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α-CGRP and β-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglion
Molecular Brain Research, 7 (1990) 299-304 Elsevier
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a-CGRP and fl-CGRP m R N A s are differentially regulated in the rat spinal cord and dorsal root ganglion Koichi Noguchi, Emiko Senba, Yasuhiro Morita, Makoto Sato and Masaya Tohyama Department Anatomy I1, Osaka University Medical School, Osaka (Japan) (Accepted 27 October 1989) Key words: a-Calcitonin gene-related peptide; fl-Calcitonin gene-related peptide; Motoneuron; Dorsal root ganglion; Axotomy; Immunohistochemistry; In situ hybridization; Northern blotting
We found an increase in calcitonin gene-related peptide (CGRP)-Iike immunoreactivity in motoneurons of rat spinal cord after peripheral axotomy. By means of in situ hybridization histochemistry and Northern blotting, we further demonstrated that this increase was the result of increased levels of a-CGRP mRNA, not fl-CGRP mRNA. The increased level of a-CGRP mRNA was maintained for at least 5 weeks, and was present on both sides. In addition, a-CGRP and fl-CGRP mRNAs had different distributions from each other in the dorsal root ganglia and levels of both were decreased after axotomy. These results indicate that a-CGRP and fl-CGRP are regulated independently and have different roles in the motor and sensory systems of the spinal cord. INTRODUCTION Calcitonin g e n e - r e l a t e d p e p t i d e ( C G R P ) is c o m p o s e d of 37 a m i n o acids, and is synthesized in neurons by an alternative transcription of the calcitonin gene 2'26. T h e r e is C G R P - l i k e i m m u n o r e a c t i v i t y ( C G R P - L I ) in the cranial and spinal m o t o n e u r o n s , sensory neurons, and enteric a u t o n o m i c n e u r o n s 6'9-11'13'26'3°'33. In the spinal m o t o n e u rons, C G R P is colocalized with acetylcholine ( A C h ) 33, and has various effects in this system; enhancing muscle contraction with a concomitant increase in c A M P 12'34'35, increasing muscle A C h receptors 4'5'19, regulating A C h r e c e p t o r p h o s p h o r y l a t i o n 16, and inhibiting the sprouting of m o t o r axon terminals 36. In p r i m a r y sensory neurons, C G R P m a y play a n e u r o m o d u l a t o r y role 17'22-24'28'29. It p o t e n t i a t e s synaptic transmission in the dorsal horn by changing calcium conductance 22'24 and by increasing the release of substance p23. f l - C G R P has been found in rat and h u m a n brain. It differs from the original a - C G R P by one amino acid in rats and by 3 amino acids in humans 1'3'27'31. The functional difference in the two different forms is unknown. It seems to be impossible to distinguish b e t w e e n these forms by a n t i g e n - a n t i b o d y methods. H o w e v e r , the m R N A s encoding these peptides can be specifically differentiated by nucleic acid hybridization. Sensory ganglia and m o t o n e u r o n s express both forms of C G R P , with a - C G R P the m o r e a b u n d a n t , but only f l - C G R P is present in enteric autonomic neurons 7'18. The
p u r p o s e of this study was to differentiate b e t w e e n a - C G R P and f l - C G R P m R N A s in the m o t o r and sensory systems of the spinal cord, with identification of their functions. We r e p o r t an increase in the C G R P - L I in spinal m o t o n e u r o n s after p e r i p h e r a l a x o t o m y which resulted from the increased level of a - C G R P m R N A . We also r e p o r t that a - and f l - C G R P m R N A had different locations in the neurons of dorsal r o o t ganglia ( D R G ) . MATERIALS AND METHODS Animals and surgery Fifty-one male Wistar rats (150-200 g) were used. For the in situ hybridization experiment, the right sciatic nerve was exposed and sectioned in the thigh of 15 rats. After postoperative survival times of 1, 3, 7, 21 or 35 days, the animals were killed and their L5 spinal cord and dorsal root ganglia (DRG) were excised. For the immunohistochemical study, 3 rats underwent the same operation. Three animals were killed to serve as normal controls. For Northern blotting and sham-operated controls (n = 3), 30 animals were killed 7 days after surgery. Oligonucleotide probes Probes used in the in situ hybridization histochemistry and Northern blotting were 35-base oligodeoxyribonucleotides synthesized with an Applied Biosystems 370A DNA synthesizer (Foster City, CA). The a-CGRP probe was complementary to bases 664-698 and the fl-CGRP probe to bases 656-690 of their cDNAs2'3'26. These probes included 3"-noncoding exons unique to each mRNA, which yielded specificities of the probes. Computer analysis by 'DNASIS' DNA homology search (Hitachi Ltd., Japan) showed that the a-CGRP probe had only 54.3% homology with the fl-CGRP mRNA sequence. The fl-CGRP probe had 54.3% homology with the a-CGRP mRNA sequence. This low homology prevented crosshybridization between a- and fl-CGRP under the conditions used for washing.
