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Neuronal localization of substance P receptors in rat neostriatum
European Journal of Pharmacology, 109 (1985) 431-432
431
Elsevier
Rapid communication NEURONAL LOCALIZATION OF SUBSTANCE P RECEPTORS IN RAT NEOSTRIATUM JOSEPH K. RITTER, DONALD R. GEHLERT,JAMES W. GIBB,JAMES K. WAMSLEYand GLEN R. HANSON * Biochemical Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112, U.S.A,
Received 6 February 1985, accepted6 February1985
Significant concentrations of substance P (SP), an undecapeptide, are present in several structures related to the basal ganglia including the corpus striatum. Morphological, biochemical, and electrophysiological studies suggest a neurotransmitter role for SP in this tissue (Bolam et al., 1983; Jones and Olpe, 1982); thus, it is not surprising that the striatum contains moderate densities of receptors for this neuropeptide (Quirion et al., 1983). The present study utilizes combined lesion and quantitative autoradiographic techniques to partially characterize the striatal SP receptors by determining their neuronal localization. The effects of three types of lesions on striatal SP binding were examined: these included 6-hydroxydopamine (6-OHDA)-induced lesions of the nigrostriatal dopamine pathway (Hanson et al., 1981), 5,7-dihydroxytryptamine (5,7-DHT)-induced lesions of the mesostriatal serotonergic projection, and kainic acid lesions of the striatum (Hanson et al., 1981). After one- (6-OHDA and 5,7-DHT treatments) or four-week (kainic acid treatment) recoveries, animals were sacrificed and their brains were removed and frozen. Slidemounted coronal brain sections through the corpus striatum were prepared and labelled with [3H]SP according to the procedure developed by Quirion et al. (1983). Specific binding of [3H]SP to tissue was defined as the component of total binding that could be displaced by a molar excess (1 #M) of unlabelled SP and represented 60-70% of total binding in all cases. Slides were dried, juxtaposed to tritium-sensitive film and exposed for 20 (6OHDA-treated) or 8 (5,7-DHT- and kainic acid-
* To whom all correspondenceshould be addressed. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishersB.V.
treated) weeks. Subsequently, the film was developed to reveal the latent autoradiograms. In order to quantitate autoradiographic grain densities on the film corresponding to specifically hound [3H]SP, computer-assisted microdensitometry was employed using a Leitz Orthoplan microscope and a DADS model 560 computer, interfaced with an MPV-Compact photometer. Neither 6-OHDA- nor 5,7-DHT-induced destruction of striatal dopaminergic or ~rotoaergic terminals, respectively, resulted in significant loss of SP receptors in corpus striatum. Fig. 1A is a representative autoradiogram from an animal that received a unilateral 6-OHDA lesion; quantitation of striatal grain densities showed no significant difference in [3H]SP binding between colatrol (right) and lesion (left) sides (30.2 + 1.5 a ~ t 29.6 + 1.7 f m o l / m g tissue, respectively). The success o/ the 6-OHDA treatment was verified in those animals used for the autoradiographic study by inducing contralateral circling behavior ( > 20 rev/min) with apomorphine administratio~ (0.5 mg/kg). Similarly, serotonergic lesions induced by 5,7-DHT (200 tag/20 #1, i.c.v.) had no effect on striatal [3H]SP binding (sham, 26.9 _+ 1.1, and lesion, 28.9 + 2.6 f m o l / m g tissue; autoradiograms not shown). Only lesioned animals whose posterior caudate concentrations of serotonin ~ d its main metabolite (as measured by HPLC-EC) were reduced more than 90% compared to control, were used for the study. In contrast, the striatal lesion induced by itainie acid, an axon-sparing neurotoxin (Kohler and Schwarcz, 1983), resulted in a dramatic toss of grains corresponding to specifically bound |3H]SP (fig. 1B), while having little effect on non-~ecifie binding (fig. 1C). Microdensitometric ektermina-
432
tion of grain densities suggest that more than 90% of the striatal receptors for this peptide (control, 25.1 _+ 2.3 f m o l / m g tissue; kainate-treated, 1.6 _+ 0.8 f m o l / m g tissue) are located on neurons that originate in this basal ganglia structure. In summary, we have used combined lesion and receptor-binding autoradiographic techniques to investigate the localization of SP receptors in rat striatum. The results reported herein suggest that the vast majority of SP receptors are associated with neurons originating in the striatum and that few, if any, SP receptors are associated with the terminals of either of the principal monoamine systems which innervate this structure. In view of the present findings, it would be interesting to examine the distribution of SP receptors in the basal ganglia of patients afflicted with Huntington's chorea, an extrapyramidal disorder characterized by marked destruction of neuronal cell bodies in the corpus striatum. References
Fig. 1. Autoradiograms of [3 H]SP-labeled striatal sections from unilaterally lesioned animals. Coronal sections through the anterior striatum (2000 /Lm anterior to bregma) from animals lesioned on the left side by (A) 6 - O H D A or (B) kainic acid, were incubated in buffer containing 1.5 nM [3HISP, dried, and apposed to tritium-sensitive film. After 20- or 8-week exposures, respectively, the film was developed to reveal the autoradiograms shown. Dark grains correspond to specifically b o u n d 13H]SP. (C) is an autoradiogram of a serial section of (B) incubated in the additional presence of 1 # M unlabeled SP to show nonspecific binding. Abbreviations are: Cx, cerebral cortex; ha, nucleus accumbens; CP, caudate-putamen.
