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Expression of the cholecystokinin gene in rat hippocampal interneurons is independent of extrinsic input
ELSEVIER
Neuroscience Letters 172 (1994) 143 146
NIUROSCIINCE LETTERS
Expression of the cholecystokinin gene in rat hippocampal interneurons is independent of extrinsic input A. Straube a, R. Bender ~, U. Otten b, M. Frotscher ~t'* "Institute of Anatomy, University 0["F?eiburg, PO Box 111, D-79001 Freihurg, FRG bInstitute of Physiology, UniversiO' o[ Basel, Basel, Switzerland Received 22 December 1993; Revised version received 9 February 1994; Accepted 14 March 1994
Abstract
In the present study we have used slice cultures of hippocampus and in situ hybridization techniques in order to study the ability of hippocampal neurons to synthesize CCK mRNA in the absence of extrinsic afferents. Our results show that very similar types of hippocampal neurons express CCK mRNA in culture as in situ. We conclude that the expression of the CCK gene in hippocampal neurons is not dependent on extrinsic afferent input. Key words'." Hippocampus; Slice culture; Nonpyramidal cell: Neuropeptide; In situ hybridization: Neuronal differentiation
The majority of hippocampal nonpyramidal neurons react with antibodies against G A B A or its synthesizing enzyme glutamate decarboxylase ( G A D ) and are assumed to be inhibitory. In addition to the inhibitory neurotransmitter m a n y of these neurons contain neuropeptides, a m o n g them cholecystokinin (CCK) [12,19]. Little is known about the functional significance of these neuropeptides and the factors determining their biosynthesis and release. Contradictory results have been reported about the functional effects of CCK. Thus, C C K has been shown to excite central neurons [1,5], but an inhibitory postsynaptic effect has also been noticed [15]. As far as the release of C C K is concerned, studies in the cortex and nucleus accumbens have provided evidence for a role of serotoninergic brain stem afferents [17,18]. These studies, as well as our own electron microscopic immunocytochemical studies on CCK-containing neurons in the hippocampus [13,14], suggest that C C K neurons are integrated in complex circuits, and one may speculate that the expression and release of C C K in cortical and hippocampal neurons is controlled by afferent fibers.
*Corresponding author. Fax: (|) (761) 203-5054. 0304-3940/94/$7.00 Cc) 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 031)4-3940(94)00231-X
In the present study, we have addressed the question of whether C C K m R N A expression in hippocampal neurons is regulated by extrinsic afferents. For this we have studied the C C K m R N A expression in neurons of the hippocampus under isolated conditions, i.e. in slice culture. Slice cultures are known to preserve the organotypic organization of the hippocampus to a great extent [7-9], allowing us to compare the location of CCK-expressing cells in slice cultures with that in the normal hippocampal formation. Slice cultures of hippocampus were prepared as described in detail elsewhere [7 9]. Four- to 5-day-old rat pups were decapitated and coronal slices of 400/Ira were cut from both hippocampi. After 5, 13 or 23 days of cultivation, the slices were fixed in 4% paraformaldehyde in phosphate buffer (PB) for 15 rain and then incubated in 20% saccharose in PB. Next, the cultures were shockfrozen on dry ice and stored at -80°C. In the present study, the expression of C C K m R N A was studied by in situ hybridization using either a radioactive method with an oligonucleotide probe or a non-radioactive approach with a digoxigenin-labeled C C K m R N A . For non-radioactive in situ hybridization a modified version of the method of Wahle and Beckh [20] was used. 15/~m cryostat sections were collected in 2 x SSC (20 x SSC is 3 M
144
A. Straube et aL/Neurosctence Letwr,~ 172 ~19!~4; 143 146