Correspondence: K. Noguchi, Department of Anatomy II, Osaka University Medical School, Nakanoshima 4, Kitaku, Osaka 530, Japan. 0169-328X/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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In situ hybridization histochemistry The in situ hybridization procedure used has been previously described2°'21. Oligonucleotide probes for hybridization were labeled with [a-3SS]dATP and terminal deoxynucleotidyl transferase, giving a specific activity of 1.0-1.5 x 10 9 dpm//~g. So that data for different animals at each time would be comparable, hybridization for all animals including normal controls was done at the same time with the same probe. The specificity of the hybridization was confirmed by Northern blotting and competition experiments. Total RNA from spinal cord and DRG hybridized with a-CGRP and fl-CGRP probes appeared as single bands. The a-CGRP and fl-CGRP mRNAs were about 1.3 kb and 1.2 kb, respectively, which has consistent with earlier results3. In the competition experiments in which sections were prehybridized with an excess of non-labeled probes, hybridization signals were virtually absent.
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Immunohistochemistry The experimental animals were perfused transcardially with Zamboni's fixative (500 ml) under sodium pentobarbital anesthesia. Ls spinal cord was excised, postfixed, placed in 30% sucrose in 0.1 M phosphate buffer overnight, and sectioned 20 /~m thick in a cryostat. The sections were immunostained with rabbit anti-CGRP antiserum (Amersham) by the peroxidase-antiperoxidase (PAP) method.
Northern blottings Fifteen rats received transection of the bilateral sciatic nerves, and 15 control rats were also used. Total cellular RNA was extracted from the L5 spinal cord and bilateral L5 DRG by ultracentrifugation with guanidium isothiocyanate/caesium chloride 15. Portions (20/~g) were denatured and separated by electrophoresis on agaroseformaldehyde gels. RNA was transferred to a nitrocellulose membrane and hybridized with 32p-labeled probes. Filters were washed in 1× SSC and 0.1% SDS at 60 °C for 1 h before being processed for autoradiography for 3 days at -80 °C. For the quantification of Northern blottings, developed X-ray films were analyzed with densitometric device Shimazu, Japan). RESULTS First, we examined the change in C G R P - L I in the m o t o n e u r o n s after sciatic nerve transection. Four days after axotomy, L 5 m o t o n e u r o n s on the operated side had more such immunoreactivity than those on the contralateral side (Fig. 1A,B). Two possible explanations for this increase were that an interruption of axonal transport caused accumulation of the peptide in the m o t o n e u r o n s , and that axotomy triggered the synthesis of the peptide in the m o t o n e u r o n s . In situ hybridization histochemistry with specific probes to a- or f l - C G R P showed that a - C G R P m R N A increased in the axotomized m o t o n e u rons 7 days after surgery (Fig. 1C,D), but that the f l - C G R P m R N A level was not increased (Fig. 1E,F). N o r t h e r n blot analysis also showed an increase of a - C G R P m R N A level but not f l - C G R P m R N A level
7d
.....................1 21 d 35 d Fig. 2. Changes in expression of a-CGRP mRNA in the motoneutons of L5 spinal cord after axotomy. Bar = 500 pm. (Fig. 1G, lanes 1 and 2). Quantitative analyses using densitometry showed that a - C G R P m R N A levels of the spinal cord of the operated animals were 191.3-339.2% (mean: 271.9%, n -- 3) of the control animals, while f l - C G R P m R N A levels in the same experiments were 42.6-93% (mean: 60.4%, n -- 3) of the control ones. These findings suggest that, the increase in C G R P - L I after axotomy was due to increased a - C G R P synthesis. Fig. 2 showed a - C G R P m R N A levels in the L 5 spinal m o t o n e u r o n s after transection of the sciatic nerve. The ipsilateral m o t o n e u r o n s had high levels of a - C G R P m R N A 1 day after surgery which persisted for at least 5 weeks, a - C G R P m R N A was also increased in the m o t o n e u r o n s on the u n o p e r a t e d side compared to control sections from normal animals that were processed in the same series of experiments, but the increase appeared later than on the operated side.
