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Evidence for the presence of l -arginine in the glial components of the peripheral nervous system
Brain Research, 559 (1991) 159-162 ~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124843D
159
BRES 24843
Evidence for the presence of --arginine in the gllal components of the peripheral nervous system Eiko Aoki, Reiji Semba and Shigeo Kashiwamata Department of Perinatology, Institute for Developmental Research, Aichi Prefecture Colony, Aichi (Japan) (Accepted 18 June 1991) Key words: Arginine; Nitric oxide; Dorsal root ganglion; Superior cervical ganglion; Cochlear ganglion; Enteric plexus; Immunocytochemistry
L-Arglnine is a precursor of nitric oxide that has been identified as an endogenous activator of soluble guanylate cyclase. We have recently reported the immunocytochemicallocalization of free L-arginine in glial cells in the central nervous system (CNS) using specific anti-arginine antibody. In the present study, we focused our attention on this particular amino acid in the peripheral nervous system (PNS). In the cochlea of the inner ear, arginine-like hnmunoreactivity was localized in satellite cells surrounding neurons of the spiral ganglion. In the dorsal root ganglia, satellite cells surrounding sensory neurons were found to be immunoreactive. In the superior cervical ganglion, L-arglulne was concentrated in satellite cells around neuronal cells. In ganglia of the enteric plexus, supporting cells that covered neuronal cells were stained. These results show that free L-arginine in the PNS is concentrated in satellite and supporting cells, both of which correspond to gliai cells in the CNS. Thus, those cells in ganglia of the PNS may support and/or control the neural activity by providing L-arginine to the neurons that they surround. Nitric oxide (NO) is considered to play an important role as a messenger molecule in the brain, as well as in the blood vessels, macrophages and neutrophiles 2's'9'1214. NO has been shown to be formed from L-arginine 12A3 by an enzyme, N O synthase 34, which is localized not only in the above mentioned tissues and cells but also in neurons of the myenteric plexus of the intestine 4'17. It has been also reported that NO is released upon stimulation of the non-adrenergic non-cholinergic nerves in the canine ileocolonic junction 5. These reports suggest the presence of an L-arginine-NO pathway in the peripheral nervous system (PNS) including the myenteric plexus. However, it is unknown how L-arginine, a precursor of NO, is supplied to the pathway. On the other hand, we raised specific anti-arginine antibody in guinea pigs and reported the immunocytochemical distribution of free L-arginine in the rat brain 1. A main locus of L-arginine in the brain was found to be astrocytes and a close correlation between neurons and astrocytes in the L-arginine-NO pathway was suggested. In the present study, we focused our attention on the localization of free L-arginine in the PNS including the myenteric plexus. Anti-arginine antibody was prepared as described before 1. Briefly, L-arginine was coupled with guinea pig serum albumin (GPSA) via glutaraldehyde (GAL) and the L-arginine-GAL-GPSA conjugate was emulsified with
an equal volume of complete Freund's adjuvant and was repeatedly injected intracutaneously into multiple sites on the back of the guinea pig. Anti-arginine antibody was purified by affinity chromatography with L-arginine immobilized on formyl ceUulofme (Seikagaku Kogyo, Japan). Specificity of the antibody was checked by a dotimmunobinding assay 1°, and the purified anti-arginine antibody was proved to be specific to the L-arginineG A L - G P S A complex 1. Anti-S-100 protein antibody was a generous gift from Dr. K. Kato of our institute. Preparation and specificity of this antibody have been described elsewhere 11. Rats of Sprague-Dawley strain of 2 months old were perfused via the heart with a mixture of 1% G A L , 4% formaldehyde, 0.2% pierie acid and 2% sucrose in 0.1 M sodium acetate buffer, p H 6.0 is. Each organ was left in the above mixture for 4-5 h, rinsed several times with 50 mM Tris-HCl buffer, p H 7.6, dehydrated with graded alcohols and embedded in paraffin. In the case of the cochlea, fixed samples were treated with sodium borohydride solution (0.4 mg/ml in final concentration) for 2 h at pH 8.8, decalcified with 20 vols. of 5% nitric acid for 16 h and processed for paraffin embedding. Sections were incubated with the purified antibody (dilution 1:500), and left overnight at room temperature. The location of arginine was visualized by the peroxidase-anti-
Correspondence: E. Aoki, Department of Perinatology, Institute for Developmental Research, Aiehi Prefecture Colony, Kasugai, Aichi 480-03, Japan.
