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Autoradiographic localization of imipramine binding in rat brain
European Journal of Pharmacology, 77 (1982) 363-364 Elsevier/North-Holland BiomedicalPress
363
Rapid communication A U T O R A D I O G R A P H I C LOCALIZATION OF IMIPRAMINE BINDING IN RAT BRAIN THOMAS C. RAINBOW *, ANAT BIEGON and BRUCE S. McEWEN The Rockefeller University, New York, N.Y. 10021, U.S.A.
Received 17 December 1981, accepted 21 December 1981
The brain possesses a specific, high-affinity binding site for the tricyclic anti-depressant, imipramine, an inhibitor of 5HT re-uptake (Langer et al., 1980; Sette et al., 1981). The high-affinity binding site for imipramine may be associated with the presynaptic uptake site for 5HT. There is a good correlation between the potency of drugs to block 5HT uptake and to compete for in vitro binding (Langer et al., 1980), and like the presynaptic uptake site, the high-affinity imipramine recognition site is depleted by lesions of 5HT cell bodies in the dorsal raphe nucleus (Sette et al., 1981). We have used the recent LKB film method of receptor autoradiography (Palacios et al., 1981; Rainbow et al., 1981) to determine the distribution of imipramine binding sites in sections of rat brain. We find that the location of imipramine binding sites parallels the known distribution of 5HT terminals in rat brain. This provides additional evidence that [3H]imipraminelabels the presym,ptic uptake site for 5HT, and offers the possibility of labeling presynaptic uptake sites in vitro for q u a l i t a t i v e autoradiography, Our procedure was to cut frozen 32/~m sections from a male rat brain, thaw-mount them onto subbed glass slides, and incubate the slides in vitro with [3H]imipramine to label binding sites. In preliminary experiments, we found that it was necessary to use a buffer with a high sodium concentration to obtain an adequate ratio of total to non-specific binding (3: 1). Our labeling procedure was to incubate sections in 50 mM Tris HC1 * To whom all correspondence should be addressed: The Rockefeller University, 1230 York Ave, New Yor, N.Y. 10021, U.S.A.
buffer, pH 7.4, containing 300 mM NaC1, for 1 h at 40. The sections were incubated with 1-10 nM of [3H]imipramine (24 or 32 Ci/mmol; New England Nuclear), with or without 100 # M cold desmethylimipramine to measure non-specific binding, or [3H]imipramine with various concentrations of unlabeled drugs to determine the pharmacological profile of the binding site. The sections were washed twice for 20 min in 40 buffer, and were either apposed against LKB Ultrofilm as described (Rainbow et al., 1981) to generate autoradiograms, or were removed from the slides with Whatman filter disks for direct measurement of radioactivity. The amount of radioactivity on the filter disks was measured by liquid scintillation counting. The binding of imipramine to the brain sections was specific, saturable and of a high affinity. Scatchard analysis of the binding indicated that [3H]imipramine labeled a single population of sites with a K D of 2 - 6 nM, and a capacity of 150 fmol/section. Imipramine was 600 times more potent than desmethylimipramine, a selective N E uptake inhibitor, in displacing the binding of [3 H]imipramine, and 60000 times more potent than the non-tricyclic antidepressant, mianserin, which does not inhibit 5HT uptake. On the basis of these results, we used a concentration of 4 n M [3H]imipramine for routine autoradiographic labeling, and measured non-specific binding by coincubation with 100/~M desmethylimipramine. The high-affinity binding sites for imipramine were distributed heterogeneously throughout the rat brain. There was little or no specific binding over white matter, or in the reticular formation and ventral thalamus. There were moderate numbers of specific binding sites in a variety of brain
0014-2999/82/0000-0000/$02.75 © 1982 Elsevier/North-HollandBiomedicalPress
364
regions, including the caudate-putamen, all layers of the cerebral cortex, and all subregions and laminae of the hippocampus. A high level o f specific binding was observed in the dorsal and medial raphe nuclei, the interpeduncular nucleus, the superficial layer of the superior colliculus, the locus coeruleus, the dorsomedial nucleus of the hypothalamus and the central grey. The distribution of high affinity imipramine binding sites in rat brain is in good agreement with the distribution of 5HT terminals (see references in Biegon et al., 1981), providing additional evidence that [3 H]imipramine labels some component of the presynaptic uptake site. This offers the possibility of applying i n v i t r o autoradiography and computerized image analysis (Palacios et al., 1981) to the study of 5HT uptake sites and terminals in brain sections.
Acknowledgements Supported by NSI7435 and NS07080. A.B. holds a Chaim Weizmann Fellowship.
References Biegon, A., T.C. Rainbow and B.S. McEwen, 1981, Quantitative autoradiography of serotonin-I receptors in the rat brain, Brain Res. (in press). Langer, S.Z., C. Moret, R. Raisman, M.L. Dubocovich and M. Briley, 1980, High-affinity [3H]-imipramine binding in rat hypothalamus is associated with the uptake of serotonin but not norepinephrine, Science 210, 1133. Palacios, J.M., D.L. Niehoff and M.J. Kuhar, 1981, Receptor autoradiography with tritium-sensitive film: Potential for computerized densitometry, Neurosci. Lett. 25, 101. Rainbow, T.C., W.V. Bleisch, A. Biegon and B.S. McEwen, 1981, Quantitative densitometry of neurotransmitter receptors, J. Neurosci. Meth. (in press). Sette, M., R. Raisman, M. Briley and S.Z. Langer, 1981, Localisation of tricyclic antidepressant binding site on serotonin nerve terminals, J. Neurochem. 37, 40.