Fig. 1. Photomicrographs of immunohistochemical staining for CGRP (A,B) and of in situ hybridization for a-CGRP mRNA (C,D) and fl-CGRP mRNA (E,F). L5 spinal motoneurons have CGRP-LI on the ipsilateral (A) and contralateral (B) sides 4 days after axotomy. Dark-field autoradiographs show neurons labeled for a-CGRP and fl-CGRP (arrow) mRNAs in the motoneurons 7 days after axotomy (C,E) or a sham operation (D,F). Bar = 100/~m. G: Northern blot hybridization of probes specific for a-CGRP (left) and fl-CGRP (right) to total cellular RNA extracted from rat L5 spinal cord (1,2) and DRG (3,4). Animals received transections of the sciatic nerves (1,3) or a sham operation (2,4) 1 week before.
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Fig. 3. Photomicrographs showing location of a-CGRP (A,C,D) and fl-CGRP (B) mRNAs in the L 5 DRG. Bright-field autoradiographs show ct-CGRP (A) and fl-CGRP (B) mRNAs in the DRG of a control rat. Dark-field autoradiographs show a-CGRP mRNAs in axotomized DRG cells (C) and contralateral DRG cells (D). Bar = 50/~m (A,B), 200 #m (C,D). E: size distribution of DRG neurons labeled for a-CGRP and fl-CGRP mRNA. Of 1000 DRG neurons, 364 were labeled for a-CGRP; of another 1000 neurons, 308 were labeled for fl-CGRP. Plotting is according to diameter. Neurons with grain densities at least 5 times that of the background density were considered to be labeled.
303 In L 5 D R G , intense signals for a- and fl-CGRP mRNAs were detected (Fig. 3A,B). These signals were more intense than those in motoneurons. Many neurons (36.4% of all D R G neurons) were densely labeled for a - C G R P m R N A , and fl-CGRP m R N A was also detected in 30.8% of D R G neurons. Large neurons (>30/~m) were labeled with the a - C G R P probe, and smaller ones were labeled with both probes (Fig. 3E). Seven days after sciatic nerve transection, levels of both mRNAs were decreased in the L 5 D R G compared to the contralateral side (Fig. 3C,D), which was consistent with the results of immunohistochemistry (data are not shown) and Northern blotting (Fig. 1G, lanes 3 and 4). Quantified Northern blot data showed that a - C G R P m R N A levels of DRGs from operated animals were 68.4% and 78.5% of the control DRGs. fl-CGRP m R N A levels were also decreased to 31.8% and 95.9% of the control ones. DISCUSSION fl-CGRP of the rat differs from a-CGRP only by the substitution of a lysine residue for glutamate at position 35 in the sequence of 37 amino acids. The present study has clearly demonstrated that a- and fl-CGRP mRNAs are successfully differentiated by the hybridization histochemistry because of the sequence divergence between the noncoding portions of these mRNAs. Increase of CGRP-like immunoreactivity following axotomy has been shown in facial motoneurons 32. However, it is not clear whether this increase is attributed to the increase of synthesis or not, or which type of CGRP this immunoreactivity represents. We have suggested in the present study that increased CGRP in the axotomized motoneurons are newly synthesized a-CGRP. That axotomy conversely decreased the fl-CGRP m R N A level in motoneurons indicates that the synthesis of a- and flREFERENCES 1 Alevizaki, M., Shiraishi, A., Rassool, F.V., Ferrier, G.J.M., MacIntyre, I. and Legon, S., The calcitonin-like sequence of the fl CGRP gene, FEBS Lett., 206 (1986) 47-52. 2 Amara, S.G., Jonas, V. and Rosenfeld, M.G., Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products, Nature (Lond.), 298 (1982) 240-244. 3 Amara, S.G., Arriza, J.L., Left, S.E., Swanson, L.W., Evans, R.M. and Rosenfeld, M.G., Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide, Science, 229 (1985) 1094-1097. 4 Fontaine, B., Klarsfeld, A., HOkfelt, T. and Changeux, J.-E, Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick myotubes, Neurosci. Lett., 71 (1986) 59-65. 5 Fontaine, B., Klarsfeld, A. and Changeux, J.-P., Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor a-subunit mRNA levels by distinct intracellular path-
CGRP in motoneurons are controlled by different mechanisms. And independent regulation of two genes provides a potential advantage for the expression of two such homologous gene products. Functional roles of these peptides in the nervous system are now open to discussion. A variety of morphological and chemical changes occur in axotomized motoneurons that are regenerating 8' 14. Enzymes and other substances required for neuronal regenerating processes are increased and those related to neural transmission are decreased 8. Our results that a-CGRP m R N A increases 1 day after surgery and remains increased for at least 5 weeks on the operated side suggest that a-CGRP has a neurotrophic action in regenerating motoneurons. Increases in this m R N A also occur in the motoneurons on the contralateral side. Similar results have already been reported for tubulin m R N A in facial motoneurons 25. Hemilateral axotomy may cause kinetic changes on movement and may influence motoneurons on the contralateral side. It is generally accepted that the size of D R G cells is closely related to the sensory receptors to which they are connected: small cells seem to mediate thermal and nociceptive information and larger ones mediate tactile and deep sensations. Immunohistochemical studies have shown that CGRP is present in both small and large D R G cells6'9'11'13. The present finding that two types of m R N A for CGRP are differently distributed in sensory neurons, with large neurons mainly producing a-CGRP, and small neurons producing both CGRPs, suggests different roles of these peptides in the primary sensory system.
