248
Neuroscience Letters, 117 (1990) 248 252 Elsevier Scientific Publishers Ireland Ltd.
NSL 07159
D2 dopamine receptor gene expression by cholinergic neurons in the rat striatum C. Le Moine, F. Tison and B. Bloch U.R.A. C.N.R.S. 1200 - Laboratoire d'Histologie-Embryologie, Universitb de Bordeaux 11, Bordeaux (France)
(Received 7 May 1990; Revised version received 2 June 1990; Accepted 5 June 1990) Key words:
D2 receptor mRNA; Cholinergic neuron; Rat striatum; In situ hybridization
In situ hybridization with D2 receptor probe and immunohistochemistry with choline acetyltransferase (CHAT) antibody performed on adjacent sections demonstrate dopamine D2 receptor gene expression in cholinergic neurons of the rat caudate-putamen and nucleus accumbens. Eighty per cent of cholinergic neurons in the striatum contain detectable D2 receptor mRNA. The other neurons without detectable D2 mRNA do not display specific localization or aspect in the eaudate-putamen and nucleus aecumbens as compared to the other eholinergic neurons. The absence of detectable I)2 mRNA in certain cholinergic neurons can be due to the limited sensitivity of the procedure that would not detect low mRNA levels, or alternatively can reflect the existence of two chotinergic cell populations in the striatum, one of which would not express the D2 receptor gene. The other forebrain cholinergic neurons do not contain D2 mRNA.
The cells expressing the D2 dopamine receptor gene have been recently identified in the central nervous system by using in situ hybridization (ISH) [13-15, 19]. Neurons containing D2 mRNA are especially abundant in the caudate-putamen, nucleus accumbens, olfactory tubercle and substantia nigra. In the caudate-putamen and nucleus accumbens, two neuronal populations expressing the D2 receptor gene are present. The most prominent one (98% of labeled cells) is a population of small neurons, identified as enkephalinergic neurons [13]. The second one is composed of few large cells (2%) having several anatomical features of cholinergic neurons [13]. This last observation is in accordance with indirect biochemical results suggesting the presence of D2 receptors on striatal interneurons of cholinergic type [5, 10, 12] and the inhibitory effect of dopamine on acetyleholine release in the striatum [1, 18]. Using an antibody against choline acetyltransferase (CHAT), a specific marker of cholinergic neurons [6], we have investi.gated the Eossibility that these large striatal neurons expressing the D2 receptor gene could be cholinergic. We report here that most of, but not all, striatal cholinergic neurons express the D2 receptor gene. Correspondence: C. Le Moine, Laboratoire d'Histologie-Embryologie, (U.F.R. II) Universit~ de Bordeaux II, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
249
Sections were prepared from 10 adult male Wistar rat brains as described [2]. Among these, serial 3/tm- or 6 pm-thick sections were selected. Each over two adjacent sections was treated either by ISH with a D~ receptor probe or by immunohistochemistry with an antibody against CHAT. The D~ receptor probe [13] was a mixture of 3 oligonucleotides labeled using [35S]dATP. It recognizes indifferently each of the two mRNAs coded by the D~ receptor gene through alternative splicing [4, 7]. ISH and autoradiography were performed as described [2] with a minor modification of the hybridization buffer (50% deionized formamide, 10% dextran sulfate, 25 pg/ml denatured salmon sperm DNA in SSC 2 x ). 0.2 ng or 0.1 ng of probe were deposited on slides. Immunohistochemistry was performed with a monoclonal antibody against rat ChAT (Boehringer Manheim) [6]. Slides were incubated in Tris saline buffer pH 7.4 (TBS) containing 3% normal goat serum (NGS) for 1 h at room temperature, and incubated with ChAT antibody (5 pg/ml in TBS, 1% NGS, 0.2% Triton X-100) for 16 h at 4°C. Slides were then processed for a double-bridge peroxidase-antiperoxidase immunolabeling using anti-rat IgG (1/100, Dakopatts) and peroxidase-antiperoxidase rat complex (1/200, Sternberger-Meyer). The large neurons containing De
O J
/
! C
D
Fig. 1. Rat caudate-putamen after in situ hybridization with 35S-labeled D2 receptor probe (A, C) and ChAT immunohistochemistry (B, D). Bright-field view of 6 pro-thick adjacent sections. Three week exposure in autoradiography for A and C. In A, two large neurons are encircled containing D: mRNA (silver grains are not visible at this magnification). In B, the same neurons contain ChAT immunoreactivity (*=corpus callosum). C, D: details of A and B show the labeling of the same large neuron with the D2 receptor probe and the ChAT antibody. The small neurons containing D2 mRNA (arrows) are unreactive with the ChAT antibody (A, B: bars = 50 pro; C, D: bars = 10/zm).