NaC1, 0.3 M sodium citrate, pH 7.0). We used a preproc h o l e c y s t o ~ n probe obtained from Dr. Jack Dixon (Purdue University, West Lafayette, USA). It was cloned into the pSPT19 vector (Pharmacia, Freiburg, FRG) which can be transcribed bidirectionally. Antisense and sense digoxigenin mRNA were made by in vitro transcription [11]. Hybrid molecules were detected with a sheep anti-digoxigenin antibody (F(ab)2-fragments) tagged with alkaline phosphatase (1:1500; Boehringer, Mannheim). We used nitroblue tetrazolium (0.35 mg/ml) and 5-bromo-4-chloro-3-indolyl-phosphate (0.18 mg/ml) which give a blue to black reaction product [20]. For identification of hippocampal cell layers and regions, the sections were stained for 5 rain in 0.002% diamidinophenylindole (DAPI; Sigma) in PB. For radioactive in situ hybridization, cryostat sections were thaw-mounted onto gelatine-coated slides and further processed according to the protocol of Burgunder and Young [2]. The antisense oligonucleotides used were complementary to bases 315-362 of the rat CCK mRNA (for details see [4,16]). The oligonucleotides were 3'-endlabeled with a system from New England Nuclear (Bad Homburg, FRG) and [35S]dATP (> 1000 Ci/mmol; Amersham, Braunschweig, FRG). Control sections with the sense probe showed only weak background labeling. After 4 weeks of exposure in nuclear emulsion (LM 1, Amersham), the sections were developed in D19 solution (Kodak) and counterstained with Cresyl violet. In Fig. 1, CCK mRNA-expressing cells in a cryostat section of perfusion-fixed hippocampus are shown using non-radioactive in situ hybridization. Heavily labeled cells are mainly found outside the pyramidal and granular layer, i.e. in strata radiatum and oriens of hippocampal region CA1 and CA3 as well as in the hilar region [3]. This distribution closely resembles the pattern found in immunocytochemical experiments using antibodies against CCK [10,12,13,19]. Similar observations were made when radioactive in situ hybridization was used (Fig. 2a,b). After all incubation periods studied, CCK mRNAexpressing cells were observed in the slice cultures. These neurons were found in all layers of the hippocampus and fascia dentata, but, like in situ, pyramidal neurons and granule cells were not labeled (Fig. 2c--e). Again, similar results were obtained with radioactive and non-radioactive in situ hybridization. The results of the present study have shown that hippocampal neurons are capable of expressing CCK m R N A under in vitro conditions. Moreover, our study of organotypic slice cultures suggests that similar types of nonprincipal cells as observed in situ express the CCK gene. Under both in situ and in vitro conditions, hippocampal principal neurons, pyramidal neurons and granule cells, were not stained above background level. Our main result is that CCK mRNA expression in hippocampal interneurons [10] is not dependent on extrinsic
Fig. 1. C C K m R N A expression in young adult rat hippocampus visualized by non-radioactive in situ hybridization. Most labeled neurons are seen in strata radiatum and oriens of the hippocampus and in the hilar region of the fascia dentata, g, gramdar layer: h, hilus: m. molecular layer of fascia dentata; sp, pyramidal layer: so, s~ratum oriens: st', stratum radiatum. Bar = 200/am.
afferents. This is in line with previous reports which have similarly shown that neocortical neurons in culture ex-
A. Strauhe el al./Nem'o,wience L e t t e r s 172 : 19~)4~ 143 140
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r Fig 2. a,b: C C K m R N A expression in the young adult rat hippocampus. Radioactive in situ hybridization. Boxed area in a shown at higher magnification in b, For abbreviations see Fig. I. Bars (a) 500 urn; (b) 50 Jam. c,d: C C K m R N A expression in a hippocampal slice culture incubated for 5 days. Boxed area in c shown at higher magnification in d. C C K mRNA-expressing cells are mainly t\mnd outside the pyramidal layer. Bars (c) 100/am; (d) 25/am. e: similar types of neurons are labeled in the slice cultures when using non-radioactixe in situ hybridization. A heavily labeled cell is seen directly underneath the pyramidal layer in ('A3 in a slice culture incubated l\~r 13 dltys. Bar = 20 lml.