Acknowledgements. This work was supported in part by a grant from the Ministry of Education, Japan.
ways, J. Cell Biol., 105 (1987) 1337-1342. 6 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulderry, P.M., Ghatei, M.A., MaGregor, G.P., Morrison, J.F.B., Kerry, 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. 7 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., lbrahim, N.B.N. and Dawson, A., Calcitonin gene-related peptide messenger RNA is expressed in sensory neurons of the dorsal root ganglia and also in spinal motoneurons in man and rat, Neurosci. Lett., 91 (1988) 283-288. 8 Hoover, D.B. and Hancock, J.C., Effect of facial nerve transection on acetylcholinesterase and (3H) quinuclidinyl benzilate binding in rat facial nuclei, Neuroscience, 15 (1985) 481-487. 9 Ju, G., H6kfelt, T., Brodin, E., Fahrenkrug, J., Fisher, J.A., Prey, 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-,
304 vasoactive intestinal polypeptide- and cholecystokinin- immunoreactive ganglion cells, Cell Tissue Res., 247 (1987) 417-431. 10 Kawai, Y., Takami, K., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S., Maclntyre, I. and Tohyama, M., Topographic localization of calcitonin gene-related peptide in the rat brain: an immunohistochemical analysis, Neuroscience, 15 (1985) 747763. 11 Kruger, L., Sternini, C., Brecha, N.C. and Mantyh, P.W., Distribution of calcitonin gene-related peptide immunoreactivity in relation to the rat central somatosensory projection, J. Cornp. Neurol., 273 (1988) 149-162. 12 Laufer, R. and Changeux, J.-P., Calcitonin gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger, EMBO J., 6 (1987) 901-906. 13 Lee, Y., Takami, K., Kawai, Y., Girgis, S., Hillyard, C.J., Maclntyre, I., Emson, P.C. and Tohyama, M., Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P, Neuroscience, 15 (1985) 1227-1235. 14 Lieberman, A.R., The axon reaction: a review of the principal features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 15 Maniatis, T., Fritsch, E.E and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982, 196 pp. 16 Miles, K., Greengard, P. and Huganir, R.L., Calcitonin generelated peptide regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes, Neuron, 2 (1989) 15171524. 17 Miletic, V. and Tan, H., Iontophoretic application of calcitonin gene-related peptide produces a slow and prolonged excitation of neurons in the cat lumbar dorsal horn, Brain Res., 446 (1988) 169-172. 18 Mulderry, P.K., Ghatei, M.A., Spokes, R.A., Spokes, R.A., Jones, P.M., Pierson, A.M., Hamid, Q.A., Kanse, S., Amara, S.G., Burrin, J.M., Legon, S., Polak, J.M. and Bloom, S.R., Differential expression of a-CGRP and fl-CGRP by primary sensory neurons and enteric autonomic neurons of the rat, Neuroscience, 25 (1988) 195-205. 19 New, H.V. and Mudge, A.W., Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis, Nature (Lond.), 323 (1986) 809-811. 20 Noguchi, K., Morita, Y., Kiyama, H., Ono, K. and Tohyama, M., A noxious stimulus induces the preprotachykinin-A gene expression in the rat dorsal root ganglion; a quantitative study using in situ hybridization histochemistry, Mol. Brain Res., 4 (1988) 31-35. 21 Noguchi, K., Morita, Y., Kiyama, H., Sato, H., Ono, K. and Tohyama, M,. Preproenkephalin gene expression in the rat spinal cord after noxious stimuli, Mol. Brain Res., 5 (1989) 227-234. 22 Nohmi, M., Gallagher, J.P., Gean, P-W., Gallagher, J.P. and Cooper, C.W., Calcitonin and calcitonin gene-related peptide enhance calcium-dependent potentials, Brain Res., 367 (1986)
