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Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection
Molecular Brain Research, 11 (1991) 187-196 © Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$03.50 ADONIS 0169328X9270329T
BRESM 70329
187
Research Reports
Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection Takanod Saika ~, Emiko Senba 2, Koichi Noguchi 2, Makoto Sato 2, Takeshi Kubo 1, Torn Matsunaga I and Masaya Tohyama 2 IDepartment of Otolaryngology and 2Department of Anatomy and Neuroscience, Osaka University Medical School, Osaka (Japan)
(Accepted 30 April 1991) Key words: Facial nerve; Motoneuron; Axotomy, In situ hybridization, a-Calcitonin gene-related peptide; fl-Calcitonin gene-related peptide; Cholecystokinin; Galanin
In situ hybridization histochemistry was used to study changes in mRNA,~ coding neuropeptides such as a-calcitonin gene-related peptide (CGRP),/I-CGRP, cholecystokinin (CCK) and galanin, in rat facial motoneurons following axotomy of the facial nerve. In control rats, 38%, 55% and 7% of the facial motoneurons expressed a-CGRP, fl-CGRP and CCK mRNAs, respectively. No galanin mRNA-containing motoneurons were observed in these animals. The levels of mRNA fcr c~-CGRP, CCK and galanin were increased while the fl-CGRP mRNA level was decreased after axotomy. The levels of mRNAs for these peptides returned to the control values by 2--4 weeks after nerve crush, whereas nerve resection had more prolonged effects. Within 3-4 weeks after injury, nerve resection had greater effects on fl-CGRP, CCK and galanin mRNAs than did nerve crush. Thus, there appear to be differences in the regulation of mRNA expression of these peptides in axotomized motoneurous.
INTRODUCTION
It is well known that axotomy causes a variety of morphological and chemical changes in motoneurons 2°. Enzymes and other substances related to neuronal transmission are decreased. For example, choline acetyltransferase and acetylcholine esterase are lower in rat facial nuclei after facial nerve transection u , and the level of a3 nicotinic acetylcholine receptor subunit messenger R N A ( m R N A ) is decreased in axotomized motoneurons 33. Glycine and muscarinic receptors are also depleted after injury 29-31. On the other hand, substances required for neuronal regeneration increase. For example, the synthesis of cytoskeletal proteins such as tubufin and actin is increased 26"42. Enhanced immunoreactivity for the transferrin receptor and a concomitant increase in iron uptake has also been reported n. Regarding neuropeptides, a marked increase in calcitonin gene-related peptide (CGRP) like immunoreactiv/ty (LI) 37 and C G R P m R N A 13 in the axotomized facial motoneurons has been reported. In the spinal motoneurons, it was shown that this increase results from increased levels of a - C G R P m R N A , not fl-CGRP m R N A 25. In
addition to CGRP, motoneurons contain galanin-LI, which is also i n a e a s e d after axotomy 24. Moreover, cholecystokinin (CCK) m R N A is expressed in the motor trigemina116'3s and facial nucleus 16. We have shown that nerve growth factor receptor (NGF-R) is expressed in facial motoneurons following axotomy, and that nerve crush and resection have different effects on N G F - R m R N A 32. N G F is one substance which may regulate expression of peptides in neurons 2t. Thus, it is of particular interest to clarify whether peptide production in regenerating facial motoneurons is also regulated by axonal regeneration. Furthermore, it is possible that changes in peptide production are similar to previously demonstrated changes in NGF-R mRNA. In the present study, we examined the expression of a- and fl-CGRP, galanin and CCK in facial motoneurons after crushing injury, which permits axonal regeneration, and also after resection, which prevents regeneration, using in situ hybridization histochemistry (ISHH). MATERIALS AND METHODS Animals and tissue preparation Forty-eight adult male Wistar rats weighing about 150 g were ~w,d. Under pentobarbital anesthesia (50 mg/kg, i.p.), the left fa-
Correspondence: T. Saika, Department of Anatomy and Neuroscience, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565, Japan.
188 cial nerve was exposed. At the point just distal to the posterior auricular branch, which diverges from the facial nerve as it leaves the stylomastoid foramen, the, main trunk was either crushed for 30 s with thin forceps or was transected with a pair of scissors. After transection, abot~t 5 mm of the distal nerve segment was removed. Following a postoperative interval of 1, 3, 7, 14, 21, 28, 42 (n 3), o r 56 days (n -- 2), the animals were deeply anesthetized with pentobarbital and killed. Untreated animals (n = 2) were al~o sacrificed to serve as normal controls. The brains were immediately excised and frozen with powdered dry ice. Then 20 ~m frozen sections were cut on a cryostat, mounted on gelatin- coated slictes, and subjected to in situ hybridization histoc~emisty.
Oligot~ucleotide probes The oligonucleotide probes were synthesized in an Applied Biosystems 381A DNA synthesizer and then purified using a Hitachi high pressure liquid chromatograph (ODS column chromatography). The probes were complementary to bases 664-698 of rat a--CGRP mRNA t, bases 517-550 of rat/~-CGRP mRNA*, bases ~07-342 of rat CCK mRNA 5 and bases 224-253 of rat galanin mRNA 46. The a- and ~-CGRP probes included 3~-non-coding exons unique to each mRNA, which yielded specificities of the probes. Computer assisted homology search showed that the a-CGRP probe had only 54.3% homology with the/~-CGRP mRNA sequence. The /~-CGRP probe had 50% homology with the a-CGRP mRNA sequence. This low homology prevented cross-hybridization between a- and/~-CGRP under the conditions used for washing.