146
A. Straube et al. /Neuroscience Letters 172 119t)4~ ~ 143 146
press CCK [16] and that hippocampal nonpyramidal neurons in slice culture express the mRNA for somatostatin and neuropeptide Y [6]. The authors wish to thank Drs B. Heimrich and C. Olenik for their help in the initial phase of this study, Prof. D.K. Meyer for helpful comments on the manuscript and U. Geiger-Denzel and M. Winter for technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 325), and is in partial fulfilment of the requirements for the degree of Dr. rer. nat. at the University of Freiburg (A.S.). [1] Boden, P. and Hill, R.G., Effects of eholecystokinin and related peptides on neuronal activity in the ventromedial nucleus of the rat hypothalamus, Br. J. Pharmaeol., 94 (1988) 246-252. [2] Burgunder, J.-M. and Young III, W.S., The distribution of thalamic projection neurons containing cholecystokinin messenger RNA, using in situ hybridization histochemistry and retrograde labeling, Mol. Brain Res,, 4 (1988) 179-189. [3] Burgunder, J.-M. and Young III, W.S., Cortical neurons expressing the cholecystokinin gene in the rat: distribution in the adult brain, 0ntogeny, and some of their projections, J. Comp. Neurol., 300 (t990) 1-21. [4] Deschenes, R.J., Haun, R.S. and Dixon, J.E., Cloning and sequence analysis of eDNA encoding rat preprocholecystokinin, Proc. Natl. Acad. Sci. USA, 81 (1984) 726-730. [5] Dodd, J. and Kelly, J.S., Excitation of CA1 pyramidal neurones of the hippocampus by the tetra- and octapeptide C-terminal fragments of cholecystokinin. J. Physiol., 295 (1979) 61-62. [6] Finsen, B.R., Tonder, N., Augood, S. and Zimmer, J., Somatostatin and neuropeptide Y in organotypic slice cultures of the rat hippocampus: An immunocytochemical and in situ hybridization study, Neuroscience, 47 (1992) 105-113. [7] Frotscher, M., Heimrich, B. and Schwegler, H., Plasticity of identified neurons in slice cultures of hippocampus: a combined Golgi/ electron microscopic and immunocytochemical study, Prog. Brain Res., 83 (1990) 323-339. [8] G/ihwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329-342. [9] G~ihwiler, B.H., Slice cultures ofcerebellar, hippocampal and hypothalamic tissue, Experientia, 40 (1984) 235-243.
[10] Handelmann, G., Meyer, D.K., Beinleld, M.C. and Oertel, W.H., CCK-containing terminals in the hippocampus are derived from intrinsic neurons: an immunohistochemical and radioimmunological study, Brain Res., 24 (1981) 180--184. [11] H61tke, H.-J. and Kessler, C., Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG): hybridization and ELISA-based detection, Nucl. Acids Res., 18 (1990) 5843-5851. [12] Kosaka, T., Kosaka, K., Tateishi, K., Harnaoka, Y, Yanaihara, N., Wu, J.-Y. and Hama, K., GABAergic neurons containing CCK-8-1ike and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus, J. Comp, Neurol., 239 (1985~ 420 430. [13] Leranth, C. and Frotscher, M., Synaptic connections of cholecystokinin-immunoreactive neurons and terminals in the rat fascia dentata: A combined light and electron microscopic study~ J. Comp. Neurol., 254 (1986) 51-64. [14] Leranth, C. and Frotscher, M., GABAergic input of cholecystokinin-immunoreactive neurons in the hilar region of the rat hippocampus. An electron microscopic double immunostaining study, Histochemistry, 86 (1987) 287-290. [15] Lopes da Silva, F.H., Witter, M.E, Boeijinga, EH. and Lohman, A.H.M., Anatomical organization and physiology of the limbic cortex, Physiol. Rev., 70 (1990)453-511. [16] Olenik, C., Heimrich, B. and Meyer, D.K., Expression of the cholecystokinin gene in organotypic slice cultures of immature rat somatosensory cortex, Neurosci. Lett., 155 (1993) 204-207. [17] Paudice, P. and Raiteri, M., Cholecystokinin release mediated by 5-HT3 receptors in rat cerebral cortex and nucleus accumbens, Br. J. Pharmacol., 103 (1991) 1790-1794. [18] Raiteri, M., Paudice, P. and Vallebuona, F., Release of cholecystokinin in the central nervous system, Neurochem. Int., 22 (1993) 519-527. [19] Somogyi, E, Hodgson, A.J., Smith, A.D., Nunzi, M.G., Gorio, A. and Wu, J.-Y., Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material, J. Neurosci., 4 0984) 2590-2603. [20] Wahle, P. and Beckh, S., A method of in situ hybridization combined with immunocytochemistry, histochemistry, and tract tracing to characterize the mRNA expressing cell types in heterogeneous neuronal populations, J. Neurosci. Methods, 41 (1992) 153--166.
