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Regional localization of the mRNA coding for the neuropeptide cholecystokinin in the rat brain studied by in situ hybridization
132
Neuroseience Letters, 93 (1988) 132 138 Elsevier Scientific Publishers Ireland Ltd.
NSL 05669
Regional localization of the mRNA coding for the neuropeptide cholecystokinin in the rat brain studied by in situ hybridization Marc Savasta, Jos6 M. Palacios and Guadalupe
Mengod
Preclinical Research, Sandoz Ltd., Basle (Switzerland)
(Received 8 June 1988; Revised version received 18 July 1988; Accepted 19 July 1988) Key words:
In situ hybridization; Synthetic oligonucleotide; Cholecystokinin; mRNA; Northern blot; Rat brain
The regional localization of mRNA coding for the neuropeptide cholecystokinin (CCK) has been studied in the rat brain by in situ hybridization using a 32p-labelledsynthetic 32 mer oligonucleotide. Autoradiograms were quantified using computer-assisted microdensitometry. High levels of hybridization were observed in the neocortex, claustrum, endopiriform nucleus, cingular cortex, amygdala, olfactory bulb, hippocampus, ventral tegmental area, geniculate nucleus, several thalamic nuclei and substantia nigra compacta. Very weak signal was detected in the striatum, the cerebellum and the brainstem. The topographic distribution of CCK neurons observed overlaps in part with that previously described by immunohistochemical techniques. However, some discrepancies were also found, particularly in the thalamus. These results show that in situ hybridization with oligonucleotide probes together with a semiquantitative method described can be used to map the expression of the CCK mRNA in rat brain sections as well as its modification after pharmacological or physiological manipulations. In the brain the c a r b o x y l t e r m i n a l o c t a p e p t i d e o f c h o l e c y s t o k i n i n ( C C K - 8 ) has been localized i m m u n o h i s t o c h e m i c a l l y in n e u r o n s in different species [3, 15]. A n involvement o f the C C K system in b r a i n p a t h o l o g y has been p r o p o s e d in neurological and psychiatric diseases such as P a r k i n s o n ' s disease, H u n t i n g t o n ' s disease a n d s c h i z o p h r e n i a [1, 2]. The structure o f the gene c o d i n g for C C K has recently been elucidated [5]. In o r d e r to m a p the d i s t r i b u t i o n o f n e u r o n s expressing C C K m R N A in the rat b r a i n we have a p p l i e d the technique o f in situ h y b r i d i z a t i o n using a synthetic o l i g o n u c l e o t i d e a n d c o m p u t e r - a s s i s t e d image analysis for the q u a n t i f i c a t i o n o f the autoradiograms. The o l i g o d e o x y r i b o n u c l e o t i d e was m a d e on a 380 A A p p l i e d Biosystems D N A synthesizer a n d purified on a n 8% p o l y a c r y l a m i d e / 8 M urea p r e p a r a t i v e sequencing gel. The rat C C K o l i g o n u c l e o t i d e was c o m p l e m e n t a r y to bases 6951 6982 o f the C C K gene [5]. T h e o l i g o m e r was labelled by using d e o x y a d e n o s i n e t r i p h o s p h a t e
Correspondence: G. Mengod, Preclinical Research, Sandoz Ltd., CH-4002 Basle, Switzerland.
