123
Neuroscience Letters, 123 (1991) 123-126
© 1991 ElsevierScientificPublishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 0304394091001034 NSL 07545
Cholecystokinin m R N A detection in rat spinal cord motoneurons but not in dorsal root ganglia neurons S.N. Schiffmann, E. Teugels, P. H a l l e u x , R. M e n u a n d J.-J. V a n d e r h a e g h e n Laboratory of Neuropathology and Neuropeptide Research, Brugmann and Erasme Academic Hospitals, UniversitbLibre de Bruxelles, Brussels (Belgium)
(Received24 August 1990;Revisedversionreceived12 November 1990;Accepted 12 November 1990) Key words: CholecystokininmRNA; Spinal cord; Dorsal root ganglia; Motoneuron; Calcitonin-generelated peptide
Cholecystokinin(CCK) mRNA has been detected by in situ hybridizationhistochemistryusing two differentoligonucleotideprobes in small to medium-sized neurons of layers II-III and X of Rexed and in large neurons of layer IX in the rat spinal cord at cervical,thoracic and lumbo-sacral levels. No labeled cells were detectedin the dorsal root ganglia. This confirmsthe previouslyestablished distribution of CCK-like immunoreactivity in layers II-III and X and indicates, in addition, that motoneurons of layer IX may expresstrue genuineCCK whilst conversely,dorsal root ganglia neurons do not.
The presence o f cholecystokinin-like immunoreactivity (CCK-LI) has been reported in nerve cell bodies and fibers in the rat spinal cord and in the dorsal root ganglia (DRG) [3, 6--9, 13, 16, 17]. However, whilst CCK-LI in layers II-III, IV, V and X of the spinal cord was recognized as true genuine CCK, it has been proposed that in large neurons of layer IX and in the D R G , CCK-LI, detected by conventional antisera raised against the carboxy-terminal part o f CCK, could represent a crossreacting detection of calcitonin-gene related peptide (CGRP) [6-8]. This cross-reaction could be explained by a sequence homology which exists between the C-terminal pentapeptides of CCK and C G R P [2, 6, 11]. The recent cloning and sequencing o f rat preproCCK c D N A [2] has permitted the localization of CCK m R N A containing neurons by in situ hybridization histochemistry (ISHH) [1, 12, 14]. Using this technique it has been demonstrated that D R G neurons expressed CCK m R N A in the guinea pig but not in the rat [15]. The purpose of this study is to investigate the real identity of CCK-LI in the rat spinal cord in addition to D R G using ISHH. Male Wistar rats (200-250 g) were decapitated and after quick removal, spinal cords and D R G taken at cerviReprint requests: S.N. Schiffmann,see address below. Correspondence: J.J. Vanderhaeghen, Laboratory of Neuropathology and Neuropeptide Research, Universit6 Libre de Bruxelles, Campus Erasme, CP 601,808 route de Lennik, 1070Brussels, Belgium.
cal, thoracic and lumbo-sacral levels were frozen in 2methylbutan cooled on dry ice. Serial 14/~m coronal sections were cut, thaw-mounted onto poly-L-lysine-coated slides and stored at - 2 0 ° C until hybridization. The sections were thawed at room temperature, fixed in 4 % paraformaldehyde for 30 min and rinsed twice in PBS. The sections were delipidated through a graded series of ethanols (60-100%) and chloroform. After air drying, the sections were incubated overnight at 42°C with approximately 8 x 105 cpm per section of aSS-labeled probe in hybridization buffer which consisted of 50% formamide, 4 x SSC (1 x SSC = 0.15 M NaC1/ 0.015 M sodium citrate, pH 7.4), 0.02 M sodium phosphate at pH 7.4, 1 x Denhardt's solution, 1% sarcosyl, 10% dextran sulfate, yeast t R N A at 500 gg/ml, salmon sperm D N A at 100/~g/ml and 60 m M dithiothreitol. After hybridization, the sections were rinsed for 4 x 15 min in 1 x SSC at 55°C, dried and covered with Hyperfilm beta-max X-ray film (Amersham) for 10 days. After development of the film, they were dipped in Kodak NTB3 emulsion and exposed for 4 weeks. The sections were stained with hematoxylin and analysed on an Axiophot Zeiss microscope under bright- and darkfield conditions. Alternatively, in order to increase the sensitivity, some sections of D R G were hybridized with a mix of the two 35S-labeled CCK probes and exposed for 8 weeks. A C C K and BCCK probes were 44 bases oligonucleotide synthesized on an Applied Biosystems 381A D N A synthesizer and were complementary, respectively, to
124
nucleotides 300-343 of rat CCK cDNA [2] corresponding to the N-terminal extended CCK8 (CCK~5), and to nucleotides 249-293 of rat CCK cDNA [2] in the N-terminal part of preproCCK. Oligonucleotides were labeled with ~-[35S] dATP ( > 1000 Ci/mmol, NEN) at the 3' end
by terminal deoxynucleotidyltransferase to a specific activity of 3-5 x 108 cpm/pg. Synthetic human tyrosine hydroxylase (TH) and rat neuropeptide Y (NPY) oligonucleotide probes with the same length, same guanosine + cytosine content and t
A
dh ,/
. :.
