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Up-regulation of GAP-43 (B50F1 ) gene expression in vestibular efferent neurons following labyrinthectomy in the rat: In situ hybridization using an alkaline phosphatase-labeled probe
HWRMC RBMRCH ELSEVIER
Hearing Research X0 (1994) 123-127
Up-regulation of GAP-43 (B50/Fl) gene expression in vestibular efferent neurons following labyrinthectomy in the rat: In situ hybridization using an alkaline phosphatase-labeled probe Koji Ohno
‘,*, Noriaki Takeda
‘, Takeshi
Kubo ‘, Hiroshi
Kiyama h
” Department of Otolaryngology, Osaka Uniwrsity Medical School, 2-2 Yamadaoka, Suita-City, Osaku 56.5. Japan h Department of Neuroanatomy. Biomedical Research Center, Osaka Unillersity Medical School, 2-2 Yamadaoka, Srtita-City. Osaka 565, Jupan Received 18 December 1993; revised 18 June 1994; accepted
4 August
1994
Abstract Growth-associated protein (GAP)-43 plays a significant role in nerve regeneration and synaptic remodeling. We examined the profiles of GAP-43 mRNA expression in vestibular efferent neurons after labyrinthcctomy in adult rats, and clearly demonstrated that labyrinthectomy increased GAP-43 expression in these neurons. This finding suggests the ability of vestibular effercnt nerves to regenerate after nerve injury. Keywords: Regeneration;
Nerve injury; GAP-43
gent;
Inner
car
1. Introduction GAP-43 (B-50,Fl) is a membrane phosphoprotein involved in the growth, regeneration and remodeling of neuronal connections (Skene, 1989; Benowitz and Perrone-Bizzozero, 1991). It is abundant in growth cones and exhibits elevated synthesis and axonal fast-transport during nerve growth. Immunohistochemical studies have shown GAP-43 immunostaining to be very intense during the period of axonal elongation, and begin to diminish at the time of end arbor and synapse formation (Dani et al., 1990). In the adult rats, therefore, GAP-43 gene expression reaches very low levels in most parts of the brain, although there are some regions where high levels of GAP-43 mRNA have been shown to persist (Yao et al., 1993). Such profiles seem related to an ongoing potential for neuronal remodeling. In peripheral nervous systems, up-regulation of GAP-43 gene expression has been demonstrated after resection of motor and sensory nerves (Woolf et al., 1990; Chong et al., 1992; Saika et al., 1993; Kobayashi et al., 19941, and it is generally accepted that GAP-43
* Corresponding
author.
Fax: + 81 (6) 879-3229.
037%5955/94/$07.00 0 1994 Elsevier SSDI 037X-SYSS(94)00125-1
Science B.V. All rights reserved
synthesis reflects the process of nerve regeneration. Recently, we reported the occurrence of galanin peptide in vestibular efferent neurons after labyrinthectomy and speculated that galanin may participate in the process of axonal regeneration (Ohno et al., 1994). However, there is no clear evidence for the regeneration of vestibular efferent nerves after chemical labyrinthectomy. In the present study, we chose GAP43 as a marker of nerve regeneration and investigated profiles of GAP-43 mRNA expression in vestibular efferent neurons after labyrinthectomy. To detect GAP-43 mRNA, we used an alkaline phosphataselabeled oligodeoxynucleotide probe. This method gives fairly high resolution (Kiyama et al., 1990) and allows the detection of mRNA in small cell groups such as vestibular efferent neurons.