250
mRNA and the neurons containing ChAT immunoreactivity were identified, mapped and counted on adjacent sections using an image analysis system (Histo 200, BioCOrn). Small neurons as well as large sized neurons were detected in the striatum by ISH with the D2 receptor probe (Figs, 1 and 2) as previously described [13]. Some of the large-sized neurons were unlabeled with the D2 probe (they contained no silver grain or a negligible number of silver grain as compared to the striatal background). ChAT-positive neurons appeared as large scattered cells throughout the extent of the striatum in agreement with results described with several antibodies against ChAT [3, 8, ! 7]. Comparison on adjacent sections showed that all the large neurons containing D2 mRNA also display ChAT immunoreactivity. Counting in two rats over 116 identified neurons demonstrated that only 80% of ChAT-immunoreactive neurons in the caudate-putamen and nucleus accumbens contained D2 mRNA (Figs. ! and 2). The other 20% of ChAT-neurons that were D2 mRNA-negative have no peculiar localization in the caudate-putamen and nucleus accumbens as compared to the other ChAT neurons. Numerous ChAT-positive neurons were detected outside the caudate-putamen and nucleus accumbens, especially in the olfactory tubercle, the diagonal band of Broca and other areas of the basal forebrain. They did not contain D2 receptor mRNA. Our results demonstrate that the population of large cells expressing D2 receptor gene in the caudate-putamen and nucleus accumbens indeed corresponds to cholinergic neurons. These results are in agreement with previous works suggesting the presence of dopamine receptors on cholinergic interneurons in the striatum [5, 10, 12] and the inhibition of striatal acetylcholine release by dopamine [1, 18]. They show that cholinergic neurons of the caudate-putamen and nucleus accumbens are under the direct influence of dopamine. In contrast, cholinergic neurons detected outside the caudate-putamen and nucleus accumbens do not express the D2 receptor gene.
Fig. 2. Rat caudate-putamen. Three/ml-thick serialsections. Immunohistochemistry with ChAT antibody (A, C), in situ hybridization with D2 receptor probe (B). Two large neurons are visible. The upper one contains ChAT immunorcactivityand D2 receptor mRNA (arrow). The lowerone onlycontainsChAT immunoreactivity(doublcarrow). (A-C: bars = I0/nn).
