” AMINES
LOCOCK,
GLEN B. BAKER, RONALD
Neurochemical Research Unit, Department of Pharmacy and Pharmaceutical Sciences,
T. COUTTS,
and WILLIAM
of Psychiatry, Faculty University of Alberta,
THE
G. DEWHURST
of Medicine and Faculty Edmonton, Alberta, Canada
(Final form, June 1984)
Abstract Displacement of Locock, Robert A., Glen B. Baker, Ronald T. Coutts and William G. Dewhurst: Prog. serotonin from binding sites in rat cortex: The effect of biogenic "trace" amines. Neuro-Psychopharmacol. & Biol. Psychiat. 1984, 8 (4-6): 701-704. 1.
2. 3.
4.
The concentrations for 50 percent inhibition of binding (IC5O's) to specific -in vitro serotonin binding sites (5-HTl and 5-HT2) of rat cerebral cortex were determined for the trace amines 2_phenylethylamine, m- and E-tyramine, tryptamine, and (+I- and (-Icr-methyltryptamine. Tryptamine gave an ICSO of 66.7 f 4.8 nM (n = 7) at the 5-HTl site and an IC50 of 3.85 f 0.16 pM (n = 7) for the 5-HT2 binding site. The IC50 values for all the other compounds were in the micromolar range and were different at the two binding sites except for g-tyramine (IC50, 5-HTl = ICSO, 5-HT2 = 17 PM. The trace amines may have different degrees of displacement of serotonin
functional roles as evidenced by their different at 5-HTl and 5-HT2 binding sites in the brain.
Keywords: serotonin receptor, binding, 2_phenylethylamine, amine, (+)-ct-methyltryptamine, (-)-cr-methyltryptamine.
_m-tyramine,
E-tyramine,
trypt-
Introduction 5-HT) is a neurotransmitter in the central nervous system Serotonin (5_hydroxytryptamine, where it elicits both inhibitory and excitatory physiological and pharmacological responses (Aghajanian, 1981). These observations suggest the existence of multiple types of receptors in vitro binding studies for serotonin and two types of "receptors" have been proposed from -The "receptors" detected in brain tis(Peroutka and Snyder, 1979; Peroutka et al., 1981). binding sites and the receptors in sue with [3H]serotonin are called 5-HTl high affinit frontal cerebral cortex preparations detected with [ JHlspiperone are designated the 5-HT2 receptors (Peroutka and Snyder, 1979; Peroutka et al., 1981). In general, 5-HT agonists bind with high affinity to the 5-HTl site, whereas antagonists have a higher affinity for the 5-HT2 site (Leysen, 1981). The term receptor for the 5-HTl binding site is inappropriate since a physiological role has not been demonstrated for this site (Leysen, 1983). Recently an increase in the 5-HT2 binding sites in the frontal cortex of suicide victims was reported (Stanley and Mann, 1983). Whether thj.s observation is related to the patient's psychiatric state or to drug treatment is an important problem in the interpretction of post mortem studies of neuropsychiatric disorders (Reynolds, 1983). A decrease in ["Hlspiperone-, labelled serotonin receptors (5-HT2) has been reported to be characteristic of antidepresSince large amounts of "trace" amines such as sant drugs (Peroutka and Snyder, 1980). 2-phenylethylamine, m- and E-tyramine and tryptamine may accumulate in brain after monoamine oxidase inhibitor antidepressant drugs (Boulton and Juorio, 19821, we have investigated the ability of these biogenic amines to interfere with the -in vitro binding of serotonin and the 5-HTl and 5-HT2 sites in rat cerebral cortex preparations.