Bolam, J.P., P. Somogyi, H. Takagi, I. Fodor and A.D. Smith, 1983, Localization of substance P-like immunoreactivity in neurons and nerve terminals in the neostriatum of the rat: a correlated light and electron microscopic study, J. Neurocytol. 12, 325. Hanson, G.R., L. Alphs, W. Wolf, R. Levine and W. Lovenberg, 1981, Haloperidol-induced reduction of nigral substance P-like immunoreactivity: a probe for the interactions between dopamine and substance P neuronal systems, J. Pharmacol. Exp. Ther. 218, 568. Jones, R.S. and H.R. Olpe, 1982, Responses of neurons in the caudate nucleus to substance P, Reg. Pept. 4, 369. Kohler, C. and R. Schwarcz, 1983, Comparison of ibotenate and kainate neurotoxicity in rat brain: a histological study, Neurosci. 8, 819. Quirion, R., C. Shuhs, T. Moody, C. Pert, T. Chase and T. O'Donohue, 1983, Autoradiographic distribution of substance P receptors in rat central nervous system, Nature 303, 714.
431
Elsevier
Rapid communication NEURONAL LOCALIZATION OF SUBSTANCE P RECEPTORS IN RAT NEOSTRIATUM JOSEPH K. RITTER, DONALD R. GEHLERT,JAMES W. GIBB,JAMES K. WAMSLEYand GLEN R. HANSON * Biochemical Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112, U.S.A,
Received 6 February 1985, accepted6 February1985
Significant concentrations of substance P (SP), an undecapeptide, are present in several structures related to the basal ganglia including the corpus striatum. Morphological, biochemical, and electrophysiological studies suggest a neurotransmitter role for SP in this tissue (Bolam et al., 1983; Jones and Olpe, 1982); thus, it is not surprising that the striatum contains moderate densities of receptors for this neuropeptide (Quirion et al., 1983). The present study utilizes combined lesion and quantitative autoradiographic techniques to partially characterize the striatal SP receptors by determining their neuronal localization. The effects of three types of lesions on striatal SP binding were examined: these included 6-hydroxydopamine (6-OHDA)-induced lesions of the nigrostriatal dopamine pathway (Hanson et al., 1981), 5,7-dihydroxytryptamine (5,7-DHT)-induced lesions of the mesostriatal serotonergic projection, and kainic acid lesions of the striatum (Hanson et al., 1981). After one- (6-OHDA and 5,7-DHT treatments) or four-week (kainic acid treatment) recoveries, animals were sacrificed and their brains were removed and frozen. Slidemounted coronal brain sections through the corpus striatum were prepared and labelled with [3H]SP according to the procedure developed by Quirion et al. (1983). Specific binding of [3H]SP to tissue was defined as the component of total binding that could be displaced by a molar excess (1 #M) of unlabelled SP and represented 60-70% of total binding in all cases. Slides were dried, juxtaposed to tritium-sensitive film and exposed for 20 (6OHDA-treated) or 8 (5,7-DHT- and kainic acid-
* To whom all correspondenceshould be addressed. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishersB.V.