In situ hybridization histochemistry These slide-mounted sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 30 rain, and rinsed 3 times (5 rain each) i~. 4 x SSC (pH 7.2) (1 x SSC contained 0.15 M sodium chloride and 0.015 M sodium citrate). The slides were then immersed in 4 x SSC and 1 x Denhardt's solution [1 x Denhardt's solution contained 0.02% bovine serum albumin (fractiost V, Sigma), 0.02% Ficoll 400 (Pharmacia), and 0.02% polyvinylpyrrolidone K-30 (Nakarai Tesque Inc.)] for I h, and dehydrated through a graded ethanol series (70-100%). The sections were treated with chloroform for 5 min to remove fat, and were immersed in 100% ethanol twice (5 min each) before being subjected to hybridization. Hybridization was performed by incubating sections with a buffer [4 x SSC, 50% deionized formamide, 0.12 M phosphate bt~er (pH 7.2), 1 x Denhardt's solution, 2.5% tRNA, 10% dextran sulfate] containing [a -35 S]dATP-labelled probes (6-9 x 106 dpndml, 0.3 ml/slide) for 1 day at 42 °C. After hybridization, the sections were rinsed in 1 x SSC (pH 7.2) for 10 min, followed by four 15 min rinses in 1 x SSC at 55 °C. The sections were then dehydrated through a graded ethanol series (70-100%) and coated with llford K-5 emulsion (diluted 1:1 with water). These sections were exposed for 4 weeks in a tightly sealed dark box at 4 °C. After being developed in D-19 (Kodak) developer, fixed with photographic fixer, and washed with tap water, the sections were counter-stained with thionin solution for morphological identification. The specificity of the hybridization was confirmed by competition experiments for each probe. In these experiments in which sections were p~hybridized with 100-fold of non-labelled probes,
Fig. 1. Dark-field photomicrographs showing facial motoneurons expressing a-CGRP mRNA hybridization signals I day (A), 1 week (B), 3 weeks (C) and 6 weeks (D) after facial nerve crushing. Bar - 1 ram.
189
Fig. 2. Dark-field photomicrographs showing facial motoneurons expressing a-CGRP mRNA hybridization signals I day (A), 1 week (B), 4 weeks (C) and 8 weeks (D) after facial nerve resection. Bar - I ram.
hybridization signals were virtually absent. Additionally, RNase A pretreatment (20 ~g/ml) just before hybridization showed no posifive signals. Computer assisted homology search also showed that these 4 probes have less than 70% homologies with any of the sequences contained in this bank.
RESULTS
gradually decreased (Fig. 1C). By the 6th week, mRNA levels had returned to the control value (Fig. 1D) and remained there at the 8th week. In the case of resection, as with crushing injury, the a-CGRP mRNA level had already increased by the first postoperative day (Fig. 2A). It remained elevated on the 3rd day, but then abruptly decreased and reached the control value one week after the procedure (Fig. 2B). From then on, mRNA increased again (Fig. 2C), and in contrast to crushing injury the increased level was maintained until at least 8 weeks after the operation (Fig. 2D).
Normal control
fl-CGRP mRNA
In the facial nucleus of control rats, 38%, 55% and 7% of motoneurons expressed a-CGRP,/I-CGRP and CCK mRNAs, respectively. Galanin mRNA was not detected.
In the case of crushing injury, the/~-CGRP mRNA level had increased by t~e first post-operative day (Fig. 3A). It then decreased aearly to zero at 3 days after the procedure (Fig. 3B). This decrease was transient, and mRNA levels were slightly increased at 7 days after the procedure (Fig. 3C). Two weeks p~t-operatively, the /~-CGRP mRNA level had returned to the control value (Fig. 3D). In the case of resection, as in crushing injury,/~-CGRP
Semi-quantitativeanalysis For semi-quantitative analysis of labelled neurons, four sections were chosen from 2 or 3 animals killed at each of the eight postsurgical intervals. Neurons with 3 times more gains than the background level were considered to be positive. The number of motoneurons was counted on Nissl-stained sections.
a-CGRP mRNA In the case of crushing injury, the a-CGRP mRNA level had already markedly increased by the first postoperative day (Fig. 1A). This was sustained at least for 2 weeks (Fig. 1B), and the a-CGRP mRNA level then
190
Fig. 3. Dark~fie|dphotomicrographsshowingfacial motoneurons on the operated side expressing/~-CGRPmRNA hybridization signals I day (A), 3 days (B), 1 week (C) and 4 weeks (D) after nerve crushing. Bar - 1 ram. mRNA was increased at 1 day after the procedure (Fig. 4A), and then decreased to zero by 3 days after the procedure (Fig. 4B). Unlike crushing injury, this lasted for 2 to 3 weeks (Fig. 4C). Three weeks after resection, the /~-CGRP mRNA level had increased but was still below the control value (Fig. 4D). CCK mRNA In the facial nucleus contralateral to the operated side, CCK mRNA positive cells were sparsely distributed throughout the nucleus. After crushing injury, there was no noticeable change in facial motoneuron CCK mRNA levels on the operated side one day after the procedure (Fig. 5A). From the 3rd day after the procedure, the level of CCK mRNA gradually increased and remained elevated for 2 weeks (Fig. 5B,C). It then decreased and returned to the control value by the 3rd week after the procedure (Fig. 5D). From then on, there was no change. In case of resection, the mRNA level was not elevated at 1 or 3 days, but then increased to a higher level than was seen after crush injury (Fig. 6A-C). In contrast to c~'ush i n j u ~ this high level of CCK mRNA was maintained for the duration of the experiment (Fig. 6D).
