Distribution of mRNA for CCK-B receptor in the brain of Mastomys natalensis: abundant expression in telencephalic neurons

BRAIN RESEARCH ELSEVIER

Brain Research 640 (1994) 81-92

Research Report

Distribution of m R N A for CCK-B receptor in the brain of Mastomys natalensis" abundant expression in telencephalic neurons Yasufumi Shigeyoshi a , , Hitoshi Okamura c, Tsutomu Inatomi c, Toshimitsu Matsui a, Mitsuhiro Ito a, Hidesuki Kaji a, Hiromi Abe a, Hirohisa Nakata b, Tsutomu Chiba b, Kazuo Chihara a a

Third Division and b Division of Gerontology, Department of Medicine, Kobe University School of Medicine, Kusunoki-cho, 7 Chome, Chuo-ku, Kobe 650, Japan c Department of Anatomy, Kyoto Prefectural University of Medicine, Kyoto, Japan (Accepted 2 November 1993)

Abstract

The distribution of chelecystokinin B (CCK-B) receptors in the Mastomys brain was studied using Northern blot analysis and in situ hybridization technique. By Northern blot analysis using 32p-labeled cDNA probe, the cortex had the highest hybridization signal of CCK-B receptor mRNA in the brain. The olfactory bulb and hippocampus showed a moderate level of signals. In situ hybridization using 35S-labeled cRNA probes revealed a wide and region-specific distribution of CCK-B receptor mRNA in the telencephalon. Throughout the cerebral cortex, labeled cells were found in all layers, with higher intensities in layers II, V and VI. Pyramidal cells of the layer II of the piriform cortex showed the highest level of signals in the brain. In the hippocampus, most of the pyramidal cells of the Ammon's horn were labeled, although labeled cells were not detected in other layers. Distinct signals were also detected in the various amygdaloid nuclei, caudate-putamen, reticular thalamic nucleus, hypothalamic ventromedial nucleus and inferior colliculus. This distribution pattern may further support the prominent existence of CCK-B receptors in the brain particularly in the telencephalon.

Key words: CCK-B receptor; Mastomys natalensis; Northern hybridization; In situ hybridization; Telencephalon; Cerebral cortex

1. Introduction

The octapeptide cholecystokinin is one of the most abundant peptides in the mammalian central nervous system. Number of studies on this peptide implicate significant roles of CCK in the brain, such as suppression of feeding behavior [30], modulation of brain dopaminergic systems [35], and antagonization to the effect of opioid analgesia [10]. Radioimmunoassay [1] as well as immunohistochemical studies [32] with the specific antibody to CCK-8 and in situ hybridization studies [3,19,28] with complementary nucleotide probes revealed the significant presence of CCK in many brain regions including the cerebral cortex, thalamus, hypothalamus, hippocampus, and brain stem nuclei.

* Corresponding author. Fax: (81) (78) 371-6468. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 15 21-4

Radio-ligand-binding studies [8,12,20,27,36] have demonstrated that CCK receptors widely exist in the mammalian central nervous system, with especially high concentrations in the neocortex, piriform cortex, hippocampus, and olfactory bulb. The development of subtype-specific antagonists facilitated the discrimination of CCK-A and CCK-B receptors [14-16,23]. Studies on the rat showed that CCK-A receptors exist in the area postrema, nucleus tractus solitarius, interpeduncular nucleus, posterior hypothalamus, and habenula, while CCK-B receptors exist in the cerebral cortex, hypothalamic ventromedial nuclei, hippocampus and amygdaloid complex. Although these studies have revealed the localization of CCK-A and CCK-B receptors in the brain to some extent, there are some limitations in this method. Radioligands detect receptors in its relatively high affinity state, and this autoradiographic binding method analyzed positive regions at gross level, but not on cellular level.

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Recently, we have isolated the CCK-B receptor cDNA from enterochromaffin-like carcinoid tumor of Mastomys natalensis [24]. In the present study, we examined what regions in the Mastomys brain express CCK-B receptor mRNA by Northern blot hybridization. In order to obtain the more precise localization of the CCK-B receptor mRNA in the CNS, we have also applied in situ hybridization technique using [35S]cRNA probe. Previous binding studies revealed wide species differences in CCK-binding sites in mammalian brain particularly in telencephalon structures [7,21,33]. Our study may help consider the species differences in the distribution of CCK-B receptors on mRNA level.