0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
133
(dATP) labelled at the 0t position with 32p ( > 3000 Ci/mmol, New England Nuclear) and terminal deoxynucleotidyltransferase (Boehringer, Mannheim) to specific activities of 0.9-3 x 104 Ci/mmol. Sprague-Dawley rats (male, 220 g) were sacrificed by decapitation and brains were rapidly removed and frozen in isopentan at -40°C. Tissue sections (10/~m in thickness) were prepared at -20°C using a microtome cryostat (Leitz 1720) and thawmounted on gelatinized slides. Tissue sections were treated following the protocol described by Hafen et al. [8]. The labelled rat CCK oligonucleotide was diluted in a buffer containing 50% formamide, 0.6 M NaC1, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 x Denhardt's solution (1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin (BSA)) and 500 /~g/ml yeast tRNA to a final concentration of 2-3 x 104 cpm/ml. The tissue on the slide was covered with 60/d of hybridization solution under a piece of parafilm to prevent evaporation. The slides were put in humid boxes and incubated at 42°C overnight. After hybridization, the parafilm was removed by floatation in a solution containing 50% formamide, 0.6 M NaC1, 20 mM Tris-HCl, pH 7.5 and 1 mM EDTA and the slides washed in the same washing buffer at 42°C for 20 h with 4 changes of buffer. Finally, the tissue sections were dehydrated by serial passages in ethanol containing 0.3 M ammonium acetate pH 7.0. Autoradiographs were prepared by apposing the slide-mounted tissue sections to ~-max sensitive film (Amersham, U.K.) for 2-3 weeks at - 70°C. The optical density of autoradiograms was quantified using an image analyzer (MCID, Imaging Research). Radioactive standards were made from serial dilutions of the corresponding labelled oligonucleotide spotted orrnylon membrane (Hybond N, Amersham). Results from film quantification are expressed in arbitrary 'CCK mRNA copies' as referred to the radioactive standards. In addition, the specificity of the hybridization localization on tissue sections was verified by Northern blot analysis. Total RNA from different brain regions was extracted according to Chomczynsky and Sacchi [4]. Approximately 10/~g of RNA for each brain area were denatured with 1 M glyoxal [I 1] and separated by electrophoresis through a 1.5% agarose gel. Transfer to a nylon membrane was carried out according to Thomas [14]. The labelled probe was hybridized for 18 h at 42°C in 50% Formamide/4 x STE solution (1 x STE: 150 mM NaC1, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (SDS), 0.5 mg/ml heparin). The blot was then washed with 2 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at 42°C and apposed to X-ray film (Fuji) for 18 h at - 70°C. In order to normalize the RNA concentration, the same blot was hybridized with an actin oligonucleotide probe complementary to bases 31-60 of the 3' untranslated region of the rat fl-actin gene [12]. The labelling of the fl-actin oligonucleotide and the hybridization conditions were the same as mentioned above. The regional distribution of neurons containing the mRNA coding for the CCK peptide was examined in various regions of the rat brain (Fig. 1, Table I). Microdensitometric measurement showed a good intra and interindividual reproducibility of the hybridization reaction. In all the animals studied the claustrum was the region
134
a
Fig. 1. Autoradiographic images showing the distribution of C C K mRNA in coronal sections of the rat brain. The images presented are bright field photomicrographs of autoradiograms generated from sections hybridized with the ~2P-synthetic oligonucleotide complementary to C C K mRNA. Acb, accumbens nucleus; ACg, anterior cingulate cortex; AOP, anterior olfactif nucleus posterior; Aq, cerebral aqueduct; AM, anteromedian thalamic nucleus; AV, anteroventral thalamic nucleus; BL, basolateral amygdaloid nucleus; CAI, CA2, CA3, CA4, field of Ammon's horn; cc, corpus callosum; Cl, claustrum; CPu, caudate-putamen; DG, dentate gyrus; DLG, dorsal lateral geniculate nucleus; En, endopiriform nucleus; Ent, entorhihal cortex; Fr, frontal cortex; G1, glomerular layer olfactory bulb; GP, globus pallidus; IC, inferior colliculus; La, lateral amygdaloid nucleus; LD, laterodorsal thalamic nucleus; LP, lateroposterior thalamic nucleus; Me, median amygdaloid nucleus; MG, median geniculate nucleus; ON, olfactory nerve layer; PO, primary olfactory cortex; Po, post thalamic nuclear group; Rt, reticular thalamic nucleus; SNC, substantia nigra compacta; VP, ventroposterior thalamic nucleus; VPM, median ventroposterior thalamic nucleus: VTA, ventral tegmental area; TT, taenia tecta (olfactory bulb).