Galanin mRNA We found no galanin mRNA in motoneurons on the unoperated side in any animals (Fig. 7A,C). Furthermore, no mRNA was detected on the 1st day after the procedure in motoneurons on the operated side. In the case of crushing injury, a faint increase of galanin mRNA was observed from the 3 days to 2 weeks after the procedure. In the case of resection, similar to crushing injury, no mRNA was detected on the 1st day and an increase in the mRNA level wa~ observed on the 3rd day after the procedure (Fig. 7B). Still, this increase was more pronounced and prolonged than that seen after crushing injury (Fig. 7D). Even 4 weeks after the procedure the mRNA level was increased, but was less pronounced than that observed 1 or 2 weeks after the procedure. In all experimental animals, there was no changv in mRNA expression for any peptide in facial motoneurons on the unoperated side.
DISCUSSION CGRP CGRP has been detected in motoneurons by immu-
191
Fig. 4. Dark-fieldphotomicrographs showingfacial motoneurons on the operated side expressing/t-CGRP mRNA hybridization signals 1 day (A), 3 days (B), 2 weeks (C) and 4 weeks (D) after nerve resection. Bar = 1 mm.
nohistochemistry ~°'~s'36'39 and by ISHH 27. This peptide has various effects on motoneuron-muscle systems: enhancing muscle contraction with a concomitant increase in cAMP ~°'4t, increasing muscle acetylcholine receptors7,s and inhibiting the sprouting of motor axon terminals43. CGRP-LI and its mRNA level are increased after axotomy in spinal 2 and facial 13'37 motoneurons. Although a-CGRP and/~-CGRP gene sequences differ by only a single amino acid among the 37 amino acids in the rat and both mRNAs are expressed in motoneurons ~, reactions of these two mRNAs to axotomy are completely different. Transcription of a-CGRP mRNA is increased whereas that of /~-CGRP mRNA is decreased after axotomy in spinal cord 25. The physiological significance of this difference is not known, but it may indicate a difference in the roles they play in motoneuron regeneration. After axotomy, enzymes and other substances required for neuronal regenerated processes are increased and those related to neuronal transmission are decreased. Therefore, it is likely in the motor neurons that a-CGRP has a t r o p h i c action, while /~-CGRP is involved in neural transmission. The present study demonstrated that after crushing
injury the increase in a-CGRP mRNAs and the decrease in/~-CGRP are transient and return to control levels as the nerves regenerate, whereas after transection these changes were sustained for at least 8 weeks. Theret0re, though the functions of these peptides may be different, their expressions parallel nerve regeneration. CCK Although CCK-LI has not been detected in motoneurons 9'19, CEK mRNA has been detected in the motor nucleus of the rat trigeminal nerve16'38 and in the nucleus of the facial nerve 16 by ISHH. The length and sequence of the oligonucleotide probe we used in the present study differ from other investigators' probes, but we also found CCK mRNA in facial motoneurons in control rats. Several explanations for the disparity between immunohistoehemistry and ISiH-I findings has been discussed 16'38, and one possibility is that ISHH is more sensitive to CCK producing cells than is immunohistochemistry. However, even after colchicine treatment 9'19, and after nerve resection (our findings, data not shown) no CCK-LI positive neurons are detected. Further studies are needed to determine whether or not
192
Fig. 5. Dark-field photomicrographs showing facial motoneurons expressing CCK mRNA hybridization signals 1 day (A), 1 week (B), 2 weeks (C) and 3 weeks (D) after facial nerve crushing. Bar = 1 mm.
CCK mRNAs are translated into peptides. In this study we show that after crushing injury in facial motoneurons, CCK mRNA levels increased from 3 days to 2 weeks postoperatively and then returned to control levels as regeneration completed. After resection, the level of CCK mRNA was increased for as long as 8 weeks. After nerve crushing injury, the function of the facial nerves seemed to be restored within 2 weeks, because recovery of movement of the whiskers occurred at that time, whereas there wag ao functional recovery following nerve resection. Therefore, the increase of CCK mRNA level appears to clo~ly parallel axon regene~-ation. Considering that CCK has trophic effects on spinal motoneurons 17, this peptide may play important roles in motoneuron regeneration. Galanin There are contradictory results regarding the presence of galanin-Ll in motoneurons under normal conditions. Several authors have reported that cranial and spinal motor neurons lack galanin-LI even after colchicine injection z~'35 and during development 34. However, Marti
et al. reported that galanin-Ll is expressed transiently in spinal motoneurons during development 22. Ch'ng et al. showed that some spinal motoneurons contain galanin-LI but cranial motoneurons do not 3, whereas Moore reported that cranial motoneurons contain galanin-LI but spinal motoneurons do not ~A. We confirmed the presence of galanin-LI in both spinal and cranial motoneurons (unpublished observation). Thus, the discrepancies among immunocytochemical studies seem to be due to the sensitivity of the antisera used, and it appears that motoneurons do contain galanin-LI, though at a low level. On the other hand, recent ISHH studies failed to demonstrate galanin mRNA in motoneurons 12. In the present study, signals for galanin mRNA in motoneurons were equivalent to the background level. It is likely that the sensitivity of ISHH is still not high enough to detect galanin mRNA. Galanin has many effects on various organs ~. In an isolated newborn rat spinal cord preparation, it depresses the monosynaptic reflex, and also depresses deporalized motor neurons 4s. Galanin is colocalized with acetylcholine in ventral basal forebrain neurons and acts as an in-
193
Fig. 6. Dark-field photomicrographs showing facial m0toneurons expressing CCK mRNA hybridization signals 1 day (A): 1 week (B), 2 weeks (C) and 8 weeks (D) after facial nerve resection. Bar - I ram.