O
B
O
~ O '
7,
""
"
:
V
g~ .
/
Fig. 1. Autoradiographs showing the hybridization signal generated by the 35S-labeled cholecystokinin ACCK (A) or BCCK (B) probe-mRNA hybrids in semi-adjacent sections of rat spinal cord at the lumbo-sacral level. Small to medium-sized CCK mRNA containing neurons in layers II-III (C) and layer X (D) of Rexed in the spinal cord and large CCK mRNA containing neurons in layer IX (E,F). Some rare large unlabeled neurons (arrow) of the ventral horn are intermingled among the labeled ones (F). No labeled neurons were present in the dorsal root ganglia (G). C,E,F: lumbo-sacral levels. D: thoracic levels, cc, central canal; dh, dorsal horn; vh, central horn. Bright- (C,D,F,G), and dark-field (E) illumination. Bar = 1 mm (A,B) and 25/zm (C~G).
125
same specific activity were used as control in adjacent sections. The observed density of grains using the human TH probe was similar to the background level using the CCK probes, i.e. on the white matter. RNAse pretreatment (1 h at 37°C) and hybridization with labeled probes and an excess of cold probes both abolished the signal. The same pattern of labeling was observed using the two CCK probes, ACCK and BCCK (Fig. 1A,B), and was different from the labeling detected after hybridization to the NPY probe. Macroscopically (Fig. 1A,B) labeling appeared in the superficial part of the dorsal horns, the lateral part of the anterior horns and around the central canal. At the microscopic level, scattered moderately labeled neurons were observed in the layer X around the central canal (Fig. 1D) and a moderate density of moderately labeled neurons was present in layers II-III of the posterior horn (Fig. 1C). In the anterior horn, numerous large neurons (25-50 pm in diameter) were moderately labeled in the layer IX (Fig. 1E,F). In this latter layer, some unlabeled large neurons were intermingled among the labeled ones (Fig. 1F). This pattern was similar at the cervical, thoracic and lumbar-sacral levels. Conversely, no labeled cells were observed in the D R G (Fig. 1G) even when hybridization was performed with a mix of the ACCK and BCCK probes and sections were exposed as long as 8 weeks. The presence of a moderate density of labeled neurons in the layers II-III and layer X of the spinal cord is in total agreement with the description of CCK-LI containing neurons in these layers [3, 6-8, 13, 16, 17]. This distribution of CCK mRNA-containing neurons confirms strongly that CCK-LI in these layers corresponds truly to genuine CCK. Conversely, the absence of any ISHH labeling in D R G confirms the previous results of Seroogy et al. [15]. Although it could never be excluded that low mRNA levels are not detected, a low level of CCK mRNA was detected in some brain areas such as the supramamillary nucleus using the same ISHH procedure [12]. Moreover, in the D R G the same result is obtained even after increase of ISHH sensitivity. This confirms then that CCK-LI detected there with conventional antisera does not represent genuine CCK. The presence of silver grains on a high percentage (6080 %) of large neurons of layer IX of the ventral horns raises the question of the nature of neuropeptide contained in these neurons. This percentage indicates that a significant proportion of motoneurons are labeled. Immunocytochemical study using antisera raised against different parts of preproCCK has suggested that in the vast majority of these neurons CCK-LI represents in fact CGRP [6] although true genuine CCK has been identified in very few of them [6]. The nucleotide sequences of