346-350. 23 Oku, R., Satoh, M., Fujii, N., Otaka, A., Yajima, H. and Takagi, H., Calcitonin gene-related peptide promotes mechanical nociception by potentiating release of substance P from the spinal dorsal horn in rats, Brain Res., 403 (1987) 350-354. 24 Oku, R., Nanayama, T. and Satoh, M., Calcitonin gene-related peptide modulates calcium mobilization in synaptosomes of rat spinal dorsal horn, Brain Res., 475 (1988) 356--360. 25 Pearson, R.C.A., Taylor, N. and Snyder, S.H., Tubulin messenger RNA: in situ hybridization reveals bilateral increases in hypoglossal and facial nuclei following nerve transection, Brain Res., 463 (1988) 245-249. 26 Rosenfeld, M.G., Mermod, J-J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing, Nature (Lond.), 304 (1983) 129-135. 27 Rosenfeld, M.G., Amara, S.G. and Evans, R.M., Alternative RNA processing: determining neuronal phenotype, Science, 225 (1984) 1315-1320. 28 Ryu, P.D., Gerber, G., Murase, K. and Randi6, M., Actions of calcitonin gene-related peptide on rat spinal dorsal horn neurons, Brain Res., 441 (1988) 357-361. 29 Ryu, P.D., Gerber, G., Murase, K. and Randi6, M., Calcitonin gene-related peptide enhances calcium current of rat dorsal root ganglion neurons and spinal excitatory synaptic transmission, Neurosci. Lett., 89 (1988) 305-312. 30 Skofitsch, G. and Jacobowitz, D.M., Calcitonin gene-related peptide; detailed immunohistochemical distribution in the central nervous system, Peptides, 6 (1985) 721-745. 31 Steenbergh, P.H., Hoppener, J.W.M., Zandberg, J., Lips, C.J.M. and Jansz, H.S., A second human calcitonin/CGRP gene, FEBS Lett., 183 (1985) 403-407. 32 Streit, W.J., Dumoulin, EL., Raivich, G. and Kreutzberg, G.W., Calcitonin gene-related peptide increases in rat facial motoneurons after peripheral nerve transection, Neurosci. Lett., 101 (1989) 143-148. 33 Takami, K., Kawai, Y., Shiosaka, S., Lee, Y., Girgis, S., Hillyard, C.J., Maclntyre, I., Emson, P.C. and Tohyama, M., Immunohistochemical evidence for the coexistence of calcitonin gene-related peptide- and choline acetyltransferase-like immunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei, Brain Res., 328 (1985) 386-389. 34 Takami, K., Kawai, Y., Uchida, S., Tohyama, M., Shiotani, Y., Yoshida, H., Emson, P.C., Girgis, S., Hillyard, C.J. and Maclntyre, I., Effect of calcitonin gene-related peptide on contraction of striated muscle in the mouse, Neurosci. Lett., 60 (1985) 227-230. 35 Takami, K., Hashimoto, K., Uchida, S., Tohyama, M. and Yoshida, H., Effect of calcitonin gene-related peptide on the cyclic AMP level of isolated mouse diaphragm, Jpn. J. Pharmacol., 42 (1986) 345-350. 36 Tsujimoto, T. and Kuno, M., Calcitonin gene-related peptide prevents disuse-induced sprouting of rat motor nerve terminals, J. Neurosci., 8 (1988) 3951-3957.