135
TABLE I Q U A N T I T A T I V E A U T O R A D I O G R A P H I C ANALYSIS O F LABELLED RAT CCK O L I G O N U C LEOTIDE H Y B R I D I Z A T I O N IN THE RAT BRAIN
The arbitrary 'CCK mRNA copy' values were calculated from optical densities obtained from the labelled oligonucleotide standards as described in the text. Data are means with S.E.M. from 5 to 10 rats. Brain regions
CCK mRNA copies
Frontal cortex Caudate-putamen Accumbens nucleus Claustrum Endopiriform nucleus Somatosensory cortex Superficial layers I, II, III Deep layers V, VI Thalamus Geniculate nucleus Substantia nigra compacta Ventral tegmental area Hippocampus Pyramidal layer CA1 Dentate gyrus granule cells Inferior colliculi Cerebellum
75.6_+ 15 4.9__+ 2 3.2+ 0.5 138.9 + 16 123.6 + 15 68.2+ 16 73.2 + 13 79.5 ___14 76.4 + 12 88.8 + 16 54.3 + 9 83.4 ___19 33.2 __+10 43.2 + 14 4.3 _+ 1
exhibiting the highest level of expression followed by the endopiriform nucleus. High densities of CCK mRNA expression were also seen in the substantia nigra pars compacta, the hippocampus, the thalamus, the geniculate nucleus (dorsal and medial parts) and throughout the neocortex. In the hippocampal formation we observed that all 4 fields (CA1, CA2, CA3 and CA4) contained CCK mRNA especially in interneurons in the pyramidal layer. The granule cells of the dentate gyrus presented lower levels of hybridization (Fig. 1F, G). Different nuclei of the thalamus including the anteroventral and anteromedian nuclei, the lateral and medial ventroposterior nuclei, the lateral and reticular nuclei and the posterior nuclear group were analyzed. Significant densities of neurons containing the CCK mRNA were found in the amygdaloid nuclei (lateral, basolateral and medial), the inferior colliculi, the entorhihal cortex, the primary olfactory cortex and in the ventral tegmental area (Fig. 1B, F-H). The expression ofCCK mRNA detected in this last region was 48% lower than in the substantia nigra pars compacta (Table I). The localization of CCK mRNA in all these regions analyzed with a cellular resolution on dipped sections confirmed our observations (data not shown). Very low levels of signal were observed in the corpus callosum, the caudate-putamen nucleus, the nucleus accumbens, the globus pallidus and the cerebellum (Fig. 1C-E; Table I). These results, except for the thalamus, are in excellent agreement with the localization of the C C K cell bodies previously described with the immunohistochemical studies [15]. Northern blot analysis of total RNA extracted from different rat brain regions,
136
1
28S
234
5
6
78
m
23S 18S 16S CCK
wO
Actin Fig. 2. Northern analysis with total RNA extracted from different brain regions: 1, brainstcm; 2, hippocampus; 3, thalamus; 4, midbrain; 5, hypothalamus; 6, cerebellum; 7, cortex; 8, striatum. Each lane was Loaded with approximately 10 pg of RNA. Lower panel: the same RNA blot was hybridized with actin oligonucleotide probe in order to normalize the RNA concentration.