2. Materials
and methods
Twenty-eight male Wistar rats (150 g> were used in the present study. Under pentobarbital anesthesia, the right bulla tympanica was opened by the retroauricular approach, the stapes footplate was removed and a small hole drilled in the vestibule. After aspiration of labyrinth fluids from the oval window, a total of 0.6 ml of 100% ethanol was perfused into the labyrinth
124
K. 0hno et (11.,i Hearrng Keseurch NO (1994) ld?.j-127
through the oval window and the drilled hole. Following postoperative periods of 1 or 3 days, 1,2,3 or 5 weeks, the animals (4 at each time point) were deeply anesthetized with ether and sacrificed. Brains were immediately removed, frozen with powdered dry ice and 20 mm-thick frozen sections (at the level of lower brain stem) were cut on a cryostat. Sections were thaw-mounted onto gelatin-coated slides and stored at
-20°C. As a control, 4 normal rats were anesthetized and also processed as described above. To visualize hybridized GAP-43 mRNA, an alkaline phosphatase-labeled oligonucleotide probe was used, complementary in sequence to bases 136-174 of the rat GAP-43 cDNA (Karns et al., 1987). The labeling method was essentially the same as that described previously (Kiyama et al., 1990). Briefly, a modified
Fig. 1. Photomicrographs showing GAP-43 mRNA expression in vestibular efferent neurons in the control animal and in the iabyrinthectomized animals on 1, 3 day, 1,3 and 5 weeks after operation. Scale bar: 50 pm.
K. Ohno et al. / Hearing Research 80 (1994) 123-127
purine with an extra amino group at the C5 position replaced thymidine and was incorporated directly into the synthesis by a DNA synthesizer. Alkaline phosphatase was cross-linked to the modified base in the oligonucleotide probe by the homobifunctional crosslinking agent disuccinimidyl suberate (DSS). The alkaline phosphatase-conjugated probe was then purified by anion exchange chromatography using HPLC. In situ hybridization histochemistry was performed as previously described (Kiyama et al., 1990). To evaluate the changes in GAP-43 mRNA expression, the sections from animals at each time point were gathered, and the following steps were carried out at the same time. Sections were quickly dried with cool air using a hair drier, and then fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at room temperature for 20 min. The sections were rinsed twice with 0.1 M PBS (pH 7.4), incubated in proteinase K solution (5 pg/ml in 50 mM Tris-HCl and 5 mM EDTA) for 2 min, then treated with 0.25% acetic anhydride in 0.1 M triethanolamine,’ 0.9% sodium chloride for 10 min at room temperature. Sections were dehydrated through a 70%, SO%, 90% and 100% ethanol series (5 min each), defatted in chloroform (10 min) and then washed with 100% ethanol for 10 min. Sections were then dried and hybridized in hybridization buffer (4 x SSC, 45% formamide, 500 Fug/ml sheared salmon testis DNA, 10% dextran sulfate and 10 x Denhardt’s solution) containing the conjugated alkaline phosphatase-labeled oligonucleotide probe at a concentration of 5 fmol/ml. Hybridization was carried out at 37°C overnight. Hybridized sections were washed in 1 X SSC once at room temperature and 4 times at 45°C for 15 min each. After a final wash in 1 X SSC for 30 min, the buffer was changed to 0.1 M Tris-HC1/0.9% NaCl (pH 7.4) and rinsed for 10 min at room temperature. Finally, sections were preincubated in 0.1 M Tris-HCl buffer (pH 9.5) containing 0.1 M NaCl and 0.05 M MgCl, for 10 min and then incubated in the same buffer containing the chromogenic substrates nitro-blue tetrazolium (NBT 340 mg/ml, Boehringer Mannheim) and 5bromo-4-chloro-3-indolyl phosphate (BCIP 170 mg/ml, Boehringer Mannheim) for two days in a dark box. Color development was stopped by incubation for 1 h in 0.1 M Tris-HCl buffer (pH 7.5) containing 10 mM EDTA and 0.9% NaCl. The sections were coverslipped in the same buffer containing 50% glycerol. Two kinds of control experiments were carried out. We prepared several slides with sections at each time point and divided into two groups. Sections in the first group were pretreated with ribonuclease A (RNase A) to digest any RNA signal on the section to check for non-specific binding to the tissue. Sections were incubated with RNase A (20 Fg/ml) before hybridization for 30 min at room temperature, and then taken through the same procedure of hybridization, wash and
125
visualization. Sections in the second group were used for a competition control with excess unlabeled probe. The excess unlabeled probe (5 pmol/pl) was added to the hybridization buffer together along with the alkaline phosphatase labeled probe (5 fmol/pl). These two control procedures abolished all specific mRNA signals.