251 T h e presence o f a significant n u m b e r o f striatal cholinergic neurons, w i t h o u t detectable D2 r e c e p t o r m R N A , can be e x p l a i n e d in two ways. E i t h e r these n e u r o n s express the D2 gene with a n u m b e r o f D2 m R N A copies t h a t is t o o low to be detected in the present technical c o n d i t i o n s o r there is a p o p u l a t i o n in striatal cholinergic neurons t h a t does n o t express the D2 r e c e p t o r gene. A t a n y rate, o u r results d e m o n s t r a t e a h e t e r o g e n e i t y in cholinergic n e u r o n s with respect to D2 r e c e p t o r gene expression. Since the striatal cholinergic n e u r o n s d o n o t b e a r Dl receptors [5], it is conceivable that the p o p u l a t i o n o f n e u r o n s that w o u l d not express the D2 gene w o u l d n o t be u n d e r d o p a m i n e influence c o n t r a r y to the 'classical' cholinergic i n t e r n e u r o n s . Interestingly, o t h e r p e c u l i a r p o p u l a t i o n s o f striatal cholinergic n e u r o n s have been recently d e m o n s t r a t e d : n e u r o n s p r o j e c t i n g outside the s t r i a t u m , especially in the cortex [8, 9, 16] a n d n e u r o n s b e a r i n g nerve g r o w t h f a c t o r ( N G F ) receptors [11] that b o t h represent I(~15% o f cholinergic n e u r o n s in the rat striatum. A p p r o p r i a t e a n a t o m i c a l investigations are n o w n e e d e d to find out w h e t h e r relations exist between n e u r o n s without d e t e c t a b l e D2 r e c e p t o r m R N A , n e u r o n s with N G F receptors a n d p r o j e c t i n g neurons, that c o u l d all relate to a circuitry distinct f r o m the one involving the nigrostriatal n e t w o r k . In an o t h e r way, the existence o f two D2 r e c e p t o r i s o f o r m s c o d e d by two m R N A s issued from a single gene by a l t e r n a t i v e splicing has been recently d e m o n s t r a t e d [4, 7]. Spatial analysis o f the cells c o n t a i n i n g these two m R N A s could also p o s s i b l y d e m o n s t r a t e a distinct a n d new heterogeneity in D2 r e c e p t o r gene expression in the striatal neurons. W e t h a n k Dr. D. T h 6 o d o s i s for her precious help a n d advice, C. V i d a u p o r t e for p h o t o g r a p h i c a l a r t w o r k a n d G. C a u n e for expert typing. This w o r k was s u p p o r t e d by the R6gion d ' A q u i t a i n e , the Minist6re de la Recherche et de l ' E n s e i g n e m e n t sup6rieur a n d the F o n d a t i o n de F r a n c e . C . L . M . was s u p p o r t e d by a fellowship from the Association France Parkinson. 1 Agid, Y., Guyenet, P., Glowinski, J., Beaujouan, J.C. and Javoy, F., Inhibitory influence of the nigrostriatal dopamine system on the striatal cholinergic neurons in the rat, Brain Res., 86 (1975) 488 492. 2 Bloch, B., Popovici, T., Le Guellec, D., Normand, E., Chouham, S., Guitteny, A.F. and Bohlen, P., In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience, J. Neurosci. Res., 16 (1986) 183 200. 3 Bolam, J.P., Wainer, B.H. and Smith, A.D., Characterization of cholinergic neurons in the rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy, Neuroscience, 12 (1984) 711 718. 4 Dal Toso, R., Sommer, B., Ewert, M., Herb, A.. Pritchett, D.B., Bach, A., Shivers, B.D. and Seeburg, P.H., The dopamine D2 receptor: two molecular forms generated by alternative splicing, EMBO J., 8 (1989) 4025~,034. 5 Dawson, V.L., Dawson, T.M., Filloux, F.M. and Wamsley, J.K., Evidence for dopamine D2 receptors on cholinergic interneurons in the rat caudate-putamen, Life Sci., 42 (1988) 1933--1939. 6 Eckenstein, F. and Thoenen, H., Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons, EMBO J., 1 (1982) 363--368. 7 Giros, B., Sokoloff, P., Martres, M.P., Riou, J.F., Emorine, L.J. and Schwartz, J.C., Alternative splicing directs the expression of two D2 dopamine receptor isoforms, Nature, 342 (1989) 923-926. 8 Ichikawa, T. and Hirata, Y., Organization of choline acetyltransferase-containing structures in the forebrain of the rat, J. Neurosci., 6 (1986) 281-292.