using the same labelled CCK oligomer as a hybridization probe, showed that the size of the CCK m R N A was about 950 nucleotides, which is in agreement with other authors [6]. A single CCK m R N A band could be detected in the hippocampus, the thalamus, the midbrain, and the cortex. No signal was detected with the cerebellum and the brainstem (Fig. 2). In contrast, we observed a hybridizing m R N A band in the hypothalamus and in the striatum where no hybridization signal could be detected in the tissue section. CCK positive cell bodies have been described in different hypothalamic nuclei but not in the caudate [15]. Our failure to visualize these cell bodies is difficult to explain. A possible explanation could be the contamination during the dissection of the brain region with neighbouring areas such as the claustrum or thalamus which are rich in CCK message. In the hypothalamus, a widespread distribution of these cell bodies with low levels of expression could account for the lack of significant autoradiographic signal. The blot was normalized for the concentration of R N A loaded per lane by hybridizing it with a labelled actin oligonucleotide probe (Fig. 2, lower panel). Our results confirm in part the distribution of CCK-like immunoreactivity previously described by immunocytochemical or radioimmunoassay techniques [3, 15]. Numerous cells containing CCK m R N A are detected in the cortex, the hippocampus, the olfactory bulb, the claustrum and the amygdala. The absence of signal observed in the striatum is in agreement with previous immunohistochemical studies where CCK cell bodies have not been observed even after direct iniection of colchicine in
137
this nucleus [7, 15]. However, the presence of CCK-immunoreactive fibers and CCK receptors in this structure supported the possibility that the CCK could act as neurotransmitter and modulate, for example, the dopaminergic input activity in this brain region. Higher levels of CCK mRNA were detected in different nuclei of the thalamus. These results do not agree with those obtained using immunohistochemical techniques where no CCK positive neuronal cell bodies could be detected. Similar results have been, however, reported by Siegel and Young [13] using riboprobes for the CCK mRNA. The presence of an elevated density of CCK mRNA in the thalamus was confirmed by Northern analysis. The absence of reported CCK containing cell bodies in immunohistochemical studies is difficult to explain. The presence of molecular forms of CCK which are not recognized by the available antibodies or differences in turnover rates should be investigated. Substantial levels of CCK mRNA were observed in the mesostriatal neurons, more precisely in the ventral tegmental area and in the substantia nigra pars compacta but not in the substantia nigra pars reticulata. The existence of CCK containing neuronal cell bodies in these regions was previously reported. Furthermore, HSkfelt et al. [9, 10] described that tyrosine hydroxylase and CCK could be colocalized in some mesencephalic neurons suggesting a possible interaction between the CCK and dopamine systems. Our observations agree with this preliminary hypothesis. In conclusion, our results illustrate the usefulness of a semiquantitative in situ hybridization approach for the determination of the levels of neuropeptide expression in the brain. This approach can now be used to analyze the effects of pharmacological treatment or other experimental manipulations which are known to alter CCK expression.
1 Agid, Y. and Javoy-Agid, F., Peptides and Parkinson's disease, Trends Neurosci., 1 (1985) 30-35. 2 Beal, M.F. and Martin, J.B., Neuropeptides in neurological disease, Ann. Neurol., 20 (1986) 547-565. 3 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. 4 Chomczynsky, P. and Sacchi, N., Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. 5 Deschenes, R.J., Lorentz, L.J., Haun, R.S., Roos, B.A., Collier, K.J. and Dixon, J.E., Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin, Proc. Natl. Acad. Sci. U.S.A., 81 (I 984) 726730. 6 Deschenes, R.J., Haun, R.S., Funckes, C.L. and Dixon, J.E., A gene encoding rat cholecystokinin: isolation nucleotide sequence and promotor activity, J. Biol. Chem., 260 (1985) 1280-1286. 7 Gilles, C., Lostra, F. and Vanderhaeghen, J.J., CCK nerve terminals in the rat striatal and limbic areas originate partly in the brainstem and partly in telencephalic structures, Life Sci., 32 (1983) 1683-1690. 8 Hafen, E., Levine, M., Garber, R.L. and Gehring, W.J., An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex, EMBO J., 2 (1983) 617~623. 9 HSkfelt, T., Rehfeld, J.F., Skirboll, L., lvemark, B., Goldstein, M. and Markey, K., Evidence for coexistence of dopamine and cholecystokinin in mesolimbic neurons, Nature (Lond.), 285 (1980) 476478.