2. Materials and methods 2.1. Radiolabeling of cDNA and cRNA probes The cDNA probe used for Northern blot analysis was made from 660-bp Mastomys CCK-B receptor cDNA containing 19 bp of 5'-untranslated region and 648 bp of coding region. They were labeled with a-[32 P]dCTP with a modification of the random priming method [11]. For in situ hybridization using cRNA probe, this cDNA (660 bp) was subcloned into the E c o R I - X h o I site of pGEM7Zf(-). For cRNA generation, the construct was linearized with either HindlIl or Xbal for antisense or sense probe, respectively. The in vitro transcription was carried out at 37°C for 60 rain in a 20-~1 reaction mixture containing 1 /zg of linearized plasmid templates and T7 or SP6 RNA polymerase in the presence of [35S]CTPaS (800 Ci/mmol, 1 Ci = 37 GBq; Amersham).

2.2. Northern blot hybridization Six brain regions from adult male Mastomys natalensis (75-85 g) were isolated, and total RNA (10/xg) was prepared from each tissue [5]. Total RNA was denatured by heating at 65°C. After fractionalization by agarose (1%) gel electrophoresis (20 mM sodium phosphate buffer, pH 7.0) and beeing transferred to nylon membranes (GeneScreenPlus, New England Nuclear), the RNA was hybridized with a cDNA probe labeled with [32p]dCTP by the random primer method. Hybridization was performed in 5 xSSPE (0.9 M NaCI, 5 mM EDTA, 50 mM NaH2PO4, pH 7.7), 1xDenhardt's solution, 50% formamide, 1% SDS, 100 /zg/ml salmon sperm DNA (sonicated and denatured), at 42°C for 16 h. After a final wash in 0.1 ×SSC (1 x = 0.15 M NaCI, and 0.015 M sodium citrate, pH 7.0) at 65°C, the hybridizing signals were visualized by autoradiography. The same filter was hybridized with 32p-labeled rat glyceraldehyde3-phosphate dehydrogenase (GAPDH) cDNA probe for internal control. The intensity of each band was quantified by densitometric scanning using the MCID system (an automated image processing system) (Imaging Research Inc., Ont., Canada).

formed by a modification of protocols utilized successfully with other probes [25,34]. Briefly, tissue sections were transferred through 4 x SSC, proteinase K (0.5 mg/ml, 0.1 M Tris buffer pH 8.0, 50 mM EDTA) for 20 min at 37°C, 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, and 4XSSC for 30 min. The sections were then incubated in the hybridization buffer (50% formamide, 10% dextran sulfate, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 0.3 M NaCI, 10 mM NaPB (pH 8.0), 0.2% N-laurylsarcosine, 500 p.g/ml yeast tRNA 0.33 mg/ml denatured salmon sperm DNA, 10 mM dithiothreitol (DTT)) containing the [!SS]CCK-B receptor cRNA at a concentration of 5x105 cpm/100 p.l for 12 h at 55°C. Following two rinses in 2 × SSC/50% formamide at 60°C for 15 min, the sections were treated with a solution containing 20 /xg/ml RNase A, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 M NaCI for 30 min at 37°C. The sections were further washed in 2 X SSC/50% formamide at 60°C for 15 rain twice, 1 x SSC (60°C, 30 min) and 0.1xSSC (60°C, 30 min). Then the sections were air-dried and dehydrated with ethanol and chloroform. The slides were placed in an X-ray cassette with /3-Max film for 5-7 days. They were then dipped in nuclear track emulsion (lllford K5) (1:1 with H20) and exposed for 4 weeks at 4°C. Following development of the tissue autoradiograms, the sections were stained with Cresyl violet and analyzed under brightfield and darkfield microscopy. The nomenclature used in this study was according to Paxinos and Watson [26], and Zilles and Wree [37].