160
Fig. 1. Photographs showing the distribution of arginine- and S-100 protein-like immunoreactivities in the cochlear ganglion of a rat. Satellite cells (arrowheads) surrounding ganglion cells (g) are immunolabeled with the anti-arginine (A) and anti-S-100 protein (B) antibodies. Bar = 20/~m.
peroxidase method 19 with diaminobenzidine as chromogen. Control sections incubated with non-immune guinea pig serum showed no positive staining. We investigated the localization of arginine-like immunoreactivity in the cochlear ganglion of the 8th cranial nerve, the spinal dorsal root ganglion, and the superior cervical ganglion and enteric plexus of the autonomic nervous system. In the cochlear ganglion, satellite cells around neurons were strongly immunolabeled with the anti-arginine antibody (Fig. 1A). The labeling pattern was very similar to that obtained with the antiS-100 protein antibody (Fig. 1B). Fig. 2 shows immunocytochemistry of the dorsal root ganglion. The perikarya and processes of satellite cells were stained with the antiarginine antibody (Fig. 2A) as well as with the anti-S100 protein antibody (Fig. 2B). In the superior cervical ganglion, both of the antibodies stained the surroundings
of ganglion cells (Fig. 3A, B). In the Auerbach's and Meissner's plexus of the intestine, arginine and S-100 protein were found to be highly concentrated in supporting cells (Fig. 4 A - D ) . It is known that structures of the sensory and sympathetic ganglia are microscopically similar to each other and that nerve cells in the ganglia are ensheathed by nucleated membranous capsules, which are formed of satellite cells TM. In the enteric plexus, neuronal cells are ensheathed by supporting cells. These satellite and supporting cells are known to contain S-100 protein 6'7 like the case of glial cells in the CNS. Thus immunolabeling with the anti-S-100 antibody was helpful in defining arginine-positive structures as satellite and supporting cells. Our previous immunocytochemical study of free L-arginine in the rat brain showed its predominant localization in glial cells, particularly in astrocytes 1. Because
Fig. 2. Photographs of arginine- and S-100 protein-like immunoreactivities in the rat spinal dorsal root ganglion. A: satellite cells (arrowheads) around ganglion cells (g) were clearly stained with the anti-arglnine antibody. B: satellite cells (arrowheads) and dorsal root ganglion cells (g) are stained with the anti-S-100 protein antibody. Bar = 100/~m.
161
Fig. 3. Photographs showing the localization of arginine- and S-100 protein-like immunoreactivities in the superior cervical ganglion of a rat. The surroundings (arrowheads) of ganglion cells (g) are labeled with the anti-arginine (A) and anti-S-100 protein (B) antibodies in the sympathetic ganglion. Bar = 100/~m.
N O synthase was localized in neurons, it was considered that in the brain L-arginine was released from astrocytes, taken up by adjacent neurons and then cleaved to form NO. In the present study, the localization of free L-arginine was examined in the sensory ganglia and autonomic nervous tissues, and arginine-like immunoreactivity was found to be selectively concentrated in the surroundings of ganglion cells. On the other hand, in the duodenum, intense immunoreactivity of N O synthase has been proved in neuronal cells of the myenteric plexus 4. Thus, it seems likely that free L-arginine is released from satellite and supporting cells, incorporated into adjacent neuronal cells and finally cleaved to form N O by N O
synthase, though the presence of N O synthase has not been reported so far in the PNS except for the myenteric plexus 4. Moreover, satellite and supporting cells may possibly control the supply of L-arginine to neurons and influence the neural activity. In conclusion, it is suggested that free L-arginine in the PNS is concentrated in satellite and supporting cells which correspond to glial cells in the CNS and that neurons are surrounded by such an L-arginine pool. These cells could have an intracellular organization to maintain a high level of free L-arginine, which is supplied to neuronal ceils and influences their activity through the metabolic conversion to NO.
~ ~ ~ii~ii!i~
Fig. 4. Photographs demonstrating the arginine- and S-100 protein-like immunoreactivities in the enteric plexus of a rat. In the Auerbach's plexus, supporting cells which cover neuronal cells (arrows) are strongly stained with the anti-arginine (A) and anti-S-100 (B) antibodies. In the Meissner's plexus, the anti-arginine (C) and anti-S-100 (D) antibodies give positive reactions to supporting cells. Bar = 20/~m.
162 A part of this research was supported by Grant (63-A) from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare and also by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Science and Culture (02670779), Japan.