701
702
R. A. Locock et al.
Methods Animals. Male Sprague-Dawley rats (ZOO-250 g) were obtained from the Small Animals Program, University of Alberta, Edmonton. [3H]Spiperone(sp act 26.3 Ci/mmol) and [3H]5-hydroxytryptaminecreatinine sulfate (2???i/mmol) were purchased from New England Nuclear, Boston, MA. 5-Hydroxytryptamine creatinine sulfate complex, 2-phenylethylaminehydrochloride, tryptamine hydrochloride, Etyramlne hydrochloride and pargyllne hydrochloride were obtained from Sigma Chemical, St. Louis, MO. Mianserin hydrochloride was a gift of Organon Canada Ltd., Westhill, Ontario. Meta-tyraminewas purchased from Vega Biotechnologies,Tucson, AZ. L-Ascorbic acid and tris (+)- and (-)(hydroxymethyl) aminomethane came from Fisher Scientific, Fairlawn, NJ. o-methyltryptaminehydrochloride were prepared by separate crystallization of the resolved tartaric acid salts of DL a-methyltryptamine hydrochloride from the Aldrich Chemical Co., Milwaukee, WI, liberation of the free base with alkali and formation of the hydrochloride salts. Tissue Preparation. Rats were decapitated and the frontal cerebral cortex removed. The tissue was homogenixed immediately in 10 volumes of 0.32 M sucrose using a teflon-glass homogenizer. The homogenate was centrifuged at 700 x g for 10 min at 4 degrees. The supernatant liquid was decanted and centrifuged at 50,000 x g for 10 min at 4 degrees. The resulting pellet was resuspended in 10 volumes of 50 mM tris-HCl buffer (pH 7.5) and was incubated at 37 degrees for 10 min and centrifuged again at 50,000 x g for 10 min. The pellet was resuspended in 50 mM tris-HCl buffer, pH 7.7, containing 4 mM calcium chloride, 10 nM pargyllne hydrochloride and 0.1% ascorbic acid. This final tissue suspension was stored on ice for no more than 4 hr before use. The final tissue concentration of frontal cortex was 10 mg/ml. Binding Assay. Binding assays were performed in triplicate. Incubation tubes contained 0.8 ml of the tissue suspension, 100 pl of the i3H]ligands and 100 pl of the various drug solutions in H 7.7 tris-HCl buffer. The ligand concentrationswere 2 nM for i3H]5-HT and 0.24 nM for JYH]spiperone. The tubes were incubated at 37 degrees: 10 min for [3H]5-HT and 15 mln for [ Hlspiperone. Rapid filtration under vacuum through Whatman CF/B filters (Whatman Inc., Clifton, NJ) with three 5 ml rinses of ice-cold 50 mM tris-HCl buffer (pH 7.7) terminated incubation. The filters were counted in 5 ml of scintillation fluor (4.0 g butyl-PBD [Sigma Chemical, St. Louis, MO] in 677 ml toluene and 333 ml Trlton X-100) after extraction for 18 hr at 4 degrees. Non-specific binding was determined by incubation in the presence of 3 uM 5-HT for 5-HTl sites and 3 uM mianserin hydrochloride for 5-HT2 sites. The data from binding assays were plotted in the form of a Hill plot, log (% bound/lOO-% bound) vs log concentration of the inhibitor. A computer program "Paired Sample Analysis" for the Hewlett Packard model 86 minicomputer (Hewlett Packard, Corvallis, OR) was used to fit the data by the method of least squares to a straight line. The concentration of the inhibitor for 50 percent inhibition of specific binding, the IC50 value, was determined by solving the equation for the line for log I (log of the concentration of inhibitor) when log (% bound/loo-% bound) was equal to zero.
Results The IC50 values are presented in Table 1. Each value includes the standard error of the mean (SE) followed by n, the number of separate determinations.
Discussion and Conclusions It has been suggested that there may be more than one population of f3H]5-HT binding sites and that agonists and antagonists may have differential effects at these sites (Leysen, 1981). Our results illustrate the marked differential effects of all the trace amines not only between the two serotonin sites but also among the trace amines themselves (structure activity relationships). The one exception to these observations is p-tyramine, which was equally potent at both 5-HTl and 5-HT2 recognition sites. Tryptamine was the most potent trace amine we studied, and our IC50 values are in reasonable agreement with values reported (Leysen and Tolleneare, 1982): 5-HTl binding, hippocampus, Ki = 197 nM, 5-HT2 binding, frontal cortex, Ki = 5.42 nM.