Neuroscience Letters 172 (1994) 143 146
NIUROSCIINCE LETTERS
Expression of the cholecystokinin gene in rat hippocampal interneurons is independent of extrinsic input A. Straube a, R. Bender ~, U. Otten b, M. Frotscher ~t'* "Institute of Anatomy, University 0["F?eiburg, PO Box 111, D-79001 Freihurg, FRG bInstitute of Physiology, UniversiO' o[ Basel, Basel, Switzerland Received 22 December 1993; Revised version received 9 February 1994; Accepted 14 March 1994
Abstract
In the present study we have used slice cultures of hippocampus and in situ hybridization techniques in order to study the ability of hippocampal neurons to synthesize CCK mRNA in the absence of extrinsic afferents. Our results show that very similar types of hippocampal neurons express CCK mRNA in culture as in situ. We conclude that the expression of the CCK gene in hippocampal neurons is not dependent on extrinsic afferent input. Key words'." Hippocampus; Slice culture; Nonpyramidal cell: Neuropeptide; In situ hybridization: Neuronal differentiation
The majority of hippocampal nonpyramidal neurons react with antibodies against G A B A or its synthesizing enzyme glutamate decarboxylase ( G A D ) and are assumed to be inhibitory. In addition to the inhibitory neurotransmitter m a n y of these neurons contain neuropeptides, a m o n g them cholecystokinin (CCK) [12,19]. Little is known about the functional significance of these neuropeptides and the factors determining their biosynthesis and release. Contradictory results have been reported about the functional effects of CCK. Thus, C C K has been shown to excite central neurons [1,5], but an inhibitory postsynaptic effect has also been noticed [15]. As far as the release of C C K is concerned, studies in the cortex and nucleus accumbens have provided evidence for a role of serotoninergic brain stem afferents [17,18]. These studies, as well as our own electron microscopic immunocytochemical studies on CCK-containing neurons in the hippocampus [13,14], suggest that C C K neurons are integrated in complex circuits, and one may speculate that the expression and release of C C K in cortical and hippocampal neurons is controlled by afferent fibers.
*Corresponding author. Fax: (|) (761) 203-5054. 0304-3940/94/$7.00 Cc) 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 031)4-3940(94)00231-X
In the present study, we have addressed the question of whether C C K m R N A expression in hippocampal neurons is regulated by extrinsic afferents. For this we have studied the C C K m R N A expression in neurons of the hippocampus under isolated conditions, i.e. in slice culture. Slice cultures are known to preserve the organotypic organization of the hippocampus to a great extent [7-9], allowing us to compare the location of CCK-expressing cells in slice cultures with that in the normal hippocampal formation. Slice cultures of hippocampus were prepared as described in detail elsewhere [7 9]. Four- to 5-day-old rat pups were decapitated and coronal slices of 400/Ira were cut from both hippocampi. After 5, 13 or 23 days of cultivation, the slices were fixed in 4% paraformaldehyde in phosphate buffer (PB) for 15 rain and then incubated in 20% saccharose in PB. Next, the cultures were shockfrozen on dry ice and stored at -80°C. In the present study, the expression of C C K m R N A was studied by in situ hybridization using either a radioactive method with an oligonucleotide probe or a non-radioactive approach with a digoxigenin-labeled C C K m R N A . For non-radioactive in situ hybridization a modified version of the method of Wahle and Beckh [20] was used. 15/~m cryostat sections were collected in 2 x SSC (20 x SSC is 3 M
144
A. Straube et aL/Neurosctence Letwr,~ 172 ~19!~4; 143 146