1 Aoki, E., Semba, R., Mikoshiba, K. and Kashiwamata, S., Predominant localization in glial cells of free L-arginine. Immunocytochemical evidence, Brain Research, 547 (1991) 190-192. 2 Bredt, D.S. and Snyder, S.H., Nitric Oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9030-9033. 3 Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 682-685. 4 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. 5 Bult, H., Boeckxstaens, G.E., Pelckmans, EA., Jordaens, EH., Van Maercke, Y.M. and Herman, A.G., Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature, 345 (1990) 346-347. 6 Cocchia, D. and Michetti, E, S-100 antigen in satellite cells of the adrenal medulla and the superior cervical ganglion of the rat, Cell Tissue Res., 215 (1981) 103-112. 7 Ferri, G.-L., Probert, L., Cocchia, D., Michetti, E, Marangos, P.J. and Polak, J.M., Evidence for the presence of S-100 protein in the glial component of the human enteric nervous system, Nature, 297 (1982) 409-410. 8 Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci. 14 (1991) 60-67. 9 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intracellular messenger in the brain, Nature, 336 (1988) 385-387. 10 Hawkes, R., Niday, E. and Gordon, J., A dot-immunobinding assay for monoclonal and other antibodies, Anal. Biochem., 119
(1982) 142-147. 11 Kimura, S., Kato, K., Semba, R. and Isobe, T., Regional distrihution of S-100a0(aa ), S-100a(a~) and S-100b(flfl) in the bovine central nervous tissue determined with a sensitive enzyme immunoassay system, Neurochem. Int., 6 (1984) 513-518. 12 Knowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5159-5162. 13 Palmer, R.M.J., Ashton, D.S. and Moncada, S., Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature, 333 (1988) 664-666. 14 Palmer, R.M.J., Ferrige, A.G. and Moncada, S., Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature, 327 (1987) 524-526. 15 Pick, J., The Autonomic Nervous System; Morphological, Comparative, Clinicaland Surgical Aspects, Lippincott, Philadelphia, 1970, 483pp. 16 Ranson, S.W. and Clark, S.L., The Anatomy of the Nervous System; its Development and Function, 10th edn., Saunders, Philadelphia, 1959, 622pp. 17 Ross, C.A., Bredt, D. and Snyder, S.H., Messenger molecules in the cerebellum, 7~ends Neurosci., 13 (1990) 216-222. 18 Schmechel, D.E., Brightman, M.W. and Marangos, EJ., Neurons switch from non-neuronal enolasc to neuron-specific enolase during differentiation, Brain Research, 190 (1980) 195-214. 19 Sternberger, L.A., The unlabeled antibody enzyme method. In L.A. Sternberger (Ed.), lmmunocytochemistry, Prentice-Hall, Englewood Cliffs, 1974, pp. 129-171.
159
BRES 24843
Evidence for the presence of --arginine in the gllal components of the peripheral nervous system Eiko Aoki, Reiji Semba and Shigeo Kashiwamata Department of Perinatology, Institute for Developmental Research, Aichi Prefecture Colony, Aichi (Japan) (Accepted 18 June 1991) Key words: Arginine; Nitric oxide; Dorsal root ganglion; Superior cervical ganglion; Cochlear ganglion; Enteric plexus; Immunocytochemistry
L-Arglnine is a precursor of nitric oxide that has been identified as an endogenous activator of soluble guanylate cyclase. We have recently reported the immunocytochemicallocalization of free L-arginine in glial cells in the central nervous system (CNS) using specific anti-arginine antibody. In the present study, we focused our attention on this particular amino acid in the peripheral nervous system (PNS). In the cochlea of the inner ear, arginine-like hnmunoreactivity was localized in satellite cells surrounding neurons of the spiral ganglion. In the dorsal root ganglia, satellite cells surrounding sensory neurons were found to be immunoreactive. In the superior cervical ganglion, L-arglulne was concentrated in satellite cells around neuronal cells. In ganglia of the enteric plexus, supporting cells that covered neuronal cells were stained. These results show that free L-arginine in the PNS is concentrated in satellite and supporting cells, both of which correspond to gliai cells in the CNS. Thus, those cells in ganglia of the PNS may support and/or control the neural activity by providing L-arginine to the neurons that they surround. Nitric oxide (NO) is considered to play an important role as a messenger molecule in the brain, as well as in the blood vessels, macrophages and neutrophiles 2's'9'1214. NO has been shown to be formed from L-arginine 12A3 by an enzyme, N O synthase 34, which is localized not only in the above mentioned tissues and cells but also in neurons of the myenteric plexus of the intestine 4'17. It has been also reported that NO is released upon stimulation of the non-adrenergic non-cholinergic nerves in the canine ileocolonic junction 5. These reports suggest the presence of an L-arginine-NO pathway in the peripheral