138 10 H6kfelt, T., Skirboll, L., Everitt, B., Meister, B., Brownstein, M., Jacobs, T., Faden, A., Kuga, S,, Goldstein, M., Markstein, R., Dockray, G. and Rehfeld, J., Distribution of cholecystokinin-like immunoreactivity in the neurons system. Co-existence with classical neurotransmitter and other neuropeptides, Ann. N.Y. Acad. Sci., 448 (1985) 255-274. 11 McMaster, G.K. and Carmichael, G.G., Analysis of single and double stranded nucleic acids on polyacrilamide and agarose gels using glyoxal and acridine orange, Proc. Natl. Acad. Sci., U.S.A., 74 (1977) 4835-4838. 12 Nudel, V., Zkut, R., Shani, M., Neuman, S., Levy, Z. and Yaffe, D., The nucleotide sequence of the rat cytoplasmic/~-actin gene, Nucleic Acids Res., 12 (1987) 1759 177 I. 13 Siegel, R.E. and Scott Young Ill, W., Detection of preprocholecystokinin and preproenkephalin A mRNAs in rat brain by hybridization histochemistry using complementary RNA probes, Neuropeptides, 6 (1985) 573 580. 14 Thomas, P.S., Hybridization of denatured RNA transferred or dotted to nitrocellulose paper, Methods Enzymol., 109 (1980) 255 266. 15 Vanderhaeghen, J.J., Neuronal cholecystokinin. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy Vol. 4, GABA and Neuropeptides in the CNS, Part 1, Elsevier, Amsterdam, 1985, pp. 406-435.
Neuroseience Letters, 93 (1988) 132 138 Elsevier Scientific Publishers Ireland Ltd.
NSL 05669
Regional localization of the mRNA coding for the neuropeptide cholecystokinin in the rat brain studied by in situ hybridization Marc Savasta, Jos6 M. Palacios and Guadalupe
Mengod
Preclinical Research, Sandoz Ltd., Basle (Switzerland)
(Received 8 June 1988; Revised version received 18 July 1988; Accepted 19 July 1988) Key words:
In situ hybridization; Synthetic oligonucleotide; Cholecystokinin; mRNA; Northern blot; Rat brain
The regional localization of mRNA coding for the neuropeptide cholecystokinin (CCK) has been studied in the rat brain by in situ hybridization using a 32p-labelledsynthetic 32 mer oligonucleotide. Autoradiograms were quantified using computer-assisted microdensitometry. High levels of hybridization were observed in the neocortex, claustrum, endopiriform nucleus, cingular cortex, amygdala, olfactory bulb, hippocampus, ventral tegmental area, geniculate nucleus, several thalamic nuclei and substantia nigra compacta. Very weak signal was detected in the striatum, the cerebellum and the brainstem. The topographic distribution of CCK neurons observed overlaps in part with that previously described by immunohistochemical techniques. However, some discrepancies were also found, particularly in the thalamus. These results show that in situ hybridization with oligonucleotide probes together with a semiquantitative method described can be used to map the expression of the CCK mRNA in rat brain sections as well as its modification after pharmacological or physiological manipulations. In the brain the c a r b o x y l t e r m i n a l o c t a p e p t i d e o f c h o l e c y s t o k i n i n ( C C K - 8 ) has been localized i m m u n o h i s t o c h e m i c a l l y in n e u r o n s in different species [3, 15]. A n involvement o f the C C K system in b r a i n p a t h o l o g y has been p r o p o s e d in neurological and psychiatric diseases such as P a r k i n s o n ' s disease, H u n t i n g t o n ' s disease a n d s c h i z o p h r e n i a [1, 2]. The structure o f the gene c o d i n g for C C K has recently been elucidated [5]. In o r d e r to m a p the d i s t r i b u t i o n o f n e u r o n s expressing C C K m R N A in the rat b r a i n we have a p p l i e d the technique o f in situ h y b r i d i z a t i o n using a synthetic o l i g o n u c l e o t i d e a n d c o m p u t e r - a s s i s t e d image analysis for the q u a n t i f i c a t i o n o f the autoradiograms. The o l i g o d e o x y r i b o n u c l e o t i d e was m a d e on a 380 A A p p l i e d Biosystems D N A synthesizer a n d purified on a n 8% p o l y a c r y l a m i d e / 8 M urea p r e p a r a t i v e sequencing gel. The rat C C K o l i g o n u c l e o t i d e was c o m p l e m e n t a r y to bases 6951 6982 o f the C C K gene [5]. T h e o l i g o m e r was labelled by using d e o x y a d e n o s i n e t r i p h o s p h a t e
Correspondence: G. Mengod, Preclinical Research, Sandoz Ltd., CH-4002 Basle, Switzerland.