the C-terminal part of CCK and CGRP are partly homologous [2, 6, 11]. Therefore, it could be possible that our ACCK probe which includes the sequence of the Cterminal pentapeptide of CCK hybridizes to the CGRP mRNA. However, the fact that a totally similar labeling was observed using a probe (BCCK) complementary to a region of the CCK mRNA which does not exhibit any homology with the CGRP mRNA, strongly suggests a specific hybridization to CCK mRNA. In addition, the absence of ISHH labeling using these two CCK probes in the D R G where CGRP-LI was abundantly detected [6-8] confirms the specificity of the hybridization. Gastrin and CCK exhibited the same C-terminal pentapeptide, but similarly, a hybridization to a gastrin mRNA could reasonably be excluded by the use of the two different, ACCK and BCCK, oligonucleotide probes. The nature of the detected CCK-LI, CGRP versus genuine CCK, in these neurons which contain also CGRP-LI [4, 6] and CGRP mRNA [5] and in the peripheral motor nerves where an anterograde transport of CCK-LI was described [10], remains to be established. The present results do not prove that CCK mRNA is really translated and do not exclude that CCK-LI detected in the majority of these neurons [6-8] represent CGRP. Indeed, it has been demonstrated that several long projection neurons such as the thalamic neurons, pyramidal neurons of the Ammon's horn and pyramidal cortical neurons [1, 12] contain CCK mRNA whereas they are devoid of detectable CCK-LI. A higher sensitivity of ISHH than immunohistochemistry in detecting CCK expressing cell bodies is the most probable, although still speculative explanation for these discrepancies [1, 12]. There are other possibilities: existence of a differential splicing of the CCK primary transcript resulting in a different peptide; existence of a highly homologous mRNA encoded by an unknown gene; differential processing of the CCK peptide precursor resulting in peptide species unrecognizable by the available antiCCK antisera and/or differences in rates of synthesis or turnover. We are thankful to J.-L. Conreur and C. Christophe for photography and oligonucleotide synthesis, respectively. Thanks are due also to M. Ludgate who carefully scrutinized the english and to Drs. M. Schalling and T. H6kfelt for training in in situ hybridization techniques. Supported by grants from Fonds de la Recherche Scientifique Medicale (3.4523-86 and 3.4574.90), Fondation Medicale Reine Elisabeth (Belgium) (Neurobiology 8690). S.N.S. is Research Assistant of Fonds National de la Recherche Scientifique (Belgium).
126 1 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. 2 Deschenes, R.J., Lorenz, 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 (1984) 726-730. 3 Fuji, K., Senba, E., Fujii, S., Nomura, I., Wu, J.Y., Ueda, Y. and Tohyama, M., Distribution, ontogeny and projections of cholecystokinin-8, vasoactive intestinal polypeptide and gamma-aminobutyrate-containing neuron systems in the rat spinal cord: an immunohistochemical analysis, Neuroscience, 14 (1985) 881-894. 4 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulderry, P.M., Ghatei, M.A., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101-3111. 5 Gibson, S.J., Polak, J.M., Giaid, A., Hamid, Q.A., Kar, S., Jones, P.M., Denny, P., Legon, S., Amara, S.G., Craig, R.K., Bloom, S.R., Penketh, R.J.A., Rodek, C., Ibraim, N.B.N. and Dawson, A., Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat, Neurosci. Lett., 91 (1988) 283-288. 6 Hrkfelt, T., Herrera-Marschitz, M., Seroogy, K., Gong, J., Staines, W.A., Holets, V., Schalling, M., Ungerstedt, U., Post, C., Rehfeld, J.F., Frey, P., Fischer, J., Dockray, G., Hamaoka, T., Walsh, J.H. and Goldstein, M., Irnmunohistochemical studies on cholecystokinin (CCK)-immunoreactive neurons in the rat using sequence specific antisera and with special reference to that caudate nucleus and primary sensory neurons, J. Chem. Neuroanat., 1 (1988) 11-52. 7 Ju, G., Hrkfelt, T., Brodin, E., Fahrenkrug, J., Fischer, J.A., Frey, P., Elde, R.P. and Brown, J.C., Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactiveganglion cells, Cell Tissue Res., 247 (1987) 417-431. 8 Ju, G., Hrkfelt, T., Fischer, J.A., Frey, P., Rehfeld, J.F. and Dockray, G.J., Does cholecystokinin-like immunoreactivity in rat prim-