703
Displacement of serotonin by biogenic amines
Table 1 Serotonin Binding Site Inhibition by Biogenic Amines
IC50, nM f SE (n) Amine 2-phenylethylamine m-tyramine $-tyramine tryptamine (+)-ormethyltryptamine (-)-cr--methyltryptamine
5-HTl 4.39 f 1.83 l 17.23 * 66.73 f 2.52 f 5.12 f
0.67 (6) 0.38 (5) 2.6 (6) 4.8 nM (7) 0.02 (5) 0.06 (4)
5-HT2 39.12 f 37.83 f 17.39 f 3.85 l 7.97 f 12.28 f
4.13 (6) 8.9 (7) 1.8 (6) 0.16 (7) 0.08 (6) 0.06 (5)
The IC50 values presented in Table 1 assume a straight line fit for all the data found for the Hill plot. A closer examination of the data for 2-phenylethylamine and rtyramine reveals that the inhibition of the 5-HT2 binding site is multiphasic. We have also observed that the Hill slopes for both these amines inhibiting both 5-HT binding sites were significantly less than unity, again indicating multiple binding sites and a complex interaction of the trace amines with serotonin binding sites. These observations are under current study and are consistent with the suggestions (Schnellmann et al., 1984) that there may be more than one type of 5-HTl binding site and that valid comparisons of the 5-HTl sites with functional receptors will be made when compounds become available for characterizationof each type of 5-HT binding site. Although a-methyltryptaminehas not been identified as an endogenous amine in the central nervous system, it has been included in this study because it represents the a-methyl analogue of tryptamine, it is a pharmacologicallyactive amine (Dewhurst and Marley, 1965), and it exists as two optical isomers. A significant difference in the IC50 values for (+)- and (-)-a-methyltryptamines was observed for both 5-HT.1and 5-HT2 binding sites. This result may indicate some stereospecificity in the serotonin receptor (recognition)site. Receptor binding studies of the trace biogenic amines and the serotonin receptor (specific binding) sites may yield important information on the functional roles of serotonin at the specific membrane receptors as well as information about the pharmacological and/or physiological functions of the trace amines themselves.
Acknowledgements This study was supported by the Medical Research Council of Canada and the Alberta Mental Health Research Fund. The authors are grateful to Dr. R. G. Micetich and Mr. J. Pyrozko for isolation of the a-methyltryptamine isomers and to Mrs. J. van Muyden for expert technical assistance.
References AGHAJANIAN, G. K. (1981) The modulatory role of serotonin at multiple receptors in the brain. In: Serotonin Neurotransmissionand Behavious, B. L. Jacobs and A. Gelperin (eds), pp. 156-185. MIT Press, Cambridge, MA. BOULTON, A. A.. and JUORIO, A. V. (1982) Brain trace amines. In: Handbook of Neurochemistry, vol. 1, A. Lathja (ed), pp. 189-222. Plenum Press, New York. DEWHURST, W. G. and MARLEY, E. (1965) The effect of e-methyl derivatives of noradrenaline, phenylethylamine and tryptamine on the central nervous system of the chicken. Brit. J. Pharmacol. 25: 682-704. LEYSEN, J. E--(1981) Serotonergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro. J. Physiol. (Paris) -77: 351-362.
704
R. A. Locock et al.
LEYSEN, .I. (1983) Serotonin binding sites: is there pharmacological or clinical significance? Med. Biol. 61: 139-143. LEYSEN, J. E. and TOLLENAERR, .I. P. (1982) Biochemical models for serotonin receptors. Ann. Reports in Med. Chem. 17: l-10. PEROUTRA, S. J., LEBOVITZ, R.%., and SNYDER, S. H. (1981) TWO distinct serotonin receptors with different physiological functions. Science 212: 827-829. PEROUTRA, S. J. and SNYDER, S. H. (1979) Multiple Gtonin receptors: differential binding of [3H]5-hydroxytryptamine,f3H]lysergic acid diethylamide and [3H]spiroperidol. Mol. Pharmacol. 16: 687-699. PEROUTRA, S. x and SNYDER, S. H. (1980) Long-term antidepressant treatment decreases spiroperidol-labeledserotonin receptor binding. Science 210: 88-90. REYNOLDS, G. P. (1983) Serotonin receptors in suicide victims. Lancet ii: 977. SCHNRLLMANN,R. G., WATERS, S. J., and NELSON, D. L. (1984) [3H15-Hydr~ytryptamine binding sites: species and tissue variation. J. Neurochem. 42: 65-70. STANLEY, M. and MANN, J. J. (1983) Increased serotoninbinding sites in frontal cortex of suicide victims. Lancet i: 214-216.
Inquiries and reprint requests should be addressed to: Dr. R. A. Locock Department of Psychiatry Clinical Sciences Building University of Alberta Edmonton, Alberta, Canada T6G 2G3
DISPLACEMENT
ROBERT A.
0
13278-5846~84 $0.00 + so 1984 ~erymor. Press Ltd.