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a-CGRP and fl-CGRP m R N A s are differentially regulated in the rat spinal cord and dorsal root ganglion Koichi Noguchi, Emiko Senba, Yasuhiro Morita, Makoto Sato and Masaya Tohyama Department Anatomy I1, Osaka University Medical School, Osaka (Japan) (Accepted 27 October 1989) Key words: a-Calcitonin gene-related peptide; fl-Calcitonin gene-related peptide; Motoneuron; Dorsal root ganglion; Axotomy; Immunohistochemistry; In situ hybridization; Northern blotting
We found an increase in calcitonin gene-related peptide (CGRP)-Iike immunoreactivity in motoneurons of rat spinal cord after peripheral axotomy. By means of in situ hybridization histochemistry and Northern blotting, we further demonstrated that this increase was the result of increased levels of a-CGRP mRNA, not fl-CGRP mRNA. The increased level of a-CGRP mRNA was maintained for at least 5 weeks, and was present on both sides. In addition, a-CGRP and fl-CGRP mRNAs had different distributions from each other in the dorsal root ganglia and levels of both were decreased after axotomy. These results indicate that a-CGRP and fl-CGRP are regulated independently and have different roles in the motor and sensory systems of the spinal cord. INTRODUCTION Calcitonin g e n e - r e l a t e d p e p t i d e ( C G R P ) is c o m p o s e d of 37 a m i n o acids, and is synthesized in neurons by an alternative transcription of the calcitonin gene 2'26. T h e r e is C G R P - l i k e i m m u n o r e a c t i v i t y ( C G R P - L I ) in the cranial and spinal m o t o n e u r o n s , sensory neurons, and enteric a u t o n o m i c n e u r o n s 6'9-11'13'26'3°'33. In the spinal m o t o n e u rons, C G R P is colocalized with acetylcholine ( A C h ) 33, and has various effects in this system; enhancing muscle contraction with a concomitant increase in c A M P 12'34'35, increasing muscle A C h receptors 4'5'19, regulating A C h r e c e p t o r p h o s p h o r y l a t i o n 16, and inhibiting the sprouting of m o t o r axon terminals 36. In p r i m a r y sensory neurons, C G R P m a y play a n e u r o m o d u l a t o r y role 17'22-24'28'29. It p o t e n t i a t e s synaptic transmission in the dorsal horn by changing calcium conductance 22'24 and by increasing the release of substance p23. f l - C G R P has been found in rat and h u m a n brain. It differs from the original a - C G R P by one amino acid in rats and by 3 amino acids in humans 1'3'27'31. The functional difference in the two different forms is unknown. It seems to be impossible to distinguish b e t w e e n these forms by a n t i g e n - a n t i b o d y methods. H o w e v e r , the m R N A s encoding these peptides can be specifically differentiated by nucleic acid hybridization. Sensory ganglia and m o t o n e u r o n s express both forms of C G R P , with a - C G R P the m o r e a b u n d a n t , but only f l - C G R P is present in enteric autonomic neurons 7'18. The
p u r p o s e of this study was to differentiate b e t w e e n a - C G R P and f l - C G R P m R N A s in the m o t o r and sensory systems of the spinal cord, with identification of their functions. We r e p o r t an increase in the C G R P - L I in spinal m o t o n e u r o n s after p e r i p h e r a l a x o t o m y which resulted from the increased level of a - C G R P m R N A . We also r e p o r t that a - and f l - C G R P m R N A had different locations in the neurons of dorsal r o o t ganglia ( D R G ) . MATERIALS AND METHODS Animals and surgery Fifty-one male Wistar rats (150-200 g) were used. For the in situ hybridization experiment, the right sciatic nerve was exposed and sectioned in the thigh of 15 rats. After postoperative survival times of 1, 3, 7, 21 or 35 days, the animals were killed and their L5 spinal cord and dorsal root ganglia (DRG) were excised. For the immunohistochemical study, 3 rats underwent the same operation. Three animals were killed to serve as normal controls. For Northern blotting and sham-operated controls (n = 3), 30 animals were killed 7 days after surgery. Oligonucleotide probes Probes used in the in situ hybridization histochemistry and Northern blotting were 35-base oligodeoxyribonucleotides synthesized with an Applied Biosystems 370A DNA synthesizer (Foster City, CA). The a-CGRP probe was complementary to bases 664-698 and the fl-CGRP probe to bases 656-690 of their cDNAs2'3'26. These probes included 3"-noncoding exons unique to each mRNA, which yielded specificities of the probes. Computer analysis by 'DNASIS' DNA homology search (Hitachi Ltd., Japan) showed that the a-CGRP probe had only 54.3% homology with the fl-CGRP mRNA sequence. The fl-CGRP probe had 54.3% homology with the a-CGRP mRNA sequence. This low homology prevented crosshybridization between a- and fl-CGRP under the conditions used for washing.
Correspondence: K. Noguchi, Department of Anatomy II, Osaka University Medical School, Nakanoshima 4, Kitaku, Osaka 530, Japan. 0169-328X/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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In situ hybridization histochemistry The in situ hybridization procedure used has been previously described2°'21. Oligonucleotide probes for hybridization were labeled with [a-3SS]dATP and terminal deoxynucleotidyl transferase, giving a specific activity of 1.0-1.5 x 10 9 dpm//~g. So that data for different animals at each time would be comparable, hybridization for all animals including normal controls was done at the same time with the same probe. The specificity of the hybridization was confirmed by Northern blotting and competition experiments. Total RNA from spinal cord and DRG hybridized with a-CGRP and fl-CGRP probes appeared as single bands. The a-CGRP and fl-CGRP mRNAs were about 1.3 kb and 1.2 kb, respectively, which has consistent with earlier results3. In the competition experiments in which sections were prehybridized with an excess of non-labeled probes, hybridization signals were virtually absent.