0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
133
(dATP) labelled at the 0t position with 32p ( > 3000 Ci/mmol, New England Nuclear) and terminal deoxynucleotidyltransferase (Boehringer, Mannheim) to specific activities of 0.9-3 x 104 Ci/mmol. Sprague-Dawley rats (male, 220 g) were sacrificed by decapitation and brains were rapidly removed and frozen in isopentan at -40°C. Tissue sections (10/~m in thickness) were prepared at -20°C using a microtome cryostat (Leitz 1720) and thawmounted on gelatinized slides. Tissue sections were treated following the protocol described by Hafen et al. [8]. The labelled rat CCK oligonucleotide was diluted in a buffer containing 50% formamide, 0.6 M NaC1, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 x Denhardt's solution (1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin (BSA)) and 500 /~g/ml yeast tRNA to a final concentration of 2-3 x 104 cpm/ml. The tissue on the slide was covered with 60/d of hybridization solution under a piece of parafilm to prevent evaporation. The slides were put in humid boxes and incubated at 42°C overnight. After hybridization, the parafilm was removed by floatation in a solution containing 50% formamide, 0.6 M NaC1, 20 mM Tris-HCl, pH 7.5 and 1 mM EDTA and the slides washed in the same washing buffer at 42°C for 20 h with 4 changes of buffer. Finally, the tissue sections were dehydrated by serial passages in ethanol containing 0.3 M ammonium acetate pH 7.0. Autoradiographs were prepared by apposing the slide-mounted tissue sections to ~-max sensitive film (Amersham, U.K.) for 2-3 weeks at - 70°C. The optical density of autoradiograms was quantified using an image analyzer (MCID, Imaging Research). Radioactive standards were made from serial dilutions of the corresponding labelled oligonucleotide spotted orrnylon membrane (Hybond N, Amersham). Results from film quantification are expressed in arbitrary 'CCK mRNA copies' as referred to the radioactive standards. In addition, the specificity of the hybridization localization on tissue sections was verified by Northern blot analysis. Total RNA from different brain regions was extracted according to Chomczynsky and Sacchi [4]. Approximately 10/~g of RNA for each brain area were denatured with 1 M glyoxal [I 1] and separated by electrophoresis through a 1.5% agarose gel. Transfer to a nylon membrane was carried out according to Thomas [14]. The labelled probe was hybridized for 18 h at 42°C in 50% Formamide/4 x STE solution (1 x STE: 150 mM NaC1, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (SDS), 0.5 mg/ml heparin). The blot was then washed with 2 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at 42°C and apposed to X-ray film (Fuji) for 18 h at - 70°C. In order to normalize the RNA concentration, the same blot was hybridized with an actin oligonucleotide probe complementary to bases 31-60 of the 3' untranslated region of the rat fl-actin gene [12]. The labelling of the fl-actin oligonucleotide and the hybridization conditions were the same as mentioned above. The regional distribution of neurons containing the mRNA coding for the CCK peptide was examined in various regions of the rat brain (Fig. 1, Table I). Microdensitometric measurement showed a good intra and interindividual reproducibility of the hybridization reaction. In all the animals studied the claustrum was the region
134
a