OF SEROTONIN FROM BINDING SITES IN RAT CORTEX: EFFECTS OF BIOGENIC “TRACE” AMINES
LOCOCK,
GLEN B. BAKER, RONALD
Neurochemical Research Unit, Department of Pharmacy and Pharmaceutical Sciences,
T. COUTTS,
and WILLIAM
of Psychiatry, Faculty University of Alberta,
THE
G. DEWHURST
of Medicine and Faculty Edmonton, Alberta, Canada
(Final form, June 1984)
Abstract Displacement of Locock, Robert A., Glen B. Baker, Ronald T. Coutts and William G. Dewhurst: Prog. serotonin from binding sites in rat cortex: The effect of biogenic "trace" amines. Neuro-Psychopharmacol. & Biol. Psychiat. 1984, 8 (4-6): 701-704. 1.
2. 3.
4.
The concentrations for 50 percent inhibition of binding (IC5O's) to specific -in vitro serotonin binding sites (5-HTl and 5-HT2) of rat cerebral cortex were determined for the trace amines 2_phenylethylamine, m- and E-tyramine, tryptamine, and (+I- and (-Icr-methyltryptamine. Tryptamine gave an ICSO of 66.7 f 4.8 nM (n = 7) at the 5-HTl site and an IC50 of 3.85 f 0.16 pM (n = 7) for the 5-HT2 binding site. The IC50 values for all the other compounds were in the micromolar range and were different at the two binding sites except for g-tyramine (IC50, 5-HTl = ICSO, 5-HT2 = 17 PM. The trace amines may have different degrees of displacement of serotonin
functional roles as evidenced by their different at 5-HTl and 5-HT2 binding sites in the brain.
Keywords: serotonin receptor, binding, 2_phenylethylamine, amine, (+)-ct-methyltryptamine, (-)-cr-methyltryptamine.
_m-tyramine,
E-tyramine,
trypt-
Introduction 5-HT) is a neurotransmitter in the central nervous system Serotonin (5_hydroxytryptamine, where it elicits both inhibitory and excitatory physiological and pharmacological responses (Aghajanian, 1981). These observations suggest the existence of multiple types of receptors in vitro binding studies for serotonin and two types of "receptors" have been proposed from -The "receptors" detected in brain tis(Peroutka and Snyder, 1979; Peroutka et al., 1981). binding sites and the receptors in sue with [3H]serotonin are called 5-HTl high affinit frontal cerebral cortex preparations detected with [ JHlspiperone are designated the 5-HT2 receptors (Peroutka and Snyder, 1979; Peroutka et al., 1981). In general, 5-HT agonists bind with high affinity to the 5-HTl site, whereas antagonists have a higher affinity for the 5-HT2 site (Leysen, 1981). The term receptor for the 5-HTl binding site is inappropriate since a physiological role has not been demonstrated for this site (Leysen, 1983). Recently an increase in the 5-HT2 binding sites in the frontal cortex of suicide victims was reported (Stanley and Mann, 1983). Whether thj.s observation is related to the patient's psychiatric state or to drug treatment is an important problem in the interpretction of post mortem studies of neuropsychiatric disorders (Reynolds, 1983). A decrease in ["Hlspiperone-, labelled serotonin receptors (5-HT2) has been reported to be characteristic of antidepresSince large amounts of "trace" amines such as sant drugs (Peroutka and Snyder, 1980). 2-phenylethylamine, m- and E-tyramine and tryptamine may accumulate in brain after monoamine oxidase inhibitor antidepressant drugs (Boulton and Juorio, 19821, we have investigated the ability of these biogenic amines to interfere with the -in vitro binding of serotonin and the 5-HTl and 5-HT2 sites in rat cerebral cortex preparations.