252 9 Jayaraman, A., Anatomical evidence for cortical projections from the striatum in the cat, Brain Res.. 195 (1980) 29-36. 10 Joyce, J.N. and Marshall, J.F., Quantitative autoradiography of dopamine D2 sites in rat caudateputamen: localization to intrinsic neurons and not to neocortical afferents, Neuroscience, 20 (1987) 773-795. 11 Kiss, J. and Patel, A.J., Characterization of neurons containing nerve growth factor receptors in the rat neostriatum, Neurosci. Lett., 105 (1989) 251-256. 12 Lehmann, J. and Langer, S.Z., The striatal cholinergic interneuron: synaptic target of dopaminergic terminals?, Neuroscience, 10 (1983) 1105-1120. 13 Le Moine, C., Normand, E., Guitteny, A.F., Fouque, B., Teoule, R. and Bloch, B., Dopamine receptor gene expression by enkephalin neurons in rat forebrain, Proc. Natl. Acad. Sci, U.S.A., 87 (1990) 230234. 14 Meador-Woodruff, J.H., Mansour, A., Bunzow, J.R., Van Tol, H.H.M., Watson, S.J. and Civelli, O., Distribution of D2 dopamine receptor mRNA in rat brain, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7625 -7628. 15 Mengod, G., Martinez-Mir, M.I., Vilaro, M.T. and Palacios, J.M., Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry, Proc. Natl. Acad. ScL U.S.A., 86 (1989) 8560-8564. 16 Parent, A., Boucher, R. and O'Reilly-Fromentin, J., Acetylcholinesterase-containing neurons in cat pallidal complex: morphological characteristics and projection towards the neocortex, Brain Res., 230 (1981) 356-361. 17 Phelps, P.E., Houser, C.R. and Vaughn, J.E., Immunocytochemical localization of choline acetyltransferase within the rat neostriatum: a correlated light and electron microscopic study of cholinergic neurons and synapses, J. Comp. Neurol., 238 (1985) 286-307. 18 Stool, J.C., Verheijden, P.F.H.M. and Leysen, J.E., Stimulation of D2 receptors in rat nucleus accumbens slices inhibits dopamine and acetylcholine release but not cyclic AMP formation, Brain Res., 423 (1987) 364-368. 19 Weiner, D.M. and Brann, M.R., The distribution of a dopamine D2 receptor mRNA in rat brain, FEBS Lett., 253 (1989) 207-213.
Neuroscience Letters, 117 (1990) 248 252 Elsevier Scientific Publishers Ireland Ltd.
NSL 07159
D2 dopamine receptor gene expression by cholinergic neurons in the rat striatum C. Le Moine, F. Tison and B. Bloch U.R.A. C.N.R.S. 1200 - Laboratoire d'Histologie-Embryologie, Universitb de Bordeaux 11, Bordeaux (France)
(Received 7 May 1990; Revised version received 2 June 1990; Accepted 5 June 1990) Key words:
D2 receptor mRNA; Cholinergic neuron; Rat striatum; In situ hybridization
In situ hybridization with D2 receptor probe and immunohistochemistry with choline acetyltransferase (CHAT) antibody performed on adjacent sections demonstrate dopamine D2 receptor gene expression in cholinergic neurons of the rat caudate-putamen and nucleus accumbens. Eighty per cent of cholinergic neurons in the striatum contain detectable D2 receptor mRNA. The other neurons without detectable D2 mRNA do not display specific localization or aspect in the eaudate-putamen and nucleus aecumbens as compared to the other eholinergic neurons. The absence of detectable I)2 mRNA in certain cholinergic neurons can be due to the limited sensitivity of the procedure that would not detect low mRNA levels, or alternatively can reflect the existence of two chotinergic cell populations in the striatum, one of which would not express the D2 receptor gene. The other forebrain cholinergic neurons do not contain D2 mRNA.