3. Results Labyrinthectomy induced the alkaline phosphatase reaction in the neuron group dorsolateral to the facial genu. In the control animals, no reaction was seen in this area (Fig. 1A). However, on the 1st day after lesion, faint signals appeared (Fig. 1B) and became noticeable on postoperative day 3 (Fig. 10. Such high signals were sustained for at least 3 weeks postoperatively (Fig. lD,E,F). The products of the alkaline phosphatase reaction filled the cytoplasm, and comparatively strong reactions could be seen in parts of the cell somata. Although the signals persisted until the end of our observation period, the strength of the alkaline phosphatase reaction slightly decreased after 3 weeks (Fig. 1F). No positive signals were observed in dendrites or axons throughout the present study. After labyrinthectomy, GAP-43 positive neurons occurred also in the area dorsomedial to the facial genu at each time point after 3 days. However, the number of positive neurons was less than 2 per a section, and we could not evaluate the profile of GAP-43 signal in this area exactly among the stages.
4. Discussion The location of vestibular efferent neurons was investigated previously in the rat (White and Warr, 1983; Schwarz et al., 1986). In the present study, neurons showing GAP-43 mRNA were found in the area dorsolateral and dorsomedial to the facial nucleus where vestibular efferent neurons are located. The shape and size of the GAP-43 positive neurons coincided well with those of vestibular efferent neurons reported before (Ohno et al., 1991), and we therefore determined that these labeled neurons were vestibular efferent neurons. The re-emergence of GAP-43 expression after nerve injury has been observed in a variety of peripheral nervous systems (Woolf et al., 1990; Doster et al., 1991; Chong et al., 1992; Saika et al.. 1993; Kobayashi et al., 19941, and this cellular response is considered a characteristic feature of axonal regeneration. In the present study, we showed that GAP-43 mRNA expression appears by one day and is sustained for at least 5 weeks in vestibular efferent neurons after labyrinthectomy.
126
K. Ohno ct ~1./ Heanng Research 80 (14941 123-127
This observation suggests that vestibular efferent nerves form growth cones and elongate after labyrinthectomy. Although the correlation of increased synthesis and fast-transport of GAP-43 with neuronal growth during nerve regeneration has been established (Skene, 1989; Benowitz and Perrone-Bizzozero, 19911, the role of GAP-43 in neural outgrowth is still obscure. Previous studies presented evidence suggestive of some possible functions, for example, interaction with calmodulin (Alexander et al., 19871, Go activation (Strittmatter et al., 1991), phospholipid metabolism (VanHoof et al., 1988) and the membrane cytoskeleton (Meiri and Gordon-weeks, 1990). However, little is known about the interrelationship of these molecular mechanisms. The expression of GAP-43 mRNA persisted in the vestibular efferent neurons until1 5 weeks postoperatively, suggesting that vestibular efferent nerves could not re-establish contact with their targets; sensory cells and afferent nerves. This seems reasonable, because labyrinthectomy could damage these target structures as well as efferent nerve fibers. However, it was unclear why GAP-43 mRNA expression showed a slight decrease after 3 weeks postoperatively. Fine structural changes in transsected vestibular nerves have been investigated previously (Gleisner and Wersall, 1975); afferent nerve endings were found only 7 days after transsection, whereas the appearance of efferent nerve endings with synaptic vesicles was comparatively late and first seen at about 5 weeks postoperatively. Taken together, the decline in GAP-43 mRNA expression might indicate the reinnervation of some efferent nerve fibers in the periphery where damage to the target structures was sufficiently slight to allow repair. To further investigate this speculation, studies will be needed to ascertain fine structural changes in the inner ear after ethanol injection. We emphasize the substantial expression of GAP-43 mRNA in vestibular efferent neurons at 5 weeks postoperatively, which probably reflected the irretrievable changes in most parts of vestibular sensory epithelia. Gene expression of GAP-43 appears to be regulated by an inhibitory retrograde-transported factor (Bisby, 1988). Vinblastine, which depolymerizes microtubules and disturbs axonal transport, induced GAP-43 mRNA expression in dorsal root ganglia (Benowitz and Perrone-Bizzozero, 1991) and the hypoglossal nucleus (Kobayashi et al., 1994) when applied to intact peripheral axons. Similarly, some neuropeptides (e.g. CGRP and galanin) were also up-regulated after axotomy (Villar et al., 1989; Saika et al., 1991) and vinblastine treatment (Kashiba et al., 1992), and their neurotrophic effects have been proposed (H&felt, 1991). Recently, we reported the occurrence of galanin-like immunoreactivity (GAL-IR) in this system after labyrinthectomy (Ohno et al., 19941. GAL-IR was first detected at postoperative day 3, reached its peak be-
tween 1 and 2 weeks and thereafter gradually decreased. In contrast to GAP-43 gene expression, the relatively short duration of GAL-IR indicates that although transcriptional regulation of each gene seems related to nerve injury, the regulating factors are different.