ary sensory neurons represent calcitonin gene-related peptide?, Neurosci. Lett., 68 (1986) 305-310. 9 Marley, P.D., Nagy, J.E., Emson, P.C. and Rehfeld, J.F., Cholecystokinin in the rat spinal cord: distribution and lack of effect of neonatal capsaicin treament and rhizotomy, Brain Res., 238 (1982) 494-498. 10 Rehfeld, J.F. and Lundberg, J.M., Cholecystokinin in feline vagal and sciatic nerves: concentration, molecular form and transport velocity, Brain Res., 275 (1983) 341-347. 11 Rosenfeld, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Swachwenko, P.E., Rivier, J., Vale, W.W. and Evans, R.H., Production of a novel neuropeptide encoded by the calcitonin gene via tissue specific RNA processing, Nature, 304 (1983) 129-135. 12 SchilTmann, S.N. and Vanderhaeghen, J.J., Distribution of cells containing mRNA encoding for cholecystokinin in the rat central nervous system, J. Comp. Neurol., in press. 13 Schroder, H.A., Localization of cholecystokinin-like immunoreactivity in the rat spinal cord, with particular reference to the autonomic innervation of the pelvic organs, J. Comp. Neurol., 217 (1983) 176-186. 14 Seroogy, K., Schalling, M., Brene, S., Dagerlind, A., Chai, S.Y., Hokfelt, T., Persson, H., Brownstein, M., Huan, R., Dixon, J., Filer, D., Schlessinger, D. and Goldstein, M., Cholecystokinin and tyrosine hydroxylase messenger RNAs in neurons of rat mesencephalon: peptide/monoamine coexistence studies using in situ hybridization combined with immunocytochemistry, Exp. Brain Res., 74 (1989) 149-162. 15 Seroogy, K.B., Mohapatra, N.K., Lund, P.K., Rethelyi, M., McGehee, D.S. and Perl, E.R., Species-specific expression ofcholecystokinin messenger RNA in rodent dorsal root ganglia, Mol. Brain Res., 7 (1990) 171-176. 16 Vanderhaeghen, J..l., Neuronal Cholecystokinin. In A. Bjrrklund and T. Hfkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 4: GABA and Neuropeptides in the CNS, Part I, Elsevier, Amsterdam, 1985, pp. 406435. 17 Vanderhaeghen, J.J., Deschepper, C., Lotstra, F., Vierendeels, G. and Schoenen, J., Immunohistochemical evidence for cholecystokinin-like peptides in neuronal cell bodies of the rat spinal cord, Cell Tissue Res., 223 (1982) 463-467.
Neuroscience Letters, 123 (1991) 123-126
© 1991 ElsevierScientificPublishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 0304394091001034 NSL 07545
Cholecystokinin m R N A detection in rat spinal cord motoneurons but not in dorsal root ganglia neurons S.N. Schiffmann, E. Teugels, P. H a l l e u x , R. M e n u a n d J.-J. V a n d e r h a e g h e n Laboratory of Neuropathology and Neuropeptide Research, Brugmann and Erasme Academic Hospitals, UniversitbLibre de Bruxelles, Brussels (Belgium)
(Received24 August 1990;Revisedversionreceived12 November 1990;Accepted 12 November 1990) Key words: CholecystokininmRNA; Spinal cord; Dorsal root ganglia; Motoneuron; Calcitonin-generelated peptide
Cholecystokinin(CCK) mRNA has been detected by in situ hybridizationhistochemistryusing two differentoligonucleotideprobes in small to medium-sized neurons of layers II-III and X of Rexed and in large neurons of layer IX in the rat spinal cord at cervical,thoracic and lumbo-sacral levels. No labeled cells were detectedin the dorsal root ganglia. This confirmsthe previouslyestablished distribution of CCK-like immunoreactivity in layers II-III and X and indicates, in addition, that motoneurons of layer IX may expresstrue genuineCCK whilst conversely,dorsal root ganglia neurons do not.