The cells expressing the D2 dopamine receptor gene have been recently identified in the central nervous system by using in situ hybridization (ISH) [13-15, 19]. Neurons containing D2 mRNA are especially abundant in the caudate-putamen, nucleus accumbens, olfactory tubercle and substantia nigra. In the caudate-putamen and nucleus accumbens, two neuronal populations expressing the D2 receptor gene are present. The most prominent one (98% of labeled cells) is a population of small neurons, identified as enkephalinergic neurons [13]. The second one is composed of few large cells (2%) having several anatomical features of cholinergic neurons [13]. This last observation is in accordance with indirect biochemical results suggesting the presence of D2 receptors on striatal interneurons of cholinergic type [5, 10, 12] and the inhibitory effect of dopamine on acetyleholine release in the striatum [1, 18]. Using an antibody against choline acetyltransferase (CHAT), a specific marker of cholinergic neurons [6], we have investi.gated the Eossibility that these large striatal neurons expressing the D2 receptor gene could be cholinergic. We report here that most of, but not all, striatal cholinergic neurons express the D2 receptor gene. Correspondence: C. Le Moine, Laboratoire d'Histologie-Embryologie, (U.F.R. II) Universit~ de Bordeaux II, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
249
Sections were prepared from 10 adult male Wistar rat brains as described [2]. Among these, serial 3/tm- or 6 pm-thick sections were selected. Each over two adjacent sections was treated either by ISH with a D~ receptor probe or by immunohistochemistry with an antibody against CHAT. The D~ receptor probe [13] was a mixture of 3 oligonucleotides labeled using [35S]dATP. It recognizes indifferently each of the two mRNAs coded by the D~ receptor gene through alternative splicing [4, 7]. ISH and autoradiography were performed as described [2] with a minor modification of the hybridization buffer (50% deionized formamide, 10% dextran sulfate, 25 pg/ml denatured salmon sperm DNA in SSC 2 x ). 0.2 ng or 0.1 ng of probe were deposited on slides. Immunohistochemistry was performed with a monoclonal antibody against rat ChAT (Boehringer Manheim) [6]. Slides were incubated in Tris saline buffer pH 7.4 (TBS) containing 3% normal goat serum (NGS) for 1 h at room temperature, and incubated with ChAT antibody (5 pg/ml in TBS, 1% NGS, 0.2% Triton X-100) for 16 h at 4°C. Slides were then processed for a double-bridge peroxidase-antiperoxidase immunolabeling using anti-rat IgG (1/100, Dakopatts) and peroxidase-antiperoxidase rat complex (1/200, Sternberger-Meyer). The large neurons containing De
O J
/
! C
D
Fig. 1. Rat caudate-putamen after in situ hybridization with 35S-labeled D2 receptor probe (A, C) and ChAT immunohistochemistry (B, D). Bright-field view of 6 pro-thick adjacent sections. Three week exposure in autoradiography for A and C. In A, two large neurons are encircled containing D: mRNA (silver grains are not visible at this magnification). In B, the same neurons contain ChAT immunoreactivity (*=corpus callosum). C, D: details of A and B show the labeling of the same large neuron with the D2 receptor probe and the ChAT antibody. The small neurons containing D2 mRNA (arrows) are unreactive with the ChAT antibody (A, B: bars = 50 pro; C, D: bars = 10/zm).
250
mRNA and the neurons containing ChAT immunoreactivity were identified, mapped and counted on adjacent sections using an image analysis system (Histo 200, BioCOrn). Small neurons as well as large sized neurons were detected in the striatum by ISH with the D2 receptor probe (Figs, 1 and 2) as previously described [13]. Some of the large-sized neurons were unlabeled with the D2 probe (they contained no silver grain or a negligible number of silver grain as compared to the striatal background). ChAT-positive neurons appeared as large scattered cells throughout the extent of the striatum in agreement with results described with several antibodies against ChAT [3, 8, ! 7]. Comparison on adjacent sections showed that all the large neurons containing D2 mRNA also display ChAT immunoreactivity. Counting in two rats over 116 identified neurons demonstrated that only 80% of ChAT-immunoreactive neurons in the caudate-putamen and nucleus accumbens contained D2 mRNA (Figs. ! and 2). The other 20% of ChAT-neurons that were D2 mRNA-negative have no peculiar localization in the caudate-putamen and nucleus accumbens as compared to the other ChAT neurons. Numerous ChAT-positive neurons were detected outside the caudate-putamen and nucleus accumbens, especially in the olfactory tubercle, the diagonal band of Broca and other areas of the basal forebrain. They did not contain D2 receptor mRNA. Our results demonstrate that the population of large cells expressing D2 receptor gene in the caudate-putamen and nucleus accumbens indeed corresponds to cholinergic neurons. These results are in agreement with previous works suggesting the presence of dopamine receptors on cholinergic interneurons in the striatum [5, 10, 12] and the inhibition of striatal acetylcholine release by dopamine [1, 18]. They show that cholinergic neurons of the caudate-putamen and nucleus accumbens are under the direct influence of dopamine. In contrast, cholinergic neurons detected outside the caudate-putamen and nucleus accumbens do not express the D2 receptor gene.