Acknowledgments
The authors wish to thank Prof. M. Tohyama for helpful suggestions. This study was supported by a Fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists.
References Alexander, K.A., Cimler, B.M., Meier, K.E. and Storm, D.R. (1987) Regulation of calmodulin binding to P-S7. _I. Biol. Chem. 263. 75447549. Benowitz, L.I. and Perrone-Bizzozero, NJ. (1991) The expression of GAP-43 in relation to neuronal growth and plasticity: when. where, how, and why? Prop. Brain Res. 89, 69-87. Bisby, M.A. (1988) Dependence of GAP-43 (BSO, Fl) transport on axonal regeneration in rat dorsal root ganglion neurons. Brain Res. 458, 157-161. Chong, M.S., Fitzgerald, M., Winter, J., Hu-Tsai, M., Emson, P.C‘.. Wiese, U. and Woolf, C.J. (lY92) GAP-43 mRNA in rat spinal cord and dorsal root ganglia neurons: developmental changes and re-expression following peripheral nerve injury. Eur. J. Neuroscr. 4, 883-895. Dani, J.W., Armstrong, D.W. and Benowitz, L.I. (IYYO)Mapping the development of the rat brain by GAP-43 immunohistochemistry. Neuroscience 40, 277-288. Doster, SK., Lozano, A.M., Aguayo, A.J. and Willard, M.B. tlY91) Expression of the growth-associated protein GAP-43 in adult rat retinal ganglion cells following axon injury. Neuron 6. 6X-647. Gleisner, L. and Werslll, J. (1975) Experimental studies on the nerve-sensory cell relationship during degeneration and regencration in ampullar nerves of the frog labyrinth. Acta Gtolaryngol. Suppl. 333, l-28. Hokfelt, T. (1991) Neuropeptides in perspective: the last ten year?;. Neuron 7, 867-879. Karns, L.R., Ng, S.C., Freeman, J.A. and Fishman. M.C. (19x7) Cloning of complementary DNA for GAP-43. a neuronal growth related protein. Science 236, 5Y7-600. Kashiba, H., Senba, E., Kawai, Y., Ueda, Y. and Tohyama. M. (lYY2) Axonal blockade induces the expression of vasoactive intestinal peptide and galanin in rat dorsal root ganglion neurons. Brain Res. 577, 19-28. Kiyama, H., Emson, PC. and Tohyama, M. (LYYOJRecent progress in the use of the technique of non-radioactive in situ hybridization histochemistry: new tools for molecular neurobiology. Ncurosci. Res. 9, 1-21. Kobayashi, N., Kiyama, H. and Tohyama, M. (1993) GAP-43 (B50/Fl) gene regulation by axonal injury of the hypoglossal nerve in the adult rat. Mol. Brain Res. 21. 9- 12. Meiri, K.F. and Gordon-Weeks. P.R. (19uOJGAP-43 in growth cones is associated with areas of membrane that arc tightly bound to substrate and is a component membrane skeleton subcellular fraction. J. Neurosci. 10, 256-266.