nervous system (PNS) including the myenteric plexus. However, it is unknown how L-arginine, a precursor of NO, is supplied to the pathway. On the other hand, we raised specific anti-arginine antibody in guinea pigs and reported the immunocytochemical distribution of free L-arginine in the rat brain 1. A main locus of L-arginine in the brain was found to be astrocytes and a close correlation between neurons and astrocytes in the L-arginine-NO pathway was suggested. In the present study, we focused our attention on the localization of free L-arginine in the PNS including the myenteric plexus. Anti-arginine antibody was prepared as described before 1. Briefly, L-arginine was coupled with guinea pig serum albumin (GPSA) via glutaraldehyde (GAL) and the L-arginine-GAL-GPSA conjugate was emulsified with
an equal volume of complete Freund's adjuvant and was repeatedly injected intracutaneously into multiple sites on the back of the guinea pig. Anti-arginine antibody was purified by affinity chromatography with L-arginine immobilized on formyl ceUulofme (Seikagaku Kogyo, Japan). Specificity of the antibody was checked by a dotimmunobinding assay 1°, and the purified anti-arginine antibody was proved to be specific to the L-arginineG A L - G P S A complex 1. Anti-S-100 protein antibody was a generous gift from Dr. K. Kato of our institute. Preparation and specificity of this antibody have been described elsewhere 11. Rats of Sprague-Dawley strain of 2 months old were perfused via the heart with a mixture of 1% G A L , 4% formaldehyde, 0.2% pierie acid and 2% sucrose in 0.1 M sodium acetate buffer, p H 6.0 is. Each organ was left in the above mixture for 4-5 h, rinsed several times with 50 mM Tris-HCl buffer, p H 7.6, dehydrated with graded alcohols and embedded in paraffin. In the case of the cochlea, fixed samples were treated with sodium borohydride solution (0.4 mg/ml in final concentration) for 2 h at pH 8.8, decalcified with 20 vols. of 5% nitric acid for 16 h and processed for paraffin embedding. Sections were incubated with the purified antibody (dilution 1:500), and left overnight at room temperature. The location of arginine was visualized by the peroxidase-anti-
Correspondence: E. Aoki, Department of Perinatology, Institute for Developmental Research, Aiehi Prefecture Colony, Kasugai, Aichi 480-03, Japan.
160
Fig. 1. Photographs showing the distribution of arginine- and S-100 protein-like immunoreactivities in the cochlear ganglion of a rat. Satellite cells (arrowheads) surrounding ganglion cells (g) are immunolabeled with the anti-arginine (A) and anti-S-100 protein (B) antibodies. Bar = 20/~m.
peroxidase method 19 with diaminobenzidine as chromogen. Control sections incubated with non-immune guinea pig serum showed no positive staining. We investigated the localization of arginine-like immunoreactivity in the cochlear ganglion of the 8th cranial nerve, the spinal dorsal root ganglion, and the superior cervical ganglion and enteric plexus of the autonomic nervous system. In the cochlear ganglion, satellite cells around neurons were strongly immunolabeled with the anti-arginine antibody (Fig. 1A). The labeling pattern was very similar to that obtained with the antiS-100 protein antibody (Fig. 1B). Fig. 2 shows immunocytochemistry of the dorsal root ganglion. The perikarya and processes of satellite cells were stained with the antiarginine antibody (Fig. 2A) as well as with the anti-S100 protein antibody (Fig. 2B). In the superior cervical ganglion, both of the antibodies stained the surroundings
of ganglion cells (Fig. 3A, B). In the Auerbach's and Meissner's plexus of the intestine, arginine and S-100 protein were found to be highly concentrated in supporting cells (Fig. 4 A - D ) . It is known that structures of the sensory and sympathetic ganglia are microscopically similar to each other and that nerve cells in the ganglia are ensheathed by nucleated membranous capsules, which are formed of satellite cells TM. In the enteric plexus, neuronal cells are ensheathed by supporting cells. These satellite and supporting cells are known to contain S-100 protein 6'7 like the case of glial cells in the CNS. Thus immunolabeling with the anti-S-100 antibody was helpful in defining arginine-positive structures as satellite and supporting cells. Our previous immunocytochemical study of free L-arginine in the rat brain showed its predominant localization in glial cells, particularly in astrocytes 1. Because
Fig. 2. Photographs of arginine- and S-100 protein-like immunoreactivities in the rat spinal dorsal root ganglion. A: satellite cells (arrowheads) around ganglion cells (g) were clearly stained with the anti-arglnine antibody. B: satellite cells (arrowheads) and dorsal root ganglion cells (g) are stained with the anti-S-100 protein antibody. Bar = 100/~m.