The presence o f cholecystokinin-like immunoreactivity (CCK-LI) has been reported in nerve cell bodies and fibers in the rat spinal cord and in the dorsal root ganglia (DRG) [3, 6--9, 13, 16, 17]. However, whilst CCK-LI in layers II-III, IV, V and X of the spinal cord was recognized as true genuine CCK, it has been proposed that in large neurons of layer IX and in the D R G , CCK-LI, detected by conventional antisera raised against the carboxy-terminal part o f CCK, could represent a crossreacting detection of calcitonin-gene related peptide (CGRP) [6-8]. This cross-reaction could be explained by a sequence homology which exists between the C-terminal pentapeptides of CCK and C G R P [2, 6, 11]. The recent cloning and sequencing o f rat preproCCK c D N A [2] has permitted the localization of CCK m R N A containing neurons by in situ hybridization histochemistry (ISHH) [1, 12, 14]. Using this technique it has been demonstrated that D R G neurons expressed CCK m R N A in the guinea pig but not in the rat [15]. The purpose of this study is to investigate the real identity of CCK-LI in the rat spinal cord in addition to D R G using ISHH. Male Wistar rats (200-250 g) were decapitated and after quick removal, spinal cords and D R G taken at cerviReprint requests: S.N. Schiffmann,see address below. Correspondence: J.J. Vanderhaeghen, Laboratory of Neuropathology and Neuropeptide Research, Universit6 Libre de Bruxelles, Campus Erasme, CP 601,808 route de Lennik, 1070Brussels, Belgium.
cal, thoracic and lumbo-sacral levels were frozen in 2methylbutan cooled on dry ice. Serial 14/~m coronal sections were cut, thaw-mounted onto poly-L-lysine-coated slides and stored at - 2 0 ° C until hybridization. The sections were thawed at room temperature, fixed in 4 % paraformaldehyde for 30 min and rinsed twice in PBS. The sections were delipidated through a graded series of ethanols (60-100%) and chloroform. After air drying, the sections were incubated overnight at 42°C with approximately 8 x 105 cpm per section of aSS-labeled probe in hybridization buffer which consisted of 50% formamide, 4 x SSC (1 x SSC = 0.15 M NaC1/ 0.015 M sodium citrate, pH 7.4), 0.02 M sodium phosphate at pH 7.4, 1 x Denhardt's solution, 1% sarcosyl, 10% dextran sulfate, yeast t R N A at 500 gg/ml, salmon sperm D N A at 100/~g/ml and 60 m M dithiothreitol. After hybridization, the sections were rinsed for 4 x 15 min in 1 x SSC at 55°C, dried and covered with Hyperfilm beta-max X-ray film (Amersham) for 10 days. After development of the film, they were dipped in Kodak NTB3 emulsion and exposed for 4 weeks. The sections were stained with hematoxylin and analysed on an Axiophot Zeiss microscope under bright- and darkfield conditions. Alternatively, in order to increase the sensitivity, some sections of D R G were hybridized with a mix of the two 35S-labeled CCK probes and exposed for 8 weeks. A C C K and BCCK probes were 44 bases oligonucleotide synthesized on an Applied Biosystems 381A D N A synthesizer and were complementary, respectively, to
124
nucleotides 300-343 of rat CCK cDNA [2] corresponding to the N-terminal extended CCK8 (CCK~5), and to nucleotides 249-293 of rat CCK cDNA [2] in the N-terminal part of preproCCK. Oligonucleotides were labeled with ~-[35S] dATP ( > 1000 Ci/mmol, NEN) at the 3' end
by terminal deoxynucleotidyltransferase to a specific activity of 3-5 x 108 cpm/pg. Synthetic human tyrosine hydroxylase (TH) and rat neuropeptide Y (NPY) oligonucleotide probes with the same length, same guanosine + cytosine content and t
A
dh ,/
. :.