3. Results

3.1. Northern hybridization Total RNA from 6 different regions (cerebellum, hippocampus, brain stem, cortex, olfactory bulb, and

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2.3. In situ hybridization histochemistry Adult male Mastomys natalensis (75-85 g) were anesthesized by pentobarbital and intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PB). Brains were postfixed in the fixative for 24 h at 4°C and transferred to 30% sucrose in PB for 48 h at 4°C. Then they were frozen at -50°C in acetone and stored at 70°C. Stored tissues were cut at 20-30 /~m on the coronal plane and collected into 4 x SSC. In situ hybridization histochemistry was per-

Fig. 1. Northern hybridization of total RNA from Mastomys brain using 32p-labeled cDNA antisense probe. 1, cerebellum; 2, hippocampus; 3, brain stem; 4, cortex; 5, olfactory bulb; 6, striatum. Discrete bands of 2.1 kb were admitted in every region examined. The strongest band was detected in the cerebral cortex. The hybridization signal of cerebellum was weak, but significantly admitted. The same filter was rehybridized with rat GAPDH cDNA probe (lower panel).

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striatum) were analyzed by Northern hybridization method using 32p-labeled Mastomys cDNA probe (Fig. 1). A discrete band of RNA with an approximated size of 2.1 kb was found in all the regions examined. Among these regions, the cerebral cortex showed the highest level. Intermediate levels were detected in hippocampus, and olfactory bulb. The lowest expression levels were admitted in the brain stem, striatum, and cerebellum, corresponding to 8-10% of the amount found in cerebral cortex. No signals were detected in the Mastomys liver (data not shown).

of Mastomys brain (data not shown). In our in situ hybridization study, most of positive signals in the brain were detected in telencephalic structures, including the cerebral cortex, hippocampus, and amygdala (Fig. 2A,C,D,E and Fig. 3). The results using smaller fragments (100-150 bp) by limited alkaline hydrolysis showed little differences from non-hydrolyzed probe. No signals were detected in sections hybridized with 35S-labeled sense probe (Fig. 2B).

3.2. In situ hybridization

CCK-B receptor mRNA-positive signals were found throughout all regions of the neocortex and paleocorrex. In the neocortex these cells were detected in all layers except layer I, but were densely localized to layers II, V, and VI (Fig. 4). Highest density of labeled cells was found in the deeper part of layer VI. In layer V, many heavily labeled pyramidal cells were detected.

3.3. Cerebral cortex

The localization of CCKoB receptor mRNA-positive signal in brain sections was analyzed by in situ hybridization using asS-labeled anti-sense RNA probes. In Northern hybridization study, this cRNA probe showed a discrete band at 2.1 kb of RNA in the cortex

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Fig. 2. Film autoradiography of in situ hybridization using asS-labeled antisense (A,C,D,E) and sense (B) probes for CCK-B receptor mRNA. A and B are photographed in sections at similar coronal level. AP, amygdalopiriform transition area; BLA, basolateral amygdaloid nucleus; CA1, 3, fields CA1 and CA3 of Ammon's horn; CCx, cerebral cortex; CP, caudate putamen; MeA, medial amygdaloid nucleus; PCoA, posterior cortical amygdaloid nucleus; Pit, piriform cortex; VMH, hypothalamic ventromedial nucleus. Bar = 1 /zm.

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Fig. 3. Schematic representation of the distribution of CCK-B receptor mRNA-expressing cells. A-F: panels are arranged in rostro-caudal order. Filled circles represent moderate to strongly labeled cells, and open circles represent weakly labeled cells. AA, anterior amygdaloid area; Acb, nucleus accumbens; AP, amygdalopiriform transition area; BLA, basolateral amygdaloid nucleus; Ca, anterior commissure; CA1, field CA1 of Ammon's horn; CA3, field CA3 of Ammon's horn; cc, corpus callosum; CeA, central amygdaloid nucleus; Cg, cingulate cortex; CP, caudate putamen; DEn, deep endopiriform nucleus; Ent, entorhinal cortex; Fr, frontal cortex; Ic, inferior colliculus; In, insular cortex; lo, lateral olfactory tract; oc, optic chiasm; Oc, occipital cortex; Par, parietal cortex; Pas, parasubiculum; Pir, piriform cortex; PCoA, posterior cortical amygdaloid nucleus; PRh, perirhinal cortex; PVT, thalamic paraventricular nucleus; RS, retrosplenial cortex; RT, reticular thalamic nucleus; S, subiculum; Te, temporal cortex; TuO, tuberculum olfactorium; VMH, hypothalamic ventromedial nucleus.