NaC1, 0.3 M sodium citrate, pH 7.0). We used a preproc h o l e c y s t o ~ n probe obtained from Dr. Jack Dixon (Purdue University, West Lafayette, USA). It was cloned into the pSPT19 vector (Pharmacia, Freiburg, FRG) which can be transcribed bidirectionally. Antisense and sense digoxigenin mRNA were made by in vitro transcription [11]. Hybrid molecules were detected with a sheep anti-digoxigenin antibody (F(ab)2-fragments) tagged with alkaline phosphatase (1:1500; Boehringer, Mannheim). We used nitroblue tetrazolium (0.35 mg/ml) and 5-bromo-4-chloro-3-indolyl-phosphate (0.18 mg/ml) which give a blue to black reaction product [20]. For identification of hippocampal cell layers and regions, the sections were stained for 5 rain in 0.002% diamidinophenylindole (DAPI; Sigma) in PB. For radioactive in situ hybridization, cryostat sections were thaw-mounted onto gelatine-coated slides and further processed according to the protocol of Burgunder and Young [2]. The antisense oligonucleotides used were complementary to bases 315-362 of the rat CCK mRNA (for details see [4,16]). The oligonucleotides were 3'-endlabeled with a system from New England Nuclear (Bad Homburg, FRG) and [35S]dATP (> 1000 Ci/mmol; Amersham, Braunschweig, FRG). Control sections with the sense probe showed only weak background labeling. After 4 weeks of exposure in nuclear emulsion (LM 1, Amersham), the sections were developed in D19 solution (Kodak) and counterstained with Cresyl violet. In Fig. 1, CCK mRNA-expressing cells in a cryostat section of perfusion-fixed hippocampus are shown using non-radioactive in situ hybridization. Heavily labeled cells are mainly found outside the pyramidal and granular layer, i.e. in strata radiatum and oriens of hippocampal region CA1 and CA3 as well as in the hilar region [3]. This distribution closely resembles the pattern found in immunocytochemical experiments using antibodies against CCK [10,12,13,19]. Similar observations were made when radioactive in situ hybridization was used (Fig. 2a,b). After all incubation periods studied, CCK mRNAexpressing cells were observed in the slice cultures. These neurons were found in all layers of the hippocampus and fascia dentata, but, like in situ, pyramidal neurons and granule cells were not labeled (Fig. 2c--e). Again, similar results were obtained with radioactive and non-radioactive in situ hybridization. The results of the present study have shown that hippocampal neurons are capable of expressing CCK m R N A under in vitro conditions. Moreover, our study of organotypic slice cultures suggests that similar types of nonprincipal cells as observed in situ express the CCK gene. Under both in situ and in vitro conditions, hippocampal principal neurons, pyramidal neurons and granule cells, were not stained above background level. Our main result is that CCK mRNA expression in hippocampal interneurons [10] is not dependent on extrinsic
Fig. 1. C C K m R N A expression in young adult rat hippocampus visualized by non-radioactive in situ hybridization. Most labeled neurons are seen in strata radiatum and oriens of the hippocampus and in the hilar region of the fascia dentata, g, gramdar layer: h, hilus: m. molecular layer of fascia dentata; sp, pyramidal layer: so, s~ratum oriens: st', stratum radiatum. Bar = 200/am.
afferents. This is in line with previous reports which have similarly shown that neocortical neurons in culture ex-
A. Strauhe el al./Nem'o,wience L e t t e r s 172 : 19~)4~ 143 140
•
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•
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r Fig 2. a,b: C C K m R N A expression in the young adult rat hippocampus. Radioactive in situ hybridization. Boxed area in a shown at higher magnification in b, For abbreviations see Fig. I. Bars (a) 500 urn; (b) 50 Jam. c,d: C C K m R N A expression in a hippocampal slice culture incubated for 5 days. Boxed area in c shown at higher magnification in d. C C K mRNA-expressing cells are mainly t\mnd outside the pyramidal layer. Bars (c) 100/am; (d) 25/am. e: similar types of neurons are labeled in the slice cultures when using non-radioactixe in situ hybridization. A heavily labeled cell is seen directly underneath the pyramidal layer in ('A3 in a slice culture incubated l\~r 13 dltys. Bar = 20 lml.