701
702
R. A. Locock et al.
Methods Animals. Male Sprague-Dawley rats (ZOO-250 g) were obtained from the Small Animals Program, University of Alberta, Edmonton. [3H]Spiperone(sp act 26.3 Ci/mmol) and [3H]5-hydroxytryptaminecreatinine sulfate (2???i/mmol) were purchased from New England Nuclear, Boston, MA. 5-Hydroxytryptamine creatinine sulfate complex, 2-phenylethylaminehydrochloride, tryptamine hydrochloride, Etyramlne hydrochloride and pargyllne hydrochloride were obtained from Sigma Chemical, St. Louis, MO. Mianserin hydrochloride was a gift of Organon Canada Ltd., Westhill, Ontario. Meta-tyraminewas purchased from Vega Biotechnologies,Tucson, AZ. L-Ascorbic acid and tris (+)- and (-)(hydroxymethyl) aminomethane came from Fisher Scientific, Fairlawn, NJ. o-methyltryptaminehydrochloride were prepared by separate crystallization of the resolved tartaric acid salts of DL a-methyltryptamine hydrochloride from the Aldrich Chemical Co., Milwaukee, WI, liberation of the free base with alkali and formation of the hydrochloride salts. Tissue Preparation. Rats were decapitated and the frontal cerebral cortex removed. The tissue was homogenixed immediately in 10 volumes of 0.32 M sucrose using a teflon-glass homogenizer. The homogenate was centrifuged at 700 x g for 10 min at 4 degrees. The supernatant liquid was decanted and centrifuged at 50,000 x g for 10 min at 4 degrees. The resulting pellet was resuspended in 10 volumes of 50 mM tris-HCl buffer (pH 7.5) and was incubated at 37 degrees for 10 min and centrifuged again at 50,000 x g for 10 min. The pellet was resuspended in 50 mM tris-HCl buffer, pH 7.7, containing 4 mM calcium chloride, 10 nM pargyllne hydrochloride and 0.1% ascorbic acid. This final tissue suspension was stored on ice for no more than 4 hr before use. The final tissue concentration of frontal cortex was 10 mg/ml. Binding Assay. Binding assays were performed in triplicate. Incubation tubes contained 0.8 ml of the tissue suspension, 100 pl of the i3H]ligands and 100 pl of the various drug solutions in H 7.7 tris-HCl buffer. The ligand concentrationswere 2 nM for i3H]5-HT and 0.24 nM for JYH]spiperone. The tubes were incubated at 37 degrees: 10 min for [3H]5-HT and 15 mln for [ Hlspiperone. Rapid filtration under vacuum through Whatman CF/B filters (Whatman Inc., Clifton, NJ) with three 5 ml rinses of ice-cold 50 mM tris-HCl buffer (pH 7.7) terminated incubation. The filters were counted in 5 ml of scintillation fluor (4.0 g butyl-PBD [Sigma Chemical, St. Louis, MO] in 677 ml toluene and 333 ml Trlton X-100) after extraction for 18 hr at 4 degrees. Non-specific binding was determined by incubation in the presence of 3 uM 5-HT for 5-HTl sites and 3 uM mianserin hydrochloride for 5-HT2 sites. The data from binding assays were plotted in the form of a Hill plot, log (% bound/lOO-% bound) vs log concentration of the inhibitor. A computer program "Paired Sample Analysis" for the Hewlett Packard model 86 minicomputer (Hewlett Packard, Corvallis, OR) was used to fit the data by the method of least squares to a straight line. The concentration of the inhibitor for 50 percent inhibition of specific binding, the IC50 value, was determined by solving the equation for the line for log I (log of the concentration of inhibitor) when log (% bound/loo-% bound) was equal to zero.
Results The IC50 values are presented in Table 1. Each value includes the standard error of the mean (SE) followed by n, the number of separate determinations.
Discussion and Conclusions It has been suggested that there may be more than one population of f3H]5-HT binding sites and that agonists and antagonists may have differential effects at these sites (Leysen, 1981). Our results illustrate the marked differential effects of all the trace amines not only between the two serotonin sites but also among the trace amines themselves (structure activity relationships). The one exception to these observations is p-tyramine, which was equally potent at both 5-HTl and 5-HT2 recognition sites. Tryptamine was the most potent trace amine we studied, and our IC50 values are in reasonable agreement with values reported (Leysen and Tolleneare, 1982): 5-HTl binding, hippocampus, Ki = 197 nM, 5-HT2 binding, frontal cortex, Ki = 5.42 nM.