Fig. 2. Rat caudate-putamen. Three/ml-thick serialsections. Immunohistochemistry with ChAT antibody (A, C), in situ hybridization with D2 receptor probe (B). Two large neurons are visible. The upper one contains ChAT immunorcactivityand D2 receptor mRNA (arrow). The lowerone onlycontainsChAT immunoreactivity(doublcarrow). (A-C: bars = I0/nn).
251 T h e presence o f a significant n u m b e r o f striatal cholinergic neurons, w i t h o u t detectable D2 r e c e p t o r m R N A , can be e x p l a i n e d in two ways. E i t h e r these n e u r o n s express the D2 gene with a n u m b e r o f D2 m R N A copies t h a t is t o o low to be detected in the present technical c o n d i t i o n s o r there is a p o p u l a t i o n in striatal cholinergic neurons t h a t does n o t express the D2 r e c e p t o r gene. A t a n y rate, o u r results d e m o n s t r a t e a h e t e r o g e n e i t y in cholinergic n e u r o n s with respect to D2 r e c e p t o r gene expression. Since the striatal cholinergic n e u r o n s d o n o t b e a r Dl receptors [5], it is conceivable that the p o p u l a t i o n o f n e u r o n s that w o u l d not express the D2 gene w o u l d n o t be u n d e r d o p a m i n e influence c o n t r a r y to the 'classical' cholinergic i n t e r n e u r o n s . Interestingly, o t h e r p e c u l i a r p o p u l a t i o n s o f striatal cholinergic n e u r o n s have been recently d e m o n s t r a t e d : n e u r o n s p r o j e c t i n g outside the s t r i a t u m , especially in the cortex [8, 9, 16] a n d n e u r o n s b e a r i n g nerve g r o w t h f a c t o r ( N G F ) receptors [11] that b o t h represent I(~15% o f cholinergic n e u r o n s in the rat striatum. A p p r o p r i a t e a n a t o m i c a l investigations are n o w n e e d e d to find out w h e t h e r relations exist between n e u r o n s without d e t e c t a b l e D2 r e c e p t o r m R N A , n e u r o n s with N G F receptors a n d p r o j e c t i n g neurons, that c o u l d all relate to a circuitry distinct f r o m the one involving the nigrostriatal n e t w o r k . In an o t h e r way, the existence o f two D2 r e c e p t o r i s o f o r m s c o d e d by two m R N A s issued from a single gene by a l t e r n a t i v e splicing has been recently d e m o n s t r a t e d [4, 7]. Spatial analysis o f the cells c o n t a i n i n g these two m R N A s could also p o s s i b l y d e m o n s t r a t e a distinct a n d new heterogeneity in D2 r e c e p t o r gene expression in the striatal neurons. W e t h a n k Dr. D. T h 6 o d o s i s for her precious help a n d advice, C. V i d a u p o r t e for p h o t o g r a p h i c a l a r t w o r k a n d G. C a u n e for expert typing. This w o r k was s u p p o r t e d by the R6gion d ' A q u i t a i n e , the Minist6re de la Recherche et de l ' E n s e i g n e m e n t sup6rieur a n d the F o n d a t i o n de F r a n c e . C . L . M . was s u p p o r t e d by a fellowship from the Association France Parkinson. 1 Agid, Y., Guyenet, P., Glowinski, J., Beaujouan, J.C. and Javoy, F., Inhibitory influence of the nigrostriatal dopamine system on the striatal cholinergic neurons in the rat, Brain Res., 86 (1975) 488 492. 