T h o u g h g e n e r a l d i s t r i b u t i o n p a t t e r n s o f t h e s e layers are c o n s i s t e n t t h r o u g h the c e r e b r a l cortex, t h e r e a r e s o m e r e g i o n a l differences. L a y e r II o f t h e s o m a t o s e n sory cortex s h o w e d fewer l a b e l e d cells c o m p a r e d with that o f o t h e r cortical r e g i o n s (Fig. 4C,D). In t h e sensor i m o t o r c o r t e x c o r r e s p o n d i n g to the f o r e l i m b a n d

h i n d l i m b areas, large-sized p y r a m i d a l cells w e r e heavily l a b e l e d (Fig. 4A, B). T h e p a l e o c o r t e x has a relatively h i g h e r density o f cells c o n t a i n i n g C C K - B r e c e p t o r m R N A t h a n t h e neocortex. In t h e g r a n u l a r insular cortex, w h e r e 6-layer s t r u c t u r e was relatively easily d e l i n e a t e d , d e n s i t y o f l a b e l e d cells in layer II, I I I a n d

Fig. 4. Darkfield (A,C,E) and brightfield (B,D,F) representation of cells containing CCK-B receptor mRNA in the cerebral cortex. A, C, and E are same sections as B, D, and F. A,B: frontal (Fr) and hindlimb area (HL) of sensorimotor cortex. Note that large-sized pyramidal cells (arrows in A) in layer V of hindlimb area were heavily labeled. C,D: parietal cortex. E,F: granular insular cortex. Bars = 200/xm.

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Fig. 5. Darkfield (A) and brightfield (B) photomicrographs

of CCK-B receptor mRNA-containing cells in the cingulate cortex. Bars = 100 sm.

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Fig. 6. Darkfield (A) and brightfield (B) photomicrograph of cells containing CCK-B receptor mRNA in the piriform cortex. I, superficial plexiform layer; II, pyramidal cell layer; III, polymorphcell layer. Bars = 100/zm.

IV was higher than that of parietal or frontal cortical areas (Fig. 4E,F). In the cingulate cortex, labeled cells were densely packed in layer I I / I I I , and they were scattered in layers IV, V, and VI (Fig. 5). Layer II of the piriform cortex showed the strongest signals in the brain (Fig. 6). Most of the pyramidal cells of this layer showed heavy isotope-labeling. Polymorphic layers also contained a moderate number of labeled cells. The retrosplenial cortex has a high density of labeled neurons in all layers.

3.4. Hippocampus

In the Ammon's horn, very heavily labeled cells were densely packed in the stratum pyramidale throughout CA1 to CA4 in both dorsal and ventral hippocampus (Fig. 7). In other layers (str. oriens, str. radiatum, and str. lacunosum moleculare) of the Ammon's horn, no labeled cells were detected. In the gyrus dentatus, no labeled cells were detected in the molecular layer, granular layer, and the hilus. In

the subiculum, many labeled cells were detected in the pyramidal layer but their signals were weaker than those of pyramidal ceils in CA1. 3.5. A m y g d a l a

The mRNA-positive cells were scattered in most of the subdivision of the amygdaloid complex. Among all, in medial amygdaloid nucleus and posterior cortical amygdaloid nucleus, many labeled cells were observed (Figs. 2D and 8B). Amygdalopiriform transition area and deep encephalitic nucleus contained a high density of labeled cells (Figs. 2E and 8A). 3.6. Striatum

Lightly labeled cells were present in the caudateputamen. Labeled cells were more frequently detected in the dorsal half of the caudate-putamen (Figs. 2A and 8E). Weakly labeled cells were also detected in the nucleus accumbens and the granular cell layer of the tuberculum olfactorium.