hibitory modulator of cholinergic function 4. After axotomy, galanin LI is increased in motoneurons u , and in the present study, we observed that the increased level of galanin mRNA lasted as long as 4 weeks after resection, though after crushing injury galanin mRNA disappeared 3 weeks after procedure. So, fike cholec- ystokinin (CCK), the increase of galanin m R N A level appears to parallel axon regeneration and it is suggested that galanin may play some role in motoneuron regeneration. NGF-R mRNA and NGF-R protein are expressed in motoneurons after axotomy 6"32"47 though they are not expressed under normal conditions. Therefore, NGF may take part in motoneuron regeneration, and one possibility is that it regulates neuropeptide synthesis. Much more NGF-R mRNA was expressed after crushing injury than after resection 32. On the other hand, there was more change in CCK m R N A and galanin m R N A after nerve resection than after nerve crush. The CCK mRNA level in the former group was about double that in the latter group 14 days after the procedure. The galanin mRNA level in the former group was 6-7 times that of the latter group at its peak (7 days after the procedure). These
findings do not coincide with the changing patterns of NGF-R mRNA after these procedures. Moreover, the delayed onset of the increase in CCK and galanin mRNAs after axotomy is different from the prompt and sharp iacrease of NGF-R mRNA. These findings, taken together, may indicate that expressions of these peptides are not regulated by NGF. The fact that galanin LI in sensory neurons is increased after axotomy 14'4s, in contrast to the decreased level of NGF-R, may also suppo~ this idea. In sensory neurons, NGF-R is expressed in adulthood. They are downregulated after axonal injury and are recovered after regeneration 44, which is completely opposite to the reaction observed in motoneurons. Levels of a- and/~-CGRP mRNA are also decreased after axotomy in dorsal root ganglia ~ and, in fact, it has been reported that NGF regulates expression of a-CGRP mRNA in adult sensory neurons, though its effect on the expression of [~-CGRP was not examined 21. But in motoneurons, the changing pattern of a- and ~-CGRP is so different, that the possibility that NGF regulates expression of a- and fl-CGRP is weak.
194
Fig. 7. Dark-field photomicrographs showing facial motoneurons expressing galanin mRNA on the operated (B,D) and unoperated (A,C) sides 3 days (A,B) and 2 weeks (C,D) after facial nerve resection. Bar = 1 mm.
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Days after procedure Fig. 8. Time course of changes in the percentage of facial motoneurons expressing mRNAs for a-CGRP,/~-CGRP, CCK and galanin on the axotomized side. O, nerve crush; o, nerve resection. Dotted line indicates percentages of positive neurons in control animals.
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(1991) 157-160. 33 Senba, E., Simmons, D.M., Wada, E., Wada, K. and Swanson, L.W., RNA levels of neuronal nicotinic acetylcholine receptor subunits are differently regulated in axotomized facial motoneutons: an in situ hybridization study, Mol. Brain Res., 8 (1990) 349-353. 34 Sizer, A.R., R6kaeus, A. and Foster, A., Analysis of the on-
196 togeny of galanin in the rat central nervous system by immunohistochemistry and radioimmunoassay, Int. J. Dev. Neurosci., 8 (1990) 81-97. 35 Skofitsch, G. and Jacobowitz, D.M., Immunol,~istochemical mapping of galanin-like neurons in the rat central nervous system, Peptides, 6 (1985) 509-546. 36 Skofitsch, G. and Jacobowitz, D.M., Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system, Peptides, 6 (1985) 721-745. 37 Streit, W.J., Dumoulin, F.L., Raivich, G. and Kreutzberg, G.W., Calcitonin gene-related peptide increases in the rat facial motoneurons after peripheral nerve transection, Neurosci. Lett., 101 (1989) 143-148. 38 Sutin, E.L. and Jacobowitz, D.M., Detection of CCK mRNA in the motor nucleus of the rat trigeminal nerve with in situ hybridization histochemistry, Mol. Brain Res., 3 (1990) 63-68. 39 Takami, K., Kawai, Y., Siosaka, S., Lee, Y., Girgis, S., Hillyard, C.J., Maclntyre, I., Emson, P.C. and Tohyama, M., !mmunohistochemical 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. 40 Takami, K., Kawai, Y., Uchida, S., Tohyama, M., Siotani, Y., Yoshida, H., Emson, EC., 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) 227-23O. 41 Takami, K., Hashbaoto, K., Uchida, S., Tohyama, M. and Yoshida, H., Effect of calcitonin gene-related peptide on the