703
Displacement of serotonin by biogenic amines
Table 1 Serotonin Binding Site Inhibition by Biogenic Amines
IC50, nM f SE (n) Amine 2-phenylethylamine m-tyramine $-tyramine tryptamine (+)-ormethyltryptamine (-)-cr--methyltryptamine
5-HTl 4.39 f 1.83 l 17.23 * 66.73 f 2.52 f 5.12 f
0.67 (6) 0.38 (5) 2.6 (6) 4.8 nM (7) 0.02 (5) 0.06 (4)
5-HT2 39.12 f 37.83 f 17.39 f 3.85 l 7.97 f 12.28 f
4.13 (6) 8.9 (7) 1.8 (6) 0.16 (7) 0.08 (6) 0.06 (5)
The IC50 values presented in Table 1 assume a straight line fit for all the data found for the Hill plot. A closer examination of the data for 2-phenylethylamine and rtyramine reveals that the inhibition of the 5-HT2 binding site is multiphasic. We have also observed that the Hill slopes for both these amines inhibiting both 5-HT binding sites were significantly less than unity, again indicating multiple binding sites and a complex interaction of the trace amines with serotonin binding sites. These observations are under current study and are consistent with the suggestions (Schnellmann et al., 1984) that there may be more than one type of 5-HTl binding site and that valid comparisons of the 5-HTl sites with functional receptors will be made when compounds become available for characterizationof each type of 5-HT binding site. Although a-methyltryptaminehas not been identified as an endogenous amine in the central nervous system, it has been included in this study because it represents the a-methyl analogue of tryptamine, it is a pharmacologicallyactive amine (Dewhurst and Marley, 1965), and it exists as two optical isomers. A significant difference in the IC50 values for (+)- and (-)-a-methyltryptamines was observed for both 5-HT.1and 5-HT2 binding sites. This result may indicate some stereospecificity in the serotonin receptor (recognition)site. Receptor binding studies of the trace biogenic amines and the serotonin receptor (specific binding) sites may yield important information on the functional roles of serotonin at the specific membrane receptors as well as information about the pharmacological and/or physiological functions of the trace amines themselves.
Acknowledgements This study was supported by the Medical Research Council of Canada and the Alberta Mental Health Research Fund. The authors are grateful to Dr. R. G. Micetich and Mr. J. Pyrozko for isolation of the a-methyltryptamine isomers and to Mrs. J. van Muyden for expert technical assistance.
References AGHAJANIAN, G. K. (1981) The modulatory role of serotonin at multiple receptors in the brain. In: Serotonin Neurotransmissionand Behavious, B. L. Jacobs and A. Gelperin (eds), pp. 156-185. MIT Press, Cambridge, MA. BOULTON, A. A.. and JUORIO, A. V. (1982) Brain trace amines. In: Handbook of Neurochemistry, vol. 1, A. Lathja (ed), pp. 189-222. Plenum Press, New York. DEWHURST, W. G. and MARLEY, E. (1965) The effect of e-methyl derivatives of noradrenaline, phenylethylamine and tryptamine on the central nervous system of the chicken. Brit. J. Pharmacol. 25: 682-704. LEYSEN, J. E--(1981) Serotonergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro. J. Physiol. (Paris) -77: 351-362.
704
R. A. Locock et al.
LEYSEN, .I. (1983) Serotonin binding sites: is there pharmacological or clinical significance? Med. Biol. 61: 139-143. LEYSEN, J. E. and TOLLENAERR, .I. P. (1982) Biochemical models for serotonin receptors. Ann. Reports in Med. Chem. 17: l-10. PEROUTRA, S. J., LEBOVITZ, R.%., and SNYDER, S. H. (1981) TWO distinct serotonin receptors with different physiological functions. Science 212: 827-829. PEROUTRA, S. J. and SNYDER, S. H. (1979) Multiple Gtonin receptors: differential binding of [3H]5-hydroxytryptamine,f3H]lysergic acid diethylamide and [3H]spiroperidol. Mol. Pharmacol. 16: 687-699. PEROUTRA, S. x and SNYDER, S. H. (1980) Long-term antidepressant treatment decreases spiroperidol-labeledserotonin receptor binding. Science 210: 88-90. REYNOLDS, G. P. (1983) Serotonin receptors in suicide victims. Lancet ii: 977. SCHNRLLMANN,R. G., WATERS, S. J., and NELSON, D. L. (1984) [3H15-Hydr~ytryptamine binding sites: species and tissue variation. J. Neurochem. 42: 65-70. STANLEY, M. and MANN, J. J. (1983) Increased serotoninbinding sites in frontal cortex of suicide victims. Lancet i: 214-216.
Inquiries and reprint requests should be addressed to: Dr. R. A. Locock Department of Psychiatry Clinical Sciences Building University of Alberta Edmonton, Alberta, Canada T6G 2G3