treated) weeks. Subsequently, the film was developed to reveal the latent autoradiograms. In order to quantitate autoradiographic grain densities on the film corresponding to specifically hound [3H]SP, computer-assisted microdensitometry was employed using a Leitz Orthoplan microscope and a DADS model 560 computer, interfaced with an MPV-Compact photometer. Neither 6-OHDA- nor 5,7-DHT-induced destruction of striatal dopaminergic or ~rotoaergic terminals, respectively, resulted in significant loss of SP receptors in corpus striatum. Fig. 1A is a representative autoradiogram from an animal that received a unilateral 6-OHDA lesion; quantitation of striatal grain densities showed no significant difference in [3H]SP binding between colatrol (right) and lesion (left) sides (30.2 + 1.5 a ~ t 29.6 + 1.7 f m o l / m g tissue, respectively). The success o/ the 6-OHDA treatment was verified in those animals used for the autoradiographic study by inducing contralateral circling behavior ( > 20 rev/min) with apomorphine administratio~ (0.5 mg/kg). Similarly, serotonergic lesions induced by 5,7-DHT (200 tag/20 #1, i.c.v.) had no effect on striatal [3H]SP binding (sham, 26.9 _+ 1.1, and lesion, 28.9 + 2.6 f m o l / m g tissue; autoradiograms not shown). Only lesioned animals whose posterior caudate concentrations of serotonin ~ d its main metabolite (as measured by HPLC-EC) were reduced more than 90% compared to control, were used for the study. In contrast, the striatal lesion induced by itainie acid, an axon-sparing neurotoxin (Kohler and Schwarcz, 1983), resulted in a dramatic toss of grains corresponding to specifically bound |3H]SP (fig. 1B), while having little effect on non-~ecifie binding (fig. 1C). Microdensitometric ektermina-
432
tion of grain densities suggest that more than 90% of the striatal receptors for this peptide (control, 25.1 _+ 2.3 f m o l / m g tissue; kainate-treated, 1.6 _+ 0.8 f m o l / m g tissue) are located on neurons that originate in this basal ganglia structure. In summary, we have used combined lesion and receptor-binding autoradiographic techniques to investigate the localization of SP receptors in rat striatum. The results reported herein suggest that the vast majority of SP receptors are associated with neurons originating in the striatum and that few, if any, SP receptors are associated with the terminals of either of the principal monoamine systems which innervate this structure. In view of the present findings, it would be interesting to examine the distribution of SP receptors in the basal ganglia of patients afflicted with Huntington's chorea, an extrapyramidal disorder characterized by marked destruction of neuronal cell bodies in the corpus striatum. References
Fig. 1. Autoradiograms of [3 H]SP-labeled striatal sections from unilaterally lesioned animals. Coronal sections through the anterior striatum (2000 /Lm anterior to bregma) from animals lesioned on the left side by (A) 6 - O H D A or (B) kainic acid, were incubated in buffer containing 1.5 nM [3HISP, dried, and apposed to tritium-sensitive film. After 20- or 8-week exposures, respectively, the film was developed to reveal the autoradiograms shown. Dark grains correspond to specifically b o u n d 13H]SP. (C) is an autoradiogram of a serial section of (B) incubated in the additional presence of 1 # M unlabeled SP to show nonspecific binding. Abbreviations are: Cx, cerebral cortex; ha, nucleus accumbens; CP, caudate-putamen.
Bolam, J.P., P. Somogyi, H. Takagi, I. Fodor and A.D. Smith, 1983, Localization of substance P-like immunoreactivity in neurons and nerve terminals in the neostriatum of the rat: a correlated light and electron microscopic study, J. Neurocytol. 12, 325. Hanson, G.R., L. Alphs, W. Wolf, R. Levine and W. Lovenberg, 1981, Haloperidol-induced reduction of nigral substance P-like immunoreactivity: a probe for the interactions between dopamine and substance P neuronal systems, J. Pharmacol. Exp. Ther. 218, 568. Jones, R.S. and H.R. Olpe, 1982, Responses of neurons in the caudate nucleus to substance P, Reg. Pept. 4, 369. Kohler, C. and R. Schwarcz, 1983, Comparison of ibotenate and kainate neurotoxicity in rat brain: a histological study, Neurosci. 8, 819. Quirion, R., C. Shuhs, T. Moody, C. Pert, T. Chase and T. O'Donohue, 1983, Autoradiographic distribution of substance P receptors in rat central nervous system, Nature 303, 714.