cyclic AMP level of isolated mouse diaphragm, Jpn..l. Pharmacol., 42 (1986) 345-350. 42 Tetzlaff, W., Bisby, M.A. and Kreutzberg, G.W., Changes in cytoskeletal proteins in the rat facial nucleus following axotomy, J. Neurosci., 8 (1988) 3181-3189. 43 Tsujimoto, T. and Kuno, M., Calcitonin gene-related peptide prevents disuse-induced sprouting of rat motor nerve terminals, J. Neurosci., 8 (1988) 3951-3957. 44 Verge, V.M.K., Riopelle, R.J. and Richardson, EM., Nerve growth factor receptors on normal and injured sensory neurons, J. Neurosci., 9 (1989) 914-922. 45 Villar, M.J., Cortes, R., Theodorsson, E., Wiesenfeld-Hallin, Z., Schalling, M., Fahrenkrug, J., Emson, P.C. and H6kfelt, T., Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin, Neuroscience, 33 (1989) 587--604. 46 Vrontakis, M.E., Peden, L.M., Duckworth, M.L. and Friesen, H.G., Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA, J. Biol. Chem., 262 (1987) 16755-16758. 47 Wood, S.J., Prltchard, J. and Sofroniew, M.V., Re-expression of nerve growth factor receptor after axonal injury recapitulates a developmental event in motor neurons: differential regulation when regeneration is allowed or prevented, Fur. J. Neurosci., 2 (1990) 650-657. 48 Yanagisawa, M., Yagi, N., Otsuka, M., Yanaihara, C. and Yanaihara, N., Inhibitory effects of galanin on the isolated spinal cord of the newborn rat, Neurosci. Lett., 70 (1986) 278-282.
BRESM 70329
187
Research Reports
Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection Takanod Saika ~, Emiko Senba 2, Koichi Noguchi 2, Makoto Sato 2, Takeshi Kubo 1, Torn Matsunaga I and Masaya Tohyama 2 IDepartment of Otolaryngology and 2Department of Anatomy and Neuroscience, Osaka University Medical School, Osaka (Japan)
(Accepted 30 April 1991) Key words: Facial nerve; Motoneuron; Axotomy, In situ hybridization, a-Calcitonin gene-related peptide; fl-Calcitonin gene-related peptide; Cholecystokinin; Galanin
In situ hybridization histochemistry was used to study changes in mRNA,~ coding neuropeptides such as a-calcitonin gene-related peptide (CGRP),/I-CGRP, cholecystokinin (CCK) and galanin, in rat facial motoneurons following axotomy of the facial nerve. In control rats, 38%, 55% and 7% of the facial motoneurons expressed a-CGRP, fl-CGRP and CCK mRNAs, respectively. No galanin mRNA-containing motoneurons were observed in these animals. The levels of mRNA fcr c~-CGRP, CCK and galanin were increased while the fl-CGRP mRNA level was decreased after axotomy. The levels of mRNAs for these peptides returned to the control values by 2--4 weeks after nerve crush, whereas nerve resection had more prolonged effects. Within 3-4 weeks after injury, nerve resection had greater effects on fl-CGRP, CCK and galanin mRNAs than did nerve crush. Thus, there appear to be differences in the regulation of mRNA expression of these peptides in axotomized motoneurous.
INTRODUCTION
It is well known that axotomy causes a variety of morphological and chemical changes in motoneurons 2°. Enzymes and other substances related to neuronal transmission are decreased. For example, choline acetyltransferase and acetylcholine esterase are lower in rat facial nuclei after facial nerve transection u , and the level of a3 nicotinic acetylcholine receptor subunit messenger R N A ( m R N A ) is decreased in axotomized motoneurons 33. Glycine and muscarinic receptors are also depleted after injury 29-31. On the other hand, substances required for neuronal regeneration increase. For example, the synthesis of cytoskeletal proteins such as tubufin and actin is increased 26"42. Enhanced immunoreactivity for the transferrin receptor and a concomitant increase in iron uptake has also been reported n. Regarding neuropeptides, a marked increase in calcitonin gene-related peptide (CGRP) like immunoreactiv/ty (LI) 37 and C G R P m R N A 13 in the axotomized facial motoneurons has been reported. In the spinal motoneurons, it was shown that this increase results from increased levels of a - C G R P m R N A , not fl-CGRP m R N A 25. In
addition to CGRP, motoneurons contain galanin-LI, which is also i n a e a s e d after axotomy 24. Moreover, cholecystokinin (CCK) m R N A is expressed in the motor trigemina116'3s and facial nucleus 16. We have shown that nerve growth factor receptor (NGF-R) is expressed in facial motoneurons following axotomy, and that nerve crush and resection have different effects on N G F - R m R N A 32. N G F is one substance which may regulate expression of peptides in neurons 2t. Thus, it is of particular interest to clarify whether peptide production in regenerating facial motoneurons is also regulated by axonal regeneration. Furthermore, it is possible that changes in peptide production are similar to previously demonstrated changes in NGF-R mRNA. In the present study, we examined the expression of a- and fl-CGRP, galanin and CCK in facial motoneurons after crushing injury, which permits axonal regeneration, and also after resection, which prevents regeneration, using in situ hybridization histochemistry (ISHH). MATERIALS AND METHODS Animals and tissue preparation Forty-eight adult male Wistar rats weighing about 150 g were ~w,d. Under pentobarbital anesthesia (50 mg/kg, i.p.), the left fa-
Correspondence: T. Saika, Department of Anatomy and Neuroscience, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565, Japan.