146
A. Straube et al. /Neuroscience Letters 172 119t)4~ ~ 143 146
press CCK [16] and that hippocampal nonpyramidal neurons in slice culture express the mRNA for somatostatin and neuropeptide Y [6]. The authors wish to thank Drs B. Heimrich and C. Olenik for their help in the initial phase of this study, Prof. D.K. Meyer for helpful comments on the manuscript and U. Geiger-Denzel and M. Winter for technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 325), and is in partial fulfilment of the requirements for the degree of Dr. rer. nat. at the University of Freiburg (A.S.). [1] Boden, P. and Hill, R.G., Effects of eholecystokinin and related peptides on neuronal activity in the ventromedial nucleus of the rat hypothalamus, Br. J. Pharmaeol., 94 (1988) 246-252. [2] Burgunder, J.-M. and Young III, W.S., The distribution of thalamic projection neurons containing cholecystokinin messenger RNA, using in situ hybridization histochemistry and retrograde labeling, Mol. Brain Res,, 4 (1988) 179-189. [3] Burgunder, J.-M. and Young III, W.S., Cortical neurons expressing the cholecystokinin gene in the rat: distribution in the adult brain, 0ntogeny, and some of their projections, J. Comp. Neurol., 300 (t990) 1-21. [4] Deschenes, R.J., Haun, R.S. and Dixon, J.E., Cloning and sequence analysis of eDNA encoding rat preprocholecystokinin, Proc. Natl. Acad. Sci. USA, 81 (1984) 726-730. [5] Dodd, J. and Kelly, J.S., Excitation of CA1 pyramidal neurones of the hippocampus by the tetra- and octapeptide C-terminal fragments of cholecystokinin. J. Physiol., 295 (1979) 61-62. [6] Finsen, B.R., Tonder, N., Augood, S. and Zimmer, J., Somatostatin and neuropeptide Y in organotypic slice cultures of the rat hippocampus: An immunocytochemical and in situ hybridization study, Neuroscience, 47 (1992) 105-113. [7] Frotscher, M., Heimrich, B. and Schwegler, H., Plasticity of identified neurons in slice cultures of hippocampus: a combined Golgi/ electron microscopic and immunocytochemical study, Prog. Brain Res., 83 (1990) 323-339. [8] G/ihwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329-342. [9] G~ihwiler, B.H., Slice cultures ofcerebellar, hippocampal and hypothalamic tissue, Experientia, 40 (1984) 235-243.
[10] Handelmann, G., Meyer, D.K., Beinleld, M.C. and Oertel, W.H., CCK-containing terminals in the hippocampus are derived from intrinsic neurons: an immunohistochemical and radioimmunological study, Brain Res., 24 (1981) 180--184. [11] H61tke, H.-J. and Kessler, C., Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG): hybridization and ELISA-based detection, Nucl. Acids Res., 18 (1990) 5843-5851. [12] Kosaka, T., Kosaka, K., Tateishi, K., Harnaoka, Y, Yanaihara, N., Wu, J.-Y. and Hama, K., GABAergic neurons containing CCK-8-1ike and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus, J. Comp, Neurol., 239 (1985~ 420 430. [13] Leranth, C. and Frotscher, M., Synaptic connections of cholecystokinin-immunoreactive neurons and terminals in the rat fascia dentata: A combined light and electron microscopic study~ J. Comp. Neurol., 254 (1986) 51-64. [14] Leranth, C. and Frotscher, M., GABAergic input of cholecystokinin-immunoreactive neurons in the hilar region of the rat hippocampus. An electron microscopic double immunostaining study, Histochemistry, 86 (1987) 287-290. [15] Lopes da Silva, F.H., Witter, M.E, Boeijinga, EH. and Lohman, A.H.M., Anatomical organization and physiology of the limbic cortex, Physiol. Rev., 70 (1990)453-511. [16] Olenik, C., Heimrich, B. and Meyer, D.K., Expression of the cholecystokinin gene in organotypic slice cultures of immature rat somatosensory cortex, Neurosci. Lett., 155 (1993) 204-207. [17] Paudice, P. and Raiteri, M., Cholecystokinin release mediated by 5-HT3 receptors in rat cerebral cortex and nucleus accumbens, Br. J. Pharmacol., 103 (1991) 1790-1794. [18] Raiteri, M., Paudice, P. and Vallebuona, F., Release of cholecystokinin in the central nervous system, Neurochem. Int., 22 (1993) 519-527. [19] Somogyi, E, Hodgson, A.J., Smith, A.D., Nunzi, M.G., Gorio, A. and Wu, J.-Y., Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material, J. Neurosci., 4 0984) 2590-2603. [20] Wahle, P. and Beckh, S., A method of in situ hybridization combined with immunocytochemistry, histochemistry, and tract tracing to characterize the mRNA expressing cell types in heterogeneous neuronal populations, J. Neurosci. Methods, 41 (1992) 153--166.