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C ld 3d
Immunohistochemistry The experimental animals were perfused transcardially with Zamboni's fixative (500 ml) under sodium pentobarbital anesthesia. Ls spinal cord was excised, postfixed, placed in 30% sucrose in 0.1 M phosphate buffer overnight, and sectioned 20 /~m thick in a cryostat. The sections were immunostained with rabbit anti-CGRP antiserum (Amersham) by the peroxidase-antiperoxidase (PAP) method.
Northern blottings Fifteen rats received transection of the bilateral sciatic nerves, and 15 control rats were also used. Total cellular RNA was extracted from the L5 spinal cord and bilateral L5 DRG by ultracentrifugation with guanidium isothiocyanate/caesium chloride 15. Portions (20/~g) were denatured and separated by electrophoresis on agaroseformaldehyde gels. RNA was transferred to a nitrocellulose membrane and hybridized with 32p-labeled probes. Filters were washed in 1× SSC and 0.1% SDS at 60 °C for 1 h before being processed for autoradiography for 3 days at -80 °C. For the quantification of Northern blottings, developed X-ray films were analyzed with densitometric device Shimazu, Japan). RESULTS First, we examined the change in C G R P - L I in the m o t o n e u r o n s after sciatic nerve transection. Four days after axotomy, L 5 m o t o n e u r o n s on the operated side had more such immunoreactivity than those on the contralateral side (Fig. 1A,B). Two possible explanations for this increase were that an interruption of axonal transport caused accumulation of the peptide in the m o t o n e u r o n s , and that axotomy triggered the synthesis of the peptide in the m o t o n e u r o n s . In situ hybridization histochemistry with specific probes to a- or f l - C G R P showed that a - C G R P m R N A increased in the axotomized m o t o n e u rons 7 days after surgery (Fig. 1C,D), but that the f l - C G R P m R N A level was not increased (Fig. 1E,F). N o r t h e r n blot analysis also showed an increase of a - C G R P m R N A level but not f l - C G R P m R N A level
7d
.....................1 21 d 35 d Fig. 2. Changes in expression of a-CGRP mRNA in the motoneutons of L5 spinal cord after axotomy. Bar = 500 pm. (Fig. 1G, lanes 1 and 2). Quantitative analyses using densitometry showed that a - C G R P m R N A levels of the spinal cord of the operated animals were 191.3-339.2% (mean: 271.9%, n -- 3) of the control animals, while f l - C G R P m R N A levels in the same experiments were 42.6-93% (mean: 60.4%, n -- 3) of the control ones. These findings suggest that, the increase in C G R P - L I after axotomy was due to increased a - C G R P synthesis. Fig. 2 showed a - C G R P m R N A levels in the L 5 spinal m o t o n e u r o n s after transection of the sciatic nerve. The ipsilateral m o t o n e u r o n s had high levels of a - C G R P m R N A 1 day after surgery which persisted for at least 5 weeks, a - C G R P m R N A was also increased in the m o t o n e u r o n s on the u n o p e r a t e d side compared to control sections from normal animals that were processed in the same series of experiments, but the increase appeared later than on the operated side.
Fig. 1. Photomicrographs of immunohistochemical staining for CGRP (A,B) and of in situ hybridization for a-CGRP mRNA (C,D) and fl-CGRP mRNA (E,F). L5 spinal motoneurons have CGRP-LI on the ipsilateral (A) and contralateral (B) sides 4 days after axotomy. Dark-field autoradiographs show neurons labeled for a-CGRP and fl-CGRP (arrow) mRNAs in the motoneurons 7 days after axotomy (C,E) or a sham operation (D,F). Bar = 100/~m. G: Northern blot hybridization of probes specific for a-CGRP (left) and fl-CGRP (right) to total cellular RNA extracted from rat L5 spinal cord (1,2) and DRG (3,4). Animals received transections of the sciatic nerves (1,3) or a sham operation (2,4) 1 week before.