188 cial nerve was exposed. At the point just distal to the posterior auricular branch, which diverges from the facial nerve as it leaves the stylomastoid foramen, the, main trunk was either crushed for 30 s with thin forceps or was transected with a pair of scissors. After transection, abot~t 5 mm of the distal nerve segment was removed. Following a postoperative interval of 1, 3, 7, 14, 21, 28, 42 (n 3), o r 56 days (n -- 2), the animals were deeply anesthetized with pentobarbital and killed. Untreated animals (n = 2) were al~o sacrificed to serve as normal controls. The brains were immediately excised and frozen with powdered dry ice. Then 20 ~m frozen sections were cut on a cryostat, mounted on gelatin- coated slictes, and subjected to in situ hybridization histoc~emisty.
Oligot~ucleotide probes The oligonucleotide probes were synthesized in an Applied Biosystems 381A DNA synthesizer and then purified using a Hitachi high pressure liquid chromatograph (ODS column chromatography). The probes were complementary to bases 664-698 of rat a--CGRP mRNA t, bases 517-550 of rat/~-CGRP mRNA*, bases ~07-342 of rat CCK mRNA 5 and bases 224-253 of rat galanin mRNA 46. The a- and ~-CGRP probes included 3~-non-coding exons unique to each mRNA, which yielded specificities of the probes. Computer assisted homology search showed that the a-CGRP probe had only 54.3% homology with the/~-CGRP mRNA sequence. The /~-CGRP probe had 50% homology with the a-CGRP mRNA sequence. This low homology prevented cross-hybridization between a- and/~-CGRP under the conditions used for washing.
In situ hybridization histochemistry These slide-mounted sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 30 rain, and rinsed 3 times (5 rain each) i~. 4 x SSC (pH 7.2) (1 x SSC contained 0.15 M sodium chloride and 0.015 M sodium citrate). The slides were then immersed in 4 x SSC and 1 x Denhardt's solution [1 x Denhardt's solution contained 0.02% bovine serum albumin (fractiost V, Sigma), 0.02% Ficoll 400 (Pharmacia), and 0.02% polyvinylpyrrolidone K-30 (Nakarai Tesque Inc.)] for I h, and dehydrated through a graded ethanol series (70-100%). The sections were treated with chloroform for 5 min to remove fat, and were immersed in 100% ethanol twice (5 min each) before being subjected to hybridization. Hybridization was performed by incubating sections with a buffer [4 x SSC, 50% deionized formamide, 0.12 M phosphate bt~er (pH 7.2), 1 x Denhardt's solution, 2.5% tRNA, 10% dextran sulfate] containing [a -35 S]dATP-labelled probes (6-9 x 106 dpndml, 0.3 ml/slide) for 1 day at 42 °C. After hybridization, the sections were rinsed in 1 x SSC (pH 7.2) for 10 min, followed by four 15 min rinses in 1 x SSC at 55 °C. The sections were then dehydrated through a graded ethanol series (70-100%) and coated with llford K-5 emulsion (diluted 1:1 with water). These sections were exposed for 4 weeks in a tightly sealed dark box at 4 °C. After being developed in D-19 (Kodak) developer, fixed with photographic fixer, and washed with tap water, the sections were counter-stained with thionin solution for morphological identification. The specificity of the hybridization was confirmed by competition experiments for each probe. In these experiments in which sections were p~hybridized with 100-fold of non-labelled probes,
Fig. 1. Dark-field photomicrographs showing facial motoneurons expressing a-CGRP mRNA hybridization signals I day (A), 1 week (B), 3 weeks (C) and 6 weeks (D) after facial nerve crushing. Bar - 1 ram.
189
Fig. 2. Dark-field photomicrographs showing facial motoneurons expressing a-CGRP mRNA hybridization signals I day (A), 1 week (B), 4 weeks (C) and 8 weeks (D) after facial nerve resection. Bar - I ram.
hybridization signals were virtually absent. Additionally, RNase A pretreatment (20 ~g/ml) just before hybridization showed no posifive signals. Computer assisted homology search also showed that these 4 probes have less than 70% homologies with any of the sequences contained in this bank.
RESULTS
gradually decreased (Fig. 1C). By the 6th week, mRNA levels had returned to the control value (Fig. 1D) and remained there at the 8th week. In the case of resection, as with crushing injury, the a-CGRP mRNA level had already increased by the first postoperative day (Fig. 2A). It remained elevated on the 3rd day, but then abruptly decreased and reached the control value one week after the procedure (Fig. 2B). From then on, mRNA increased again (Fig. 2C), and in contrast to crushing injury the increased level was maintained until at least 8 weeks after the operation (Fig. 2D).
Normal control
fl-CGRP mRNA
In the facial nucleus of control rats, 38%, 55% and 7% of motoneurons expressed a-CGRP,/I-CGRP and CCK mRNAs, respectively. Galanin mRNA was not detected.