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Fig. 8. Darkfield (A,B,C,E) and brightfield (D,F) representations of CCK-B mRNA-expressing cells in various rat brain areas. A: amygdalopiriform transition area (AP) and posterior cortical amygdaloid nucleus (PCOA). B: basolateral amygdaloid nucleus (BLA). C: reticular thalamic nucleus (RT). D: hypothalamic ventromedial nucleus (VMH). E: caudate putamen. F: inferior colliculus (IC). Note that positive signals in RT, VMH, caudate-putamen, and inferior colliculus were weak compared with those in cortical structures (see Figs. 4, 5, 6, or panels A and B in this figure). 3V, third ventricle; aq, aqueduct of the midbrain; Hipp, hippocampus; ic, internal capsule; IC, inferior colliculus; ot, optic tract. Bars = 200/zm.

3. 7. Diencephalon, mesencephalon and metencephalon C o n t r a r y to the t e l e n c e p h a l o n , o t h e r b r a i n regions express very few signals of C C K - B r e c e p t o r m R N A . I n

the d i e n c e p h a l o n , the t h a l a m i c reticular n u c l e u s expressed distinct signals of C C K - B r e c e p t o r m R N A (Fig. 8C). I n o t h e r regions of the thalamus, n o l a b e l e d cells were d e t e c t e d except weakly l a b e l e d cells in the

Fig. 7. CCK-B receptor mRNA-containing cells in the dorsal (A) and ventral (B) hippocampus. Note positive signals were only found in stratum pyramidale, and most pyramidal cells express CCK-B receptor mRNA. cc, corpus callosum; fi, fimbria hippocampi; GD, gyrus dentatus; if, rhinal fissure. Bars = 500/zm.

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paraventricular nucleus. Weakly labeled cells were detected in the hypothalamic ventromedial nucleus (Figs. 2D and 8D). In other regions of the hypothalamus, a few weakly labeled cells were scattered. Except cells expressing weak signals in the inferior colliculus (Fig. 8F), no labeled cells were detected in the mesencephalon including substantia nigra, and in the pons. In the medulla oblongata, weakly labeled cells were only detected in the prepositus hypoglossal nucleus. In the cerebellar cortex, weak signals were detected in the granular layer with the CCK-B receptor antisense probe. However, we judged them as non-specific reaction, since we observed the same degree of signals using CCK-B receptor sense probe.

4. Discussion

We applied the Northern and in situ hybridization techniques to analyze the regional expression of CCK-B receptor mRNA in the brain of Mastomys natalensis. This study has firstly demonstrated the abundant expression of CCK-B receptor mRNA signals in neurons of the telencephalon. Northern blot analysis on the distribution of CCK-B receptor mRNA in the Mastorays brain using the 32p-labeled cDNA fragment of the CCK-B receptor showed a discrete band of RNA with an approximated size of 2.1 kb, which is in agreement with our former study [24]. Among various brain regions, the cerebral cortex exhibited a single strongest band. The olfactory bulb and hippocampus also showed strong bands. These results are compatible with our in situ hybridization studies since we admitted strong hybridization signal-containing cells in these structures. On the other hand, the Northern hybridization showed weak CCK-B receptor mRNA signals in the cerebellum, where no specific signals were found by in situ hybridization study. The reason of this discrepancy is not clear. Existence of numerous cells containing a low amount of mRNA for each cell may cause this discrepancy. Our results confirmed the distribution of CCK receptor sites previously described by the autoradiography using radio-labeled ligands [8,14,27,36]. In their autoradiographic binding studies on the rat brain with selective antagonists for the CCK-B receptor, Hills et al. [16]. demonstrated that the cerebral cortex, hippocampus, amygdala, olfactory bulb and hypothalamic ventromedial nucleus (VMH) have high density of CCK-B receptors. We also admitted significant amount of CCK-B receptor mRNA in these structures. From the methodological point of view, autoradiographic study using radioligands visualize CCK-B receptors not only on the intrinsic cells but also on the afferent fibers from other regions of the brain, while in situ hybridization technique exhibits CCK-B receptor mRNA-con-