Fig. 1. Autoradiographic images showing the distribution of C C K mRNA in coronal sections of the rat brain. The images presented are bright field photomicrographs of autoradiograms generated from sections hybridized with the ~2P-synthetic oligonucleotide complementary to C C K mRNA. Acb, accumbens nucleus; ACg, anterior cingulate cortex; AOP, anterior olfactif nucleus posterior; Aq, cerebral aqueduct; AM, anteromedian thalamic nucleus; AV, anteroventral thalamic nucleus; BL, basolateral amygdaloid nucleus; CAI, CA2, CA3, CA4, field of Ammon's horn; cc, corpus callosum; Cl, claustrum; CPu, caudate-putamen; DG, dentate gyrus; DLG, dorsal lateral geniculate nucleus; En, endopiriform nucleus; Ent, entorhihal cortex; Fr, frontal cortex; G1, glomerular layer olfactory bulb; GP, globus pallidus; IC, inferior colliculus; La, lateral amygdaloid nucleus; LD, laterodorsal thalamic nucleus; LP, lateroposterior thalamic nucleus; Me, median amygdaloid nucleus; MG, median geniculate nucleus; ON, olfactory nerve layer; PO, primary olfactory cortex; Po, post thalamic nuclear group; Rt, reticular thalamic nucleus; SNC, substantia nigra compacta; VP, ventroposterior thalamic nucleus; VPM, median ventroposterior thalamic nucleus: VTA, ventral tegmental area; TT, taenia tecta (olfactory bulb).
135
TABLE I Q U A N T I T A T I V E A U T O R A D I O G R A P H I C ANALYSIS O F LABELLED RAT CCK O L I G O N U C LEOTIDE H Y B R I D I Z A T I O N IN THE RAT BRAIN
The arbitrary 'CCK mRNA copy' values were calculated from optical densities obtained from the labelled oligonucleotide standards as described in the text. Data are means with S.E.M. from 5 to 10 rats. Brain regions
CCK mRNA copies
Frontal cortex Caudate-putamen Accumbens nucleus Claustrum Endopiriform nucleus Somatosensory cortex Superficial layers I, II, III Deep layers V, VI Thalamus Geniculate nucleus Substantia nigra compacta Ventral tegmental area Hippocampus Pyramidal layer CA1 Dentate gyrus granule cells Inferior colliculi Cerebellum
75.6_+ 15 4.9__+ 2 3.2+ 0.5 138.9 + 16 123.6 + 15 68.2+ 16 73.2 + 13 79.5 ___14 76.4 + 12 88.8 + 16 54.3 + 9 83.4 ___19 33.2 __+10 43.2 + 14 4.3 _+ 1
exhibiting the highest level of expression followed by the endopiriform nucleus. High densities of CCK mRNA expression were also seen in the substantia nigra pars compacta, the hippocampus, the thalamus, the geniculate nucleus (dorsal and medial parts) and throughout the neocortex. In the hippocampal formation we observed that all 4 fields (CA1, CA2, CA3 and CA4) contained CCK mRNA especially in interneurons in the pyramidal layer. The granule cells of the dentate gyrus presented lower levels of hybridization (Fig. 1F, G). Different nuclei of the thalamus including the anteroventral and anteromedian nuclei, the lateral and medial ventroposterior nuclei, the lateral and reticular nuclei and the posterior nuclear group were analyzed. Significant densities of neurons containing the CCK mRNA were found in the amygdaloid nuclei (lateral, basolateral and medial), the inferior colliculi, the entorhihal cortex, the primary olfactory cortex and in the ventral tegmental area (Fig. 1B, F-H). The expression ofCCK mRNA detected in this last region was 48% lower than in the substantia nigra pars compacta (Table I). The localization of CCK mRNA in all these regions analyzed with a cellular resolution on dipped sections confirmed our observations (data not shown). Very low levels of signal were observed in the corpus callosum, the caudate-putamen nucleus, the nucleus accumbens, the globus pallidus and the cerebellum (Fig. 1C-E; Table I). These results, except for the thalamus, are in excellent agreement with the localization of the C C K cell bodies previously described with the immunohistochemical studies [15]. Northern blot analysis of total RNA extracted from different rat brain regions,
136
1
28S
234
5
6
78
m
23S 18S 16S CCK
wO
Actin Fig. 2. Northern analysis with total RNA extracted from different brain regions: 1, brainstcm; 2, hippocampus; 3, thalamus; 4, midbrain; 5, hypothalamus; 6, cerebellum; 7, cortex; 8, striatum. Each lane was Loaded with approximately 10 pg of RNA. Lower panel: the same RNA blot was hybridized with actin oligonucleotide probe in order to normalize the RNA concentration.