K. Ohno et al. /Hearing Ohno, K., Takeda, N., Yamano, M., Matsunaga, T. and Tohyama, M. (1991) Coexistence of acetylcholine and calcitonin gene-related peptide in the vestibular efferent neurons in the rat. Brain Res. 566, 103-107. Ohno, K., Takeda. N., Kiyama H., Kubo T. and Tohyama, M. (19Y4) Occurrence of galanin-like immunoreactivity in vestibular and cochlear efferent neurons after labyrinthectomy in the rat. Brain Res. 644. 1355143. Saika, T.. Senba, E.. Noguchi, K., Sato, M., Kubo, T., Matsunaga, T. and Tohyama. M. (1991) Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection. Mol. Brain Res. I I. 187-196. Saika, T., Kiyama, H., Tohyama. M. and Matsunaga, T. (1903) GAP-43 mRNA expression in facial motoneurons during regeneration: in situ hybridization histochemistry study using alkaline phosphatase-labelled probe. Acta Otolaryngol. Suppl. 501, 80-84. Schwarz. D.W.F., Satoh, K., Schwarz, I.E., Hu, K. and Fibiger, H.C. (1986) Cholinergic innervation of the rat’s labyrinth. Exp. Brain Res. 64. 10-26. Skene. J.Ll.P. (1989) Axonal growth-associated proteins. Annu. Rev. Neurosci. I?, 127-156.
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Strittmatter, S.M., Valenzuela, D.. Sudo, Y., Linder. M.E. and Fishman, M.C. (199 I) An intracellular guanine nucleotide release protein for Go. GAP-43. J. Biol. Chem. 266, 22465-22471. VanHoof, CO., DeGraan, P.N., Oestreicher. A.B. and Gispen, W.H. (1988) B-50 phosphorylation and phosphatide metabolism in nerve growth cone membranes. J. Neurobiol. X. 17855 1795. Villar, M.J.. Cartes, R.. Theodorson. E., Wiesenfeld-Hallin. 2.. Schalling, M., Fahrenkrug, J.. Emson, P.C. and Hokfelt, T. (lY8Y) Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience 33. 587-604. White, J.S. and Warr. W.B. (1983) The dual origins of the olivocochlear bundle in the albino rat. J. Comp. Neurol. 2lY. 203-214. Woolf. C.J.. Molander, C., Reynolds. M. and Benowitz, L.I. (IYYO) GAP-43 appears in the rat dorsal horn following peripheral nerve injury. Neuroscience 34, 465-478. Yao. G.L., Kiyama. H. and Tohyama, M. (19Y3) Distribution of GAP-43 (BSO/FlJ mRNA in the adult brain by in situ hyhridization using an alkaline phosphatase labeled probe. Mol. Brain Res. 1X, l-16.