In the case of crushing injury, the/~-CGRP mRNA level had increased by t~e first post-operative day (Fig. 3A). It then decreased aearly to zero at 3 days after the procedure (Fig. 3B). This decrease was transient, and mRNA levels were slightly increased at 7 days after the procedure (Fig. 3C). Two weeks p~t-operatively, the /~-CGRP mRNA level had returned to the control value (Fig. 3D). In the case of resection, as in crushing injury,/~-CGRP
Semi-quantitativeanalysis For semi-quantitative analysis of labelled neurons, four sections were chosen from 2 or 3 animals killed at each of the eight postsurgical intervals. Neurons with 3 times more gains than the background level were considered to be positive. The number of motoneurons was counted on Nissl-stained sections.
a-CGRP mRNA In the case of crushing injury, the a-CGRP mRNA level had already markedly increased by the first postoperative day (Fig. 1A). This was sustained at least for 2 weeks (Fig. 1B), and the a-CGRP mRNA level then
190
Fig. 3. Dark~fie|dphotomicrographsshowingfacial motoneurons on the operated side expressing/~-CGRPmRNA hybridization signals I day (A), 3 days (B), 1 week (C) and 4 weeks (D) after nerve crushing. Bar - 1 ram. mRNA was increased at 1 day after the procedure (Fig. 4A), and then decreased to zero by 3 days after the procedure (Fig. 4B). Unlike crushing injury, this lasted for 2 to 3 weeks (Fig. 4C). Three weeks after resection, the /~-CGRP mRNA level had increased but was still below the control value (Fig. 4D). CCK mRNA In the facial nucleus contralateral to the operated side, CCK mRNA positive cells were sparsely distributed throughout the nucleus. After crushing injury, there was no noticeable change in facial motoneuron CCK mRNA levels on the operated side one day after the procedure (Fig. 5A). From the 3rd day after the procedure, the level of CCK mRNA gradually increased and remained elevated for 2 weeks (Fig. 5B,C). It then decreased and returned to the control value by the 3rd week after the procedure (Fig. 5D). From then on, there was no change. In case of resection, the mRNA level was not elevated at 1 or 3 days, but then increased to a higher level than was seen after crush injury (Fig. 6A-C). In contrast to c~'ush i n j u ~ this high level of CCK mRNA was maintained for the duration of the experiment (Fig. 6D).
Galanin mRNA We found no galanin mRNA in motoneurons on the unoperated side in any animals (Fig. 7A,C). Furthermore, no mRNA was detected on the 1st day after the procedure in motoneurons on the operated side. In the case of crushing injury, a faint increase of galanin mRNA was observed from the 3 days to 2 weeks after the procedure. In the case of resection, similar to crushing injury, no mRNA was detected on the 1st day and an increase in the mRNA level wa~ observed on the 3rd day after the procedure (Fig. 7B). Still, this increase was more pronounced and prolonged than that seen after crushing injury (Fig. 7D). Even 4 weeks after the procedure the mRNA level was increased, but was less pronounced than that observed 1 or 2 weeks after the procedure. In all experimental animals, there was no changv in mRNA expression for any peptide in facial motoneurons on the unoperated side.
DISCUSSION CGRP CGRP has been detected in motoneurons by immu-
191
Fig. 4. Dark-fieldphotomicrographs showingfacial motoneurons on the operated side expressing/t-CGRP mRNA hybridization signals 1 day (A), 3 days (B), 2 weeks (C) and 4 weeks (D) after nerve resection. Bar = 1 mm.
nohistochemistry ~°'~s'36'39 and by ISHH 27. This peptide has various effects on motoneuron-muscle systems: enhancing muscle contraction with a concomitant increase in cAMP ~°'4t, increasing muscle acetylcholine receptors7,s and inhibiting the sprouting of motor axon terminals43. CGRP-LI and its mRNA level are increased after axotomy in spinal 2 and facial 13'37 motoneurons. Although a-CGRP and/~-CGRP gene sequences differ by only a single amino acid among the 37 amino acids in the rat and both mRNAs are expressed in motoneurons ~, reactions of these two mRNAs to axotomy are completely different. Transcription of a-CGRP mRNA is increased whereas that of /~-CGRP mRNA is decreased after axotomy in spinal cord 25. The physiological significance of this difference is not known, but it may indicate a difference in the roles they play in motoneuron regeneration. After axotomy, enzymes and other substances required for neuronal regenerated processes are increased and those related to neuronal transmission are decreased. Therefore, it is likely in the motor neurons that a-CGRP has a t r o p h i c action, while /~-CGRP is involved in neural transmission. The present study demonstrated that after crushing
injury the increase in a-CGRP mRNAs and the decrease in/~-CGRP are transient and return to control levels as the nerves regenerate, whereas after transection these changes were sustained for at least 8 weeks. Theret0re, though the functions of these peptides may be different, their expressions parallel nerve regeneration. CCK Although CCK-LI has not been detected in motoneurons 9'19, CEK mRNA has been detected in the motor nucleus of the rat trigeminal nerve16'38 and in the nucleus of the facial nerve 16 by ISHH. The length and sequence of the oligonucleotide probe we used in the present study differ from other investigators' probes, but we also found CCK mRNA in facial motoneurons in control rats. Several explanations for the disparity between immunohistoehemistry and ISiH-I findings has been discussed 16'38, and one possibility is that ISHH is more sensitive to CCK producing cells than is immunohistochemistry. However, even after colchicine treatment 9'19, and after nerve resection (our findings, data not shown) no CCK-LI positive neurons are detected. Further studies are needed to determine whether or not
192
Fig. 5. Dark-field photomicrographs showing facial motoneurons expressing CCK mRNA hybridization signals 1 day (A), 1 week (B), 2 weeks (C) and 3 weeks (D) after facial nerve crushing. Bar = 1 mm.