using the same labelled CCK oligomer as a hybridization probe, showed that the size of the CCK m R N A was about 950 nucleotides, which is in agreement with other authors [6]. A single CCK m R N A band could be detected in the hippocampus, the thalamus, the midbrain, and the cortex. No signal was detected with the cerebellum and the brainstem (Fig. 2). In contrast, we observed a hybridizing m R N A band in the hypothalamus and in the striatum where no hybridization signal could be detected in the tissue section. CCK positive cell bodies have been described in different hypothalamic nuclei but not in the caudate [15]. Our failure to visualize these cell bodies is difficult to explain. A possible explanation could be the contamination during the dissection of the brain region with neighbouring areas such as the claustrum or thalamus which are rich in CCK message. In the hypothalamus, a widespread distribution of these cell bodies with low levels of expression could account for the lack of significant autoradiographic signal. The blot was normalized for the concentration of R N A loaded per lane by hybridizing it with a labelled actin oligonucleotide probe (Fig. 2, lower panel). Our results confirm in part the distribution of CCK-like immunoreactivity previously described by immunocytochemical or radioimmunoassay techniques [3, 15]. Numerous cells containing CCK m R N A are detected in the cortex, the hippocampus, the olfactory bulb, the claustrum and the amygdala. The absence of signal observed in the striatum is in agreement with previous immunohistochemical studies where CCK cell bodies have not been observed even after direct iniection of colchicine in
137
this nucleus [7, 15]. However, the presence of CCK-immunoreactive fibers and CCK receptors in this structure supported the possibility that the CCK could act as neurotransmitter and modulate, for example, the dopaminergic input activity in this brain region. Higher levels of CCK mRNA were detected in different nuclei of the thalamus. These results do not agree with those obtained using immunohistochemical techniques where no CCK positive neuronal cell bodies could be detected. Similar results have been, however, reported by Siegel and Young [13] using riboprobes for the CCK mRNA. The presence of an elevated density of CCK mRNA in the thalamus was confirmed by Northern analysis. The absence of reported CCK containing cell bodies in immunohistochemical studies is difficult to explain. The presence of molecular forms of CCK which are not recognized by the available antibodies or differences in turnover rates should be investigated. Substantial levels of CCK mRNA were observed in the mesostriatal neurons, more precisely in the ventral tegmental area and in the substantia nigra pars compacta but not in the substantia nigra pars reticulata. The existence of CCK containing neuronal cell bodies in these regions was previously reported. Furthermore, HSkfelt et al. [9, 10] described that tyrosine hydroxylase and CCK could be colocalized in some mesencephalic neurons suggesting a possible interaction between the CCK and dopamine systems. Our observations agree with this preliminary hypothesis. In conclusion, our results illustrate the usefulness of a semiquantitative in situ hybridization approach for the determination of the levels of neuropeptide expression in the brain. This approach can now be used to analyze the effects of pharmacological treatment or other experimental manipulations which are known to alter CCK expression.
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