302
E (n) c~- CGRP /~ - C G R P F"
20
n 10 II
20
30
40
(H m )
Fig. 3. Photomicrographs showing location of a-CGRP (A,C,D) and fl-CGRP (B) mRNAs in the L 5 DRG. Bright-field autoradiographs show ct-CGRP (A) and fl-CGRP (B) mRNAs in the DRG of a control rat. Dark-field autoradiographs show a-CGRP mRNAs in axotomized DRG cells (C) and contralateral DRG cells (D). Bar = 50/~m (A,B), 200 #m (C,D). E: size distribution of DRG neurons labeled for a-CGRP and fl-CGRP mRNA. Of 1000 DRG neurons, 364 were labeled for a-CGRP; of another 1000 neurons, 308 were labeled for fl-CGRP. Plotting is according to diameter. Neurons with grain densities at least 5 times that of the background density were considered to be labeled.
303 In L 5 D R G , intense signals for a- and fl-CGRP mRNAs were detected (Fig. 3A,B). These signals were more intense than those in motoneurons. Many neurons (36.4% of all D R G neurons) were densely labeled for a - C G R P m R N A , and fl-CGRP m R N A was also detected in 30.8% of D R G neurons. Large neurons (>30/~m) were labeled with the a - C G R P probe, and smaller ones were labeled with both probes (Fig. 3E). Seven days after sciatic nerve transection, levels of both mRNAs were decreased in the L 5 D R G compared to the contralateral side (Fig. 3C,D), which was consistent with the results of immunohistochemistry (data are not shown) and Northern blotting (Fig. 1G, lanes 3 and 4). Quantified Northern blot data showed that a - C G R P m R N A levels of DRGs from operated animals were 68.4% and 78.5% of the control DRGs. fl-CGRP m R N A levels were also decreased to 31.8% and 95.9% of the control ones. DISCUSSION fl-CGRP of the rat differs from a-CGRP only by the substitution of a lysine residue for glutamate at position 35 in the sequence of 37 amino acids. The present study has clearly demonstrated that a- and fl-CGRP mRNAs are successfully differentiated by the hybridization histochemistry because of the sequence divergence between the noncoding portions of these mRNAs. Increase of CGRP-like immunoreactivity following axotomy has been shown in facial motoneurons 32. However, it is not clear whether this increase is attributed to the increase of synthesis or not, or which type of CGRP this immunoreactivity represents. We have suggested in the present study that increased CGRP in the axotomized motoneurons are newly synthesized a-CGRP. That axotomy conversely decreased the fl-CGRP m R N A level in motoneurons indicates that the synthesis of a- and flREFERENCES 1 Alevizaki, M., Shiraishi, A., Rassool, F.V., Ferrier, G.J.M., MacIntyre, I. and Legon, S., The calcitonin-like sequence of the fl CGRP gene, FEBS Lett., 206 (1986) 47-52. 2 Amara, S.G., Jonas, V. and Rosenfeld, M.G., Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products, Nature (Lond.), 298 (1982) 240-244. 3 Amara, S.G., Arriza, J.L., Left, S.E., Swanson, L.W., Evans, R.M. and Rosenfeld, M.G., Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide, Science, 229 (1985) 1094-1097. 4 Fontaine, B., Klarsfeld, A., HOkfelt, T. and Changeux, J.-E, Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick myotubes, Neurosci. Lett., 71 (1986) 59-65. 5 Fontaine, B., Klarsfeld, A. and Changeux, J.-P., Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor a-subunit mRNA levels by distinct intracellular path-
CGRP in motoneurons are controlled by different mechanisms. And independent regulation of two genes provides a potential advantage for the expression of two such homologous gene products. Functional roles of these peptides in the nervous system are now open to discussion. A variety of morphological and chemical changes occur in axotomized motoneurons that are regenerating 8' 14. Enzymes and other substances required for neuronal regenerating processes are increased and those related to neural transmission are decreased 8. Our results that a-CGRP m R N A increases 1 day after surgery and remains increased for at least 5 weeks on the operated side suggest that a-CGRP has a neurotrophic action in regenerating motoneurons. Increases in this m R N A also occur in the motoneurons on the contralateral side. Similar results have already been reported for tubulin m R N A in facial motoneurons 25. Hemilateral axotomy may cause kinetic changes on movement and may influence motoneurons on the contralateral side. It is generally accepted that the size of D R G cells is closely related to the sensory receptors to which they are connected: small cells seem to mediate thermal and nociceptive information and larger ones mediate tactile and deep sensations. Immunohistochemical studies have shown that CGRP is present in both small and large D R G cells6'9'11'13. The present finding that two types of m R N A for CGRP are differently distributed in sensory neurons, with large neurons mainly producing a-CGRP, and small neurons producing both CGRPs, suggests different roles of these peptides in the primary sensory system.
Acknowledgements. This work was supported in part by a grant from the Ministry of Education, Japan.
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