CCK mRNAs are translated into peptides. In this study we show that after crushing injury in facial motoneurons, CCK mRNA levels increased from 3 days to 2 weeks postoperatively and then returned to control levels as regeneration completed. After resection, the level of CCK mRNA was increased for as long as 8 weeks. After nerve crushing injury, the function of the facial nerves seemed to be restored within 2 weeks, because recovery of movement of the whiskers occurred at that time, whereas there wag ao functional recovery following nerve resection. Therefore, the increase of CCK mRNA level appears to clo~ly parallel axon regene~-ation. Considering that CCK has trophic effects on spinal motoneurons 17, this peptide may play important roles in motoneuron regeneration. Galanin There are contradictory results regarding the presence of galanin-Ll in motoneurons under normal conditions. Several authors have reported that cranial and spinal motor neurons lack galanin-LI even after colchicine injection z~'35 and during development 34. However, Marti
et al. reported that galanin-Ll is expressed transiently in spinal motoneurons during development 22. Ch'ng et al. showed that some spinal motoneurons contain galanin-LI but cranial motoneurons do not 3, whereas Moore reported that cranial motoneurons contain galanin-LI but spinal motoneurons do not ~A. We confirmed the presence of galanin-LI in both spinal and cranial motoneurons (unpublished observation). Thus, the discrepancies among immunocytochemical studies seem to be due to the sensitivity of the antisera used, and it appears that motoneurons do contain galanin-LI, though at a low level. On the other hand, recent ISHH studies failed to demonstrate galanin mRNA in motoneurons 12. In the present study, signals for galanin mRNA in motoneurons were equivalent to the background level. It is likely that the sensitivity of ISHH is still not high enough to detect galanin mRNA. Galanin has many effects on various organs ~. In an isolated newborn rat spinal cord preparation, it depresses the monosynaptic reflex, and also depresses deporalized motor neurons 4s. Galanin is colocalized with acetylcholine in ventral basal forebrain neurons and acts as an in-
193
Fig. 6. Dark-field photomicrographs showing facial m0toneurons expressing CCK mRNA hybridization signals 1 day (A): 1 week (B), 2 weeks (C) and 8 weeks (D) after facial nerve resection. Bar - I ram.
hibitory modulator of cholinergic function 4. After axotomy, galanin LI is increased in motoneurons u , and in the present study, we observed that the increased level of galanin mRNA lasted as long as 4 weeks after resection, though after crushing injury galanin mRNA disappeared 3 weeks after procedure. So, fike cholec- ystokinin (CCK), the increase of galanin m R N A level appears to parallel axon regeneration and it is suggested that galanin may play some role in motoneuron regeneration. NGF-R mRNA and NGF-R protein are expressed in motoneurons after axotomy 6"32"47 though they are not expressed under normal conditions. Therefore, NGF may take part in motoneuron regeneration, and one possibility is that it regulates neuropeptide synthesis. Much more NGF-R mRNA was expressed after crushing injury than after resection 32. On the other hand, there was more change in CCK m R N A and galanin m R N A after nerve resection than after nerve crush. The CCK mRNA level in the former group was about double that in the latter group 14 days after the procedure. The galanin mRNA level in the former group was 6-7 times that of the latter group at its peak (7 days after the procedure). These
findings do not coincide with the changing patterns of NGF-R mRNA after these procedures. Moreover, the delayed onset of the increase in CCK and galanin mRNAs after axotomy is different from the prompt and sharp iacrease of NGF-R mRNA. These findings, taken together, may indicate that expressions of these peptides are not regulated by NGF. The fact that galanin LI in sensory neurons is increased after axotomy 14'4s, in contrast to the decreased level of NGF-R, may also suppo~ this idea. In sensory neurons, NGF-R is expressed in adulthood. They are downregulated after axonal injury and are recovered after regeneration 44, which is completely opposite to the reaction observed in motoneurons. Levels of a- and/~-CGRP mRNA are also decreased after axotomy in dorsal root ganglia ~ and, in fact, it has been reported that NGF regulates expression of a-CGRP mRNA in adult sensory neurons, though its effect on the expression of [~-CGRP was not examined 21. But in motoneurons, the changing pattern of a- and ~-CGRP is so different, that the possibility that NGF regulates expression of a- and fl-CGRP is weak.
194
Fig. 7. Dark-field photomicrographs showing facial motoneurons expressing galanin mRNA on the operated (B,D) and unoperated (A,C) sides 3 days (A,B) and 2 weeks (C,D) after facial nerve resection. Bar = 1 mm.
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Days after procedure Fig. 8. Time course of changes in the percentage of facial motoneurons expressing mRNAs for a-CGRP,/~-CGRP, CCK and galanin on the axotomized side. O, nerve crush; o, nerve resection. Dotted line indicates percentages of positive neurons in control animals.
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