363
Rapid communication A U T O R A D I O G R A P H I C LOCALIZATION OF IMIPRAMINE BINDING IN RAT BRAIN THOMAS C. RAINBOW *, ANAT BIEGON and BRUCE S. McEWEN The Rockefeller University, New York, N.Y. 10021, U.S.A.
Received 17 December 1981, accepted 21 December 1981
The brain possesses a specific, high-affinity binding site for the tricyclic anti-depressant, imipramine, an inhibitor of 5HT re-uptake (Langer et al., 1980; Sette et al., 1981). The high-affinity binding site for imipramine may be associated with the presynaptic uptake site for 5HT. There is a good correlation between the potency of drugs to block 5HT uptake and to compete for in vitro binding (Langer et al., 1980), and like the presynaptic uptake site, the high-affinity imipramine recognition site is depleted by lesions of 5HT cell bodies in the dorsal raphe nucleus (Sette et al., 1981). We have used the recent LKB film method of receptor autoradiography (Palacios et al., 1981; Rainbow et al., 1981) to determine the distribution of imipramine binding sites in sections of rat brain. We find that the location of imipramine binding sites parallels the known distribution of 5HT terminals in rat brain. This provides additional evidence that [3H]imipraminelabels the presym,ptic uptake site for 5HT, and offers the possibility of labeling presynaptic uptake sites in vitro for q u a l i t a t i v e autoradiography, Our procedure was to cut frozen 32/~m sections from a male rat brain, thaw-mount them onto subbed glass slides, and incubate the slides in vitro with [3H]imipramine to label binding sites. In preliminary experiments, we found that it was necessary to use a buffer with a high sodium concentration to obtain an adequate ratio of total to non-specific binding (3: 1). Our labeling procedure was to incubate sections in 50 mM Tris HC1 * To whom all correspondence should be addressed: The Rockefeller University, 1230 York Ave, New Yor, N.Y. 10021, U.S.A.
buffer, pH 7.4, containing 300 mM NaC1, for 1 h at 40. The sections were incubated with 1-10 nM of [3H]imipramine (24 or 32 Ci/mmol; New England Nuclear), with or without 100 # M cold desmethylimipramine to measure non-specific binding, or [3H]imipramine with various concentrations of unlabeled drugs to determine the pharmacological profile of the binding site. The sections were washed twice for 20 min in 40 buffer, and were either apposed against LKB Ultrofilm as described (Rainbow et al., 1981) to generate autoradiograms, or were removed from the slides with Whatman filter disks for direct measurement of radioactivity. The amount of radioactivity on the filter disks was measured by liquid scintillation counting. The binding of imipramine to the brain sections was specific, saturable and of a high affinity. Scatchard analysis of the binding indicated that [3H]imipramine labeled a single population of sites with a K D of 2 - 6 nM, and a capacity of 150 fmol/section. Imipramine was 600 times more potent than desmethylimipramine, a selective N E uptake inhibitor, in displacing the binding of [3 H]imipramine, and 60000 times more potent than the non-tricyclic antidepressant, mianserin, which does not inhibit 5HT uptake. On the basis of these results, we used a concentration of 4 n M [3H]imipramine for routine autoradiographic labeling, and measured non-specific binding by coincubation with 100/~M desmethylimipramine. The high-affinity binding sites for imipramine were distributed heterogeneously throughout the rat brain. There was little or no specific binding over white matter, or in the reticular formation and ventral thalamus. There were moderate numbers of specific binding sites in a variety of brain
0014-2999/82/0000-0000/$02.75 © 1982 Elsevier/North-HollandBiomedicalPress
364
regions, including the caudate-putamen, all layers of the cerebral cortex, and all subregions and laminae of the hippocampus. A high level o f specific binding was observed in the dorsal and medial raphe nuclei, the interpeduncular nucleus, the superficial layer of the superior colliculus, the locus coeruleus, the dorsomedial nucleus of the hypothalamus and the central grey. The distribution of high affinity imipramine binding sites in rat brain is in good agreement with the distribution of 5HT terminals (see references in Biegon et al., 1981), providing additional evidence that [3 H]imipramine labels some component of the presynaptic uptake site. This offers the possibility of applying i n v i t r o autoradiography and computerized image analysis (Palacios et al., 1981) to the study of 5HT uptake sites and terminals in brain sections.
Acknowledgements Supported by NSI7435 and NS07080. A.B. holds a Chaim Weizmann Fellowship.
References Biegon, A., T.C. Rainbow and B.S. McEwen, 1981, Quantitative autoradiography of serotonin-I receptors in the rat brain, Brain Res. (in press). Langer, S.Z., C. Moret, R. Raisman, M.L. Dubocovich and M. Briley, 1980, High-affinity [3H]-imipramine binding in rat hypothalamus is associated with the uptake of serotonin but not norepinephrine, Science 210, 1133. Palacios, J.M., D.L. Niehoff and M.J. Kuhar, 1981, Receptor autoradiography with tritium-sensitive film: Potential for computerized densitometry, Neurosci. Lett. 25, 101. Rainbow, T.C., W.V. Bleisch, A. Biegon and B.S. McEwen, 1981, Quantitative densitometry of neurotransmitter receptors, J. Neurosci. Meth. (in press). Sette, M., R. Raisman, M. Briley and S.Z. Langer, 1981, Localisation of tricyclic antidepressant binding site on serotonin nerve terminals, J. Neurochem. 37, 40.