Hearing Research X0 (1994) 123-127
Up-regulation of GAP-43 (B50/Fl) gene expression in vestibular efferent neurons following labyrinthectomy in the rat: In situ hybridization using an alkaline phosphatase-labeled probe Koji Ohno
‘,*, Noriaki Takeda
‘, Takeshi
Kubo ‘, Hiroshi
Kiyama h
” Department of Otolaryngology, Osaka Uniwrsity Medical School, 2-2 Yamadaoka, Suita-City, Osaku 56.5. Japan h Department of Neuroanatomy. Biomedical Research Center, Osaka Unillersity Medical School, 2-2 Yamadaoka, Srtita-City. Osaka 565, Jupan Received 18 December 1993; revised 18 June 1994; accepted
4 August
1994
Abstract Growth-associated protein (GAP)-43 plays a significant role in nerve regeneration and synaptic remodeling. We examined the profiles of GAP-43 mRNA expression in vestibular efferent neurons after labyrinthcctomy in adult rats, and clearly demonstrated that labyrinthectomy increased GAP-43 expression in these neurons. This finding suggests the ability of vestibular effercnt nerves to regenerate after nerve injury. Keywords: Regeneration;
Nerve injury; GAP-43
gent;
Inner
car
1. Introduction GAP-43 (B-50,Fl) is a membrane phosphoprotein involved in the growth, regeneration and remodeling of neuronal connections (Skene, 1989; Benowitz and Perrone-Bizzozero, 1991). It is abundant in growth cones and exhibits elevated synthesis and axonal fast-transport during nerve growth. Immunohistochemical studies have shown GAP-43 immunostaining to be very intense during the period of axonal elongation, and begin to diminish at the time of end arbor and synapse formation (Dani et al., 1990). In the adult rats, therefore, GAP-43 gene expression reaches very low levels in most parts of the brain, although there are some regions where high levels of GAP-43 mRNA have been shown to persist (Yao et al., 1993). Such profiles seem related to an ongoing potential for neuronal remodeling. In peripheral nervous systems, up-regulation of GAP-43 gene expression has been demonstrated after resection of motor and sensory nerves (Woolf et al., 1990; Chong et al., 1992; Saika et al., 1993; Kobayashi et al., 19941, and it is generally accepted that GAP-43
* Corresponding
author.
Fax: + 81 (6) 879-3229.
037%5955/94/$07.00 0 1994 Elsevier SSDI 037X-SYSS(94)00125-1
Science B.V. All rights reserved
synthesis reflects the process of nerve regeneration. Recently, we reported the occurrence of galanin peptide in vestibular efferent neurons after labyrinthectomy and speculated that galanin may participate in the process of axonal regeneration (Ohno et al., 1994). However, there is no clear evidence for the regeneration of vestibular efferent nerves after chemical labyrinthectomy. In the present study, we chose GAP43 as a marker of nerve regeneration and investigated profiles of GAP-43 mRNA expression in vestibular efferent neurons after labyrinthectomy. To detect GAP-43 mRNA, we used an alkaline phosphataselabeled oligodeoxynucleotide probe. This method gives fairly high resolution (Kiyama et al., 1990) and allows the detection of mRNA in small cell groups such as vestibular efferent neurons.
2. Materials
and methods
Twenty-eight male Wistar rats (150 g> were used in the present study. Under pentobarbital anesthesia, the right bulla tympanica was opened by the retroauricular approach, the stapes footplate was removed and a small hole drilled in the vestibule. After aspiration of labyrinth fluids from the oval window, a total of 0.6 ml of 100% ethanol was perfused into the labyrinth
124
K. 0hno et (11.,i Hearrng Keseurch NO (1994) ld?.j-127
through the oval window and the drilled hole. Following postoperative periods of 1 or 3 days, 1,2,3 or 5 weeks, the animals (4 at each time point) were deeply anesthetized with ether and sacrificed. Brains were immediately removed, frozen with powdered dry ice and 20 mm-thick frozen sections (at the level of lower brain stem) were cut on a cryostat. Sections were thaw-mounted onto gelatin-coated slides and stored at
-20°C. As a control, 4 normal rats were anesthetized and also processed as described above. To visualize hybridized GAP-43 mRNA, an alkaline phosphatase-labeled oligonucleotide probe was used, complementary in sequence to bases 136-174 of the rat GAP-43 cDNA (Karns et al., 1987). The labeling method was essentially the same as that described previously (Kiyama et al., 1990). Briefly, a modified
Fig. 1. Photomicrographs showing GAP-43 mRNA expression in vestibular efferent neurons in the control animal and in the iabyrinthectomized animals on 1, 3 day, 1,3 and 5 weeks after operation. Scale bar: 50 pm.