2 Bloch, B., Popovici, T., Le Guellec, D., Normand, E., Chouham, S., Guitteny, A.F. and Bohlen, P., In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience, J. Neurosci. Res., 16 (1986) 183 200. 3 Bolam, J.P., Wainer, B.H. and Smith, A.D., Characterization of cholinergic neurons in the rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy, Neuroscience, 12 (1984) 711 718. 4 Dal Toso, R., Sommer, B., Ewert, M., Herb, A.. Pritchett, D.B., Bach, A., Shivers, B.D. and Seeburg, P.H., The dopamine D2 receptor: two molecular forms generated by alternative splicing, EMBO J., 8 (1989) 4025~,034. 5 Dawson, V.L., Dawson, T.M., Filloux, F.M. and Wamsley, J.K., Evidence for dopamine D2 receptors on cholinergic interneurons in the rat caudate-putamen, Life Sci., 42 (1988) 1933--1939. 6 Eckenstein, F. and Thoenen, H., Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons, EMBO J., 1 (1982) 363--368. 7 Giros, B., Sokoloff, P., Martres, M.P., Riou, J.F., Emorine, L.J. and Schwartz, J.C., Alternative splicing directs the expression of two D2 dopamine receptor isoforms, Nature, 342 (1989) 923-926. 8 Ichikawa, T. and Hirata, Y., Organization of choline acetyltransferase-containing structures in the forebrain of the rat, J. Neurosci., 6 (1986) 281-292.
252 9 Jayaraman, A., Anatomical evidence for cortical projections from the striatum in the cat, Brain Res.. 195 (1980) 29-36. 10 Joyce, J.N. and Marshall, J.F., Quantitative autoradiography of dopamine D2 sites in rat caudateputamen: localization to intrinsic neurons and not to neocortical afferents, Neuroscience, 20 (1987) 773-795. 11 Kiss, J. and Patel, A.J., Characterization of neurons containing nerve growth factor receptors in the rat neostriatum, Neurosci. Lett., 105 (1989) 251-256. 12 Lehmann, J. and Langer, S.Z., The striatal cholinergic interneuron: synaptic target of dopaminergic terminals?, Neuroscience, 10 (1983) 1105-1120. 13 Le Moine, C., Normand, E., Guitteny, A.F., Fouque, B., Teoule, R. and Bloch, B., Dopamine receptor gene expression by enkephalin neurons in rat forebrain, Proc. Natl. Acad. Sci, U.S.A., 87 (1990) 230234. 14 Meador-Woodruff, J.H., Mansour, A., Bunzow, J.R., Van Tol, H.H.M., Watson, S.J. and Civelli, O., Distribution of D2 dopamine receptor mRNA in rat brain, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7625 -7628. 15 Mengod, G., Martinez-Mir, M.I., Vilaro, M.T. and Palacios, J.M., Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry, Proc. Natl. Acad. ScL U.S.A., 86 (1989) 8560-8564. 16 Parent, A., Boucher, R. and O'Reilly-Fromentin, J., Acetylcholinesterase-containing neurons in cat pallidal complex: morphological characteristics and projection towards the neocortex, Brain Res., 230 (1981) 356-361. 17 Phelps, P.E., Houser, C.R. and Vaughn, J.E., Immunocytochemical localization of choline acetyltransferase within the rat neostriatum: a correlated light and electron microscopic study of cholinergic neurons and synapses, J. Comp. Neurol., 238 (1985) 286-307. 18 Stool, J.C., Verheijden, P.F.H.M. and Leysen, J.E., Stimulation of D2 receptors in rat nucleus accumbens slices inhibits dopamine and acetylcholine release but not cyclic AMP formation, Brain Res., 423 (1987) 364-368. 19 Weiner, D.M. and Brann, M.R., The distribution of a dopamine D2 receptor mRNA in rat brain, FEBS Lett., 253 (1989) 207-213.