O
B
O
~ O '
7,
""
"
:
V
g~ .
/
Fig. 1. Autoradiographs showing the hybridization signal generated by the 35S-labeled cholecystokinin ACCK (A) or BCCK (B) probe-mRNA hybrids in semi-adjacent sections of rat spinal cord at the lumbo-sacral level. Small to medium-sized CCK mRNA containing neurons in layers II-III (C) and layer X (D) of Rexed in the spinal cord and large CCK mRNA containing neurons in layer IX (E,F). Some rare large unlabeled neurons (arrow) of the ventral horn are intermingled among the labeled ones (F). No labeled neurons were present in the dorsal root ganglia (G). C,E,F: lumbo-sacral levels. D: thoracic levels, cc, central canal; dh, dorsal horn; vh, central horn. Bright- (C,D,F,G), and dark-field (E) illumination. Bar = 1 mm (A,B) and 25/zm (C~G).
125
same specific activity were used as control in adjacent sections. The observed density of grains using the human TH probe was similar to the background level using the CCK probes, i.e. on the white matter. RNAse pretreatment (1 h at 37°C) and hybridization with labeled probes and an excess of cold probes both abolished the signal. The same pattern of labeling was observed using the two CCK probes, ACCK and BCCK (Fig. 1A,B), and was different from the labeling detected after hybridization to the NPY probe. Macroscopically (Fig. 1A,B) labeling appeared in the superficial part of the dorsal horns, the lateral part of the anterior horns and around the central canal. At the microscopic level, scattered moderately labeled neurons were observed in the layer X around the central canal (Fig. 1D) and a moderate density of moderately labeled neurons was present in layers II-III of the posterior horn (Fig. 1C). In the anterior horn, numerous large neurons (25-50 pm in diameter) were moderately labeled in the layer IX (Fig. 1E,F). In this latter layer, some unlabeled large neurons were intermingled among the labeled ones (Fig. 1F). This pattern was similar at the cervical, thoracic and lumbar-sacral levels. Conversely, no labeled cells were observed in the D R G (Fig. 1G) even when hybridization was performed with a mix of the ACCK and BCCK probes and sections were exposed as long as 8 weeks. The presence of a moderate density of labeled neurons in the layers II-III and layer X of the spinal cord is in total agreement with the description of CCK-LI containing neurons in these layers [3, 6-8, 13, 16, 17]. This distribution of CCK mRNA-containing neurons confirms strongly that CCK-LI in these layers corresponds truly to genuine CCK. Conversely, the absence of any ISHH labeling in D R G confirms the previous results of Seroogy et al. [15]. Although it could never be excluded that low mRNA levels are not detected, a low level of CCK mRNA was detected in some brain areas such as the supramamillary nucleus using the same ISHH procedure [12]. Moreover, in the D R G the same result is obtained even after increase of ISHH sensitivity. This confirms then that CCK-LI detected there with conventional antisera does not represent genuine CCK. The presence of silver grains on a high percentage (6080 %) of large neurons of layer IX of the ventral horns raises the question of the nature of neuropeptide contained in these neurons. This percentage indicates that a significant proportion of motoneurons are labeled. Immunocytochemical study using antisera raised against different parts of preproCCK has suggested that in the vast majority of these neurons CCK-LI represents in fact CGRP [6] although true genuine CCK has been identified in very few of them [6]. The nucleotide sequences of