K. Ohno et al. / Hearing Research 80 (1994) 123-127
purine with an extra amino group at the C5 position replaced thymidine and was incorporated directly into the synthesis by a DNA synthesizer. Alkaline phosphatase was cross-linked to the modified base in the oligonucleotide probe by the homobifunctional crosslinking agent disuccinimidyl suberate (DSS). The alkaline phosphatase-conjugated probe was then purified by anion exchange chromatography using HPLC. In situ hybridization histochemistry was performed as previously described (Kiyama et al., 1990). To evaluate the changes in GAP-43 mRNA expression, the sections from animals at each time point were gathered, and the following steps were carried out at the same time. Sections were quickly dried with cool air using a hair drier, and then fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at room temperature for 20 min. The sections were rinsed twice with 0.1 M PBS (pH 7.4), incubated in proteinase K solution (5 pg/ml in 50 mM Tris-HCl and 5 mM EDTA) for 2 min, then treated with 0.25% acetic anhydride in 0.1 M triethanolamine,’ 0.9% sodium chloride for 10 min at room temperature. Sections were dehydrated through a 70%, SO%, 90% and 100% ethanol series (5 min each), defatted in chloroform (10 min) and then washed with 100% ethanol for 10 min. Sections were then dried and hybridized in hybridization buffer (4 x SSC, 45% formamide, 500 Fug/ml sheared salmon testis DNA, 10% dextran sulfate and 10 x Denhardt’s solution) containing the conjugated alkaline phosphatase-labeled oligonucleotide probe at a concentration of 5 fmol/ml. Hybridization was carried out at 37°C overnight. Hybridized sections were washed in 1 X SSC once at room temperature and 4 times at 45°C for 15 min each. After a final wash in 1 X SSC for 30 min, the buffer was changed to 0.1 M Tris-HC1/0.9% NaCl (pH 7.4) and rinsed for 10 min at room temperature. Finally, sections were preincubated in 0.1 M Tris-HCl buffer (pH 9.5) containing 0.1 M NaCl and 0.05 M MgCl, for 10 min and then incubated in the same buffer containing the chromogenic substrates nitro-blue tetrazolium (NBT 340 mg/ml, Boehringer Mannheim) and 5bromo-4-chloro-3-indolyl phosphate (BCIP 170 mg/ml, Boehringer Mannheim) for two days in a dark box. Color development was stopped by incubation for 1 h in 0.1 M Tris-HCl buffer (pH 7.5) containing 10 mM EDTA and 0.9% NaCl. The sections were coverslipped in the same buffer containing 50% glycerol. Two kinds of control experiments were carried out. We prepared several slides with sections at each time point and divided into two groups. Sections in the first group were pretreated with ribonuclease A (RNase A) to digest any RNA signal on the section to check for non-specific binding to the tissue. Sections were incubated with RNase A (20 Fg/ml) before hybridization for 30 min at room temperature, and then taken through the same procedure of hybridization, wash and
125
visualization. Sections in the second group were used for a competition control with excess unlabeled probe. The excess unlabeled probe (5 pmol/pl) was added to the hybridization buffer together along with the alkaline phosphatase labeled probe (5 fmol/pl). These two control procedures abolished all specific mRNA signals.
3. Results Labyrinthectomy induced the alkaline phosphatase reaction in the neuron group dorsolateral to the facial genu. In the control animals, no reaction was seen in this area (Fig. 1A). However, on the 1st day after lesion, faint signals appeared (Fig. 1B) and became noticeable on postoperative day 3 (Fig. 10. Such high signals were sustained for at least 3 weeks postoperatively (Fig. lD,E,F). The products of the alkaline phosphatase reaction filled the cytoplasm, and comparatively strong reactions could be seen in parts of the cell somata. Although the signals persisted until the end of our observation period, the strength of the alkaline phosphatase reaction slightly decreased after 3 weeks (Fig. 1F). No positive signals were observed in dendrites or axons throughout the present study. After labyrinthectomy, GAP-43 positive neurons occurred also in the area dorsomedial to the facial genu at each time point after 3 days. However, the number of positive neurons was less than 2 per a section, and we could not evaluate the profile of GAP-43 signal in this area exactly among the stages.