taining cell bodies. Thus the almost complete identical distribution of CCK-B ligand-binding sites and CCK mRNA-expressing cells suggests that many of CCK-B receptors are expressed in cell bodies and their nearby neuronal processes. The moderate to high densities of hybridization signals present in areas of the brain containing dopaminergic terminals such as the frontal cortex, cingulate cortex, and caudate-putamen, indicates the interaction between CCK-B receptors and dopamine systems in the brain. Previous immunohistochemical studies [17,29] exhibited the co-existence of dopamine and CCK in several brain regions, with highest frequency in the ventral tegmental area, i.e. the A10 dopamine groups. Seroogy et al. [29] demonstrated in their study using retrograde neuronal tracer and immunohistochemistry that dopamine-CCK neurons in the ventral tegmental area project to the caudate-putamen, nucleus accumbens, prefrontal cortex, and amygdala. In our study, these structures demonstrated a significant amount of signals of CCK-B receptor mRNA suggesting that released CCK from these dopamine-CCK neurons might act on its receptor through CCK-B type. However, it is known that the striatum also receives dense CCK projections from the cortex, amygdala and claustrum [22,23]. Thus it is possible that many CCK-B mRNA-expressing cells in the striatum also transduct CCK information from these structures. The cerebral cortex showed the most high density of CCK-B receptor mRNA positive cells in the brain. Particularly, we found numerous large-sized pyramidal cells in the motor cortical area expressing CCK-B mRNA signals. Since CCK mRNA-expressing cells and CCK-immunoreactive cells and fibers [3,32] are abundantly distributed in all layers of the cerebral cortex, these cells may project to surrounding CCK-B receptor-expressing cells. Interhemispheric projection of CCK-neurons [6] may also be the source of CCK to these receptors, particularly those in deep layer VI. This cortical CCK not only modulates cortical neuronal activity [4] but also may protect glutamate-induced cytotoxicity through CCK-B receptors [31]. Projection from the CCK neurons in lower brain nuclei may also contribute to CCK source to the cortex. In particular, a large number of CCK mRNA-containing neurons were demonstrated by in situ hybridization in the majority of thalamic nuclei except habenula, paraventricular, reticular, lateroventral geniculate, and parafascicular nuclei [2,19,28]. These neurons are known to project to the cerebral cortex, and may terminate in CCK-B receptor mRNA-expressing cells in layer III and IV. However, it is still doubtful that these large number of thalamic CCKmRNA-containing cells really express CCK peptide, since almost no CCK-immunoreactive cells are detected by immunocytochemical techniques [32].

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In the present study, we found prominent CCK-B receptor mRNA signals in the pyramidal cells of the hippocampus. By immunohistochemical studies, it has been demonstrated that CCK-8 immunoreactivity is present in neurons in the stratum oriens, stratum pyramidale, and stratum radiatum [13]. The demonstration of CCK-B receptor mRNA in the pyramidal cells may suggests that CCK acts on pyramidal cells through CCK-B receptors, and deporalizes these cells accompanied by a marked increase in neuronal excitability [9]. Very recently, rat CCK-B receptor mRNA-expressing cells were mapped by Honda et al. [18]. Their distributions grossly paralelled our present findings in Mastomys brain, suggesting that no remarkable species differences were noted between the two rodents. Although marked species differences of CCK-B binding sites were noted between rat and guinea pig [7,21,33], the discrepancy should be reexamined at their mRNA levels. Acknowledgements. This work was supported in part by research grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan.