the C-terminal part of CCK and CGRP are partly homologous [2, 6, 11]. Therefore, it could be possible that our ACCK probe which includes the sequence of the Cterminal pentapeptide of CCK hybridizes to the CGRP mRNA. However, the fact that a totally similar labeling was observed using a probe (BCCK) complementary to a region of the CCK mRNA which does not exhibit any homology with the CGRP mRNA, strongly suggests a specific hybridization to CCK mRNA. In addition, the absence of ISHH labeling using these two CCK probes in the D R G where CGRP-LI was abundantly detected [6-8] confirms the specificity of the hybridization. Gastrin and CCK exhibited the same C-terminal pentapeptide, but similarly, a hybridization to a gastrin mRNA could reasonably be excluded by the use of the two different, ACCK and BCCK, oligonucleotide probes. The nature of the detected CCK-LI, CGRP versus genuine CCK, in these neurons which contain also CGRP-LI [4, 6] and CGRP mRNA [5] and in the peripheral motor nerves where an anterograde transport of CCK-LI was described [10], remains to be established. The present results do not prove that CCK mRNA is really translated and do not exclude that CCK-LI detected in the majority of these neurons [6-8] represent CGRP. Indeed, it has been demonstrated that several long projection neurons such as the thalamic neurons, pyramidal neurons of the Ammon's horn and pyramidal cortical neurons [1, 12] contain CCK mRNA whereas they are devoid of detectable CCK-LI. A higher sensitivity of ISHH than immunohistochemistry in detecting CCK expressing cell bodies is the most probable, although still speculative explanation for these discrepancies [1, 12]. There are other possibilities: existence of a differential splicing of the CCK primary transcript resulting in a different peptide; existence of a highly homologous mRNA encoded by an unknown gene; differential processing of the CCK peptide precursor resulting in peptide species unrecognizable by the available antiCCK antisera and/or differences in rates of synthesis or turnover. We are thankful to J.-L. Conreur and C. Christophe for photography and oligonucleotide synthesis, respectively. Thanks are due also to M. Ludgate who carefully scrutinized the english and to Drs. M. Schalling and T. H6kfelt for training in in situ hybridization techniques. Supported by grants from Fonds de la Recherche Scientifique Medicale (3.4523-86 and 3.4574.90), Fondation Medicale Reine Elisabeth (Belgium) (Neurobiology 8690). S.N.S. is Research Assistant of Fonds National de la Recherche Scientifique (Belgium).
126 1 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. 2 Deschenes, R.J., Lorenz, 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 (1984) 726-730. 3 Fuji, K., Senba, E., Fujii, S., Nomura, I., Wu, J.Y., Ueda, Y. and Tohyama, M., Distribution, ontogeny and projections of cholecystokinin-8, vasoactive intestinal polypeptide and gamma-aminobutyrate-containing neuron systems in the rat spinal cord: an immunohistochemical analysis, Neuroscience, 14 (1985) 881-894. 4 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulderry, P.M., Ghatei, M.A., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101-3111. 5 Gibson, S.J., Polak, J.M., Giaid, A., Hamid, Q.A., Kar, S., Jones, P.M., Denny, P., Legon, S., Amara, S.G., Craig, R.K., Bloom, S.R., Penketh, R.J.A., Rodek, C., Ibraim, N.B.N. and Dawson, A., Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat, Neurosci. Lett., 91 (1988) 283-288. 6 Hrkfelt, T., Herrera-Marschitz, M., Seroogy, K., Gong, J., Staines, W.A., Holets, V., Schalling, M., Ungerstedt, U., Post, C., Rehfeld, J.F., Frey, P., Fischer, J., Dockray, G., Hamaoka, T., Walsh, J.H. and Goldstein, M., Irnmunohistochemical studies on cholecystokinin (CCK)-immunoreactive neurons in the rat using sequence specific antisera and with special reference to that caudate nucleus and primary sensory neurons, J. Chem. Neuroanat., 1 (1988) 11-52. 7 Ju, G., Hrkfelt, T., Brodin, E., Fahrenkrug, J., Fischer, J.A., Frey, P., Elde, R.P. and Brown, J.C., Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactiveganglion cells, Cell Tissue Res., 247 (1987) 417-431. 8 Ju, G., Hrkfelt, T., Fischer, J.A., Frey, P., Rehfeld, J.F. and Dockray, G.J., Does cholecystokinin-like immunoreactivity in rat prim-
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