4. Discussion The location of vestibular efferent neurons was investigated previously in the rat (White and Warr, 1983; Schwarz et al., 1986). In the present study, neurons showing GAP-43 mRNA were found in the area dorsolateral and dorsomedial to the facial nucleus where vestibular efferent neurons are located. The shape and size of the GAP-43 positive neurons coincided well with those of vestibular efferent neurons reported before (Ohno et al., 1991), and we therefore determined that these labeled neurons were vestibular efferent neurons. The re-emergence of GAP-43 expression after nerve injury has been observed in a variety of peripheral nervous systems (Woolf et al., 1990; Doster et al., 1991; Chong et al., 1992; Saika et al.. 1993; Kobayashi et al., 19941, and this cellular response is considered a characteristic feature of axonal regeneration. In the present study, we showed that GAP-43 mRNA expression appears by one day and is sustained for at least 5 weeks in vestibular efferent neurons after labyrinthectomy.
126
K. Ohno ct ~1./ Heanng Research 80 (14941 123-127
This observation suggests that vestibular efferent nerves form growth cones and elongate after labyrinthectomy. Although the correlation of increased synthesis and fast-transport of GAP-43 with neuronal growth during nerve regeneration has been established (Skene, 1989; Benowitz and Perrone-Bizzozero, 19911, the role of GAP-43 in neural outgrowth is still obscure. Previous studies presented evidence suggestive of some possible functions, for example, interaction with calmodulin (Alexander et al., 19871, Go activation (Strittmatter et al., 1991), phospholipid metabolism (VanHoof et al., 1988) and the membrane cytoskeleton (Meiri and Gordon-weeks, 1990). However, little is known about the interrelationship of these molecular mechanisms. The expression of GAP-43 mRNA persisted in the vestibular efferent neurons until1 5 weeks postoperatively, suggesting that vestibular efferent nerves could not re-establish contact with their targets; sensory cells and afferent nerves. This seems reasonable, because labyrinthectomy could damage these target structures as well as efferent nerve fibers. However, it was unclear why GAP-43 mRNA expression showed a slight decrease after 3 weeks postoperatively. Fine structural changes in transsected vestibular nerves have been investigated previously (Gleisner and Wersall, 1975); afferent nerve endings were found only 7 days after transsection, whereas the appearance of efferent nerve endings with synaptic vesicles was comparatively late and first seen at about 5 weeks postoperatively. Taken together, the decline in GAP-43 mRNA expression might indicate the reinnervation of some efferent nerve fibers in the periphery where damage to the target structures was sufficiently slight to allow repair. To further investigate this speculation, studies will be needed to ascertain fine structural changes in the inner ear after ethanol injection. We emphasize the substantial expression of GAP-43 mRNA in vestibular efferent neurons at 5 weeks postoperatively, which probably reflected the irretrievable changes in most parts of vestibular sensory epithelia. Gene expression of GAP-43 appears to be regulated by an inhibitory retrograde-transported factor (Bisby, 1988). Vinblastine, which depolymerizes microtubules and disturbs axonal transport, induced GAP-43 mRNA expression in dorsal root ganglia (Benowitz and Perrone-Bizzozero, 1991) and the hypoglossal nucleus (Kobayashi et al., 1994) when applied to intact peripheral axons. Similarly, some neuropeptides (e.g. CGRP and galanin) were also up-regulated after axotomy (Villar et al., 1989; Saika et al., 1991) and vinblastine treatment (Kashiba et al., 1992), and their neurotrophic effects have been proposed (H&felt, 1991). Recently, we reported the occurrence of galanin-like immunoreactivity (GAL-IR) in this system after labyrinthectomy (Ohno et al., 19941. GAL-IR was first detected at postoperative day 3, reached its peak be-
tween 1 and 2 weeks and thereafter gradually decreased. In contrast to GAP-43 gene expression, the relatively short duration of GAL-IR indicates that although transcriptional regulation of each gene seems related to nerve injury, the regulating factors are different.
Acknowledgments
The authors wish to thank Prof. M. Tohyama for helpful suggestions. This study was supported by a Fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists.
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