5. References [1] Beinfeld, M.C., Meyer, D.K., Eskay, R.L., Jensen, R.T. and Brownstein, M.J., The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay, Brain Res., 212 (1981) 51-57. [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 cholecystyokinin gene in the rat: distribution in the adult brain, ontogeny, and some of their projections, J. Comp. Neurol., 300 (1990) 26-46. [4] Chiodo, L.A. and Bunney, B.S., Proglumide: selective antagonism of excitatory effects of cholecystokinin in central nervous system, Science, 219 (1983) 1449-1451. [5] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal, Biochem., 162 (1987) 156-159. [6] Cho, H.J., Shiotani, Y., Shiosaka, S., Inagaki, S., Kubota, Y., Kiyama, H., Umegaki, K., Tateishi, K., Hashimura, E., Hamaoka, T. and Tohyama, M., Ontogeny of cholecystokinin-8-containing neuron system of the rat: an immnohistochemical analysis. I. Forebrain and upper brainstem, J. Comp. Neurol., 218 (1983) 25-41. [7] Dietle, M.M. and Palacios, J.M., The distribution of cholecystokinin receptors in the vertebrate brain: species differences studied by receptor autoradiography, J. Chem. Neuroanat., 2 (1989) 149-161. [8] Dijk, A.V., Richards, J.G., Trzeciak, A., Gillessen, D. and M/Shier, H., Cholecystokinin receptors: biochemical demonstration and autoradiographical localization in rat brain and pancreas using [3H]Cholecystokinin-8 as radioligand, J. Neurosci., 4 (1984) 1021-1033. [9] Dodd, J. and Kelly, J.S., The actions of cholecystokinin and

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related peptides on pyramidal neurons of the mammalian hippocampus, Brain Res., 205 (1981) 337-350. [10] Faris, P.L., Opiate antagonistic function of cholecystokinin in analgesia and energy balance systems, Ann. N.Y. Acad. Sci., 448 (1985) 437-447. [11] Feinberg, A.P. and Vogelstein, B., A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity, Anal Biochem., 132 (1983) 6-13. [12] Gaudreau, P., Quirion, R., St-Pierre, S. and Pert, C.B., Characterization and visualization of cholecystokinin receptors in rat brain using [3H]pentagastrin, Peptides, 4 (1983) 755-762. [13] Harris, K.M., Marshall, P.E. and Landis, D.M.D., Ultrastructural study of cholecystokinin-immunoreactive cells and processes in area CA1 of the rat hippocampus, J. Comp. Neurol., 223 (1985) 147-58. [14] Hill, D.R., Campbell, N.J., Shaw, T.M. and Woodruff, G.N., Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective nonpeptide CCK antagonists, J. Neurosci., 7 (1987) 2967-2976. [15] Hill, D.R. and Woodruff, G.N., Differentiation of central cholecystokinin receptor binding sites using the non-peptide antagonists MK-329 and L-365,260, Brain Res., 526 (1990) 276-283. [16] Hill, D.R., Singh, L., Boden, P., Pinnock, R., Woodruff, G.N. and Huges, J., Detection of CCK receptor subtypes in mammalian brain using highly selective non-peptide antagonists. In C.T. Dourish, S.J. Cooper, S.D. Iversen and L.L. Iversen (Eds.), Multiple Cholecystokinin Receptors in the CNS, Oxford University Press, New York, 1992, pp. 57-76. [17] H6kfelt, T., Rehfeld, J.F., Skirboll, L., Ivemark, B., Goldstein, M. and Markey, K., Evidence for coexistence of dopamine and CCK in meso-limbic neurons, Nature, 285 (1980) 476-478. [18] Honda, T., Wada, E., Battey, J.F. and Wank, S.A., Differential gene expression of CCK-A and CCK-B receptors in the rat brain, Mol. Cell. Neurosci., 4 (1993) 143-154. [19] Ingram, S.M., Krause II, R.G., Baldino Jr., F., Skeen, L.C. and Lewis, M.E., Neuronal localization of cholecystokinin mRNA in the rat brain by using in situ hybridization histochemistry, J. Comp. Neurol., 287 (1989) 260-272. [20] Innis, R.B. and Snyder, S.H., Cholecystokinin receptor binding in brain and pancreas: regulation of pancreatic binding by cyclic and acyclic guanine nucleotides, Eur. J. Pharmacol., 65 (1980) 123-124. [21] Mantyh, C.R. and Mantyh, P.W., Differential localization of cholecystokinin-8 binding sites in the rat vs. the guinea pig brain, Eur. J. PharmacoL, 113 (1985) 137-139. [22] Meyer, D.K. and Protopapas, Z., Studies of cholecystokinin containing neuronal pathways in rat cerebral cortex and striatum, Ann. N.Y. Acad. Sci., 448 (1985) 133-143. [23] Moran, T.H. and Mchugh, P.R., Cholecystokinin receptors. In A. B/Sklund, T. H/Skfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, Vol. 7, Neuropeptides in the CNS, Part II, Elsevier, Amsterdam, 1990, pp. 455-476. [24] Nakata, H., Matsui, T., Ito, M., Taniguchi, T., Naribayashi, Y., Arima, N., Nakamura, A., Kinoshita, Y., Chihara, K., Hosoda, S. and Chiba, T., Cloning and characterization of gastrin receptor from ECL carcinoid tumor of Mastomys natalensis, Biochem. Biophys. Res. Commun., 187 (1992) 1151-1157. [25] Okamura, H., Abitbol, M., Julien, J.F., Dumas, S., Berod, A., Geffard, M., Kitahama, K., Bobillier, P., Mallet, J. and Wiklund, L., Neurons containing messenger RNA encoding glutamate decarboxylase in rat hypothalamus demonstrated by in situ hybridization, with special emphasis on cell groups in medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus, Neuroscience, 39 (1990) 675-699. [26] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd. edn., Academic Press, Sydney, 1986.

92

Y. Shigeyoshi et al. / Brain Research 640 (1994) 81-92

[27] Saito, A., Sankaran, H., Goidfine, I.D. and Williams, J.A., Cholecystokinin receptors in the brain: characterization and distribution, Science, 208 (1980) 1155-1156. [28] Savasta, M., Palacios, J.M. and Mengod, G., Regional localization of the mRNA coding for the neuropeptide cholecystokinin in the rat brain studied by in situ hybridization, Neurosci. Lett., 93 (1988) 132-138. [29] Seroogy, K.B. Dangaran, K., Lim, S., Haycock, J.W. and Fallon, J.H., Ventral mesencephalic neurons containing both cholecystokinin- and tyrosine hydroxylase-like immunoreactivities project to forebrain regions, J. Comp. Neurol., 279 (1989) 397-414. [30] Smith, G.P. and Gibbs, J., The satiety effect of cholecystokinin. Recent progress and current problems, Ann. N.Y. Acad. Sci., 448 (1985) 417-423. [31] Tamura, Y., Sato, Y., Akaike, A. and Shiomi, H., Mechanisms of cholecystokinin-induced protection of cultured cortical neurons against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity, Brain Res., 592 (1992) 317-325. [32] Vanderhaeghen, J.J., Neuronal cholecystokinin. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy,

[33]

[34]

[35]

[36]

[37]

Vol. 4, GABA and Neuropeptieds in the CNS, Part I, Elsevier, Amsterdam, 1985, pp. 406-435. Williams, J.A., Gryson, K.A. and McChesney, D.J., Brain CCK receptors: species differences in regional distribution and selectivity, Peptides, 7 (1986) 293-296. Yamada, K., Sakai, M., Okamura, H., Ibata, Y. and Nagatsu, L, Detection of tyrosine hydroxylase and phenylethanolamime-Nmethyltransferase messenger RNAs in the mouse adrenal gland and the brain by in situ hybridization, Histochemistry, 97 (1992) 201-206. Yim, C.C. and Mogenson, G.J., Electrophysiological evidence of modulatory interaction between dopamine and cholecystokinin in the nucleus accumbens, Brain Res., 541 (1991) 12-20. Zarbin, M.A., Innis, R.B., Wamsley, J.K., Snyder, S.H. and Kuhar, M.J., Autoradiographic localization of cholecystokinin receptors in rodent brain, J. Neurosci., 3 (1983) 877-906. Zilles, K. and Wree, A , Cortex: areal and laminar structure. In G. Paximos (Ed.), The Rat Nervous System, Vol. 1, Forbrain and Midbrain, Academic Press, Sydney, 1985, pp. 375-415.