161
Fig. 3. Photographs showing the localization of arginine- and S-100 protein-like immunoreactivities in the superior cervical ganglion of a rat. The surroundings (arrowheads) of ganglion cells (g) are labeled with the anti-arginine (A) and anti-S-100 protein (B) antibodies in the sympathetic ganglion. Bar = 100/~m.
N O synthase was localized in neurons, it was considered that in the brain L-arginine was released from astrocytes, taken up by adjacent neurons and then cleaved to form NO. In the present study, the localization of free L-arginine was examined in the sensory ganglia and autonomic nervous tissues, and arginine-like immunoreactivity was found to be selectively concentrated in the surroundings of ganglion cells. On the other hand, in the duodenum, intense immunoreactivity of N O synthase has been proved in neuronal cells of the myenteric plexus 4. Thus, it seems likely that free L-arginine is released from satellite and supporting cells, incorporated into adjacent neuronal cells and finally cleaved to form N O by N O
synthase, though the presence of N O synthase has not been reported so far in the PNS except for the myenteric plexus 4. Moreover, satellite and supporting cells may possibly control the supply of L-arginine to neurons and influence the neural activity. In conclusion, it is suggested that free L-arginine in the PNS is concentrated in satellite and supporting cells which correspond to glial cells in the CNS and that neurons are surrounded by such an L-arginine pool. These cells could have an intracellular organization to maintain a high level of free L-arginine, which is supplied to neuronal ceils and influences their activity through the metabolic conversion to NO.
~ ~ ~ii~ii!i~
Fig. 4. Photographs demonstrating the arginine- and S-100 protein-like immunoreactivities in the enteric plexus of a rat. In the Auerbach's plexus, supporting cells which cover neuronal cells (arrows) are strongly stained with the anti-arginine (A) and anti-S-100 (B) antibodies. In the Meissner's plexus, the anti-arginine (C) and anti-S-100 (D) antibodies give positive reactions to supporting cells. Bar = 20/~m.
162 A part of this research was supported by Grant (63-A) from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare and also by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Science and Culture (02670779), Japan.
1 Aoki, E., Semba, R., Mikoshiba, K. and Kashiwamata, S., Predominant localization in glial cells of free L-arginine. Immunocytochemical evidence, Brain Research, 547 (1991) 190-192. 2 Bredt, D.S. and Snyder, S.H., Nitric Oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9030-9033. 3 Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 682-685. 4 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. 5 Bult, H., Boeckxstaens, G.E., Pelckmans, EA., Jordaens, EH., Van Maercke, Y.M. and Herman, A.G., Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature, 345 (1990) 346-347. 6 Cocchia, D. and Michetti, E, S-100 antigen in satellite cells of the adrenal medulla and the superior cervical ganglion of the rat, Cell Tissue Res., 215 (1981) 103-112. 7 Ferri, G.-L., Probert, L., Cocchia, D., Michetti, E, Marangos, P.J. and Polak, J.M., Evidence for the presence of S-100 protein in the glial component of the human enteric nervous system, Nature, 297 (1982) 409-410. 8 Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci. 14 (1991) 60-67. 9 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intracellular messenger in the brain, Nature, 336 (1988) 385-387. 10 Hawkes, R., Niday, E. and Gordon, J., A dot-immunobinding assay for monoclonal and other antibodies, Anal. Biochem., 119
(1982) 142-147. 11 Kimura, S., Kato, K., Semba, R. and Isobe, T., Regional distrihution of S-100a0(aa ), S-100a(a~) and S-100b(flfl) in the bovine central nervous tissue determined with a sensitive enzyme immunoassay system, Neurochem. Int., 6 (1984) 513-518. 12 Knowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5159-5162. 13 Palmer, R.M.J., Ashton, D.S. and Moncada, S., Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature, 333 (1988) 664-666. 14 Palmer, R.M.J., Ferrige, A.G. and Moncada, S., Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature, 327 (1987) 524-526. 15 Pick, J., The Autonomic Nervous System; Morphological, Comparative, Clinicaland Surgical Aspects, Lippincott, Philadelphia, 1970, 483pp. 16 Ranson, S.W. and Clark, S.L., The Anatomy of the Nervous System; its Development and Function, 10th edn., Saunders, Philadelphia, 1959, 622pp. 17 Ross, C.A., Bredt, D. and Snyder, S.H., Messenger molecules in the cerebellum, 7~ends Neurosci., 13 (1990) 216-222. 18 Schmechel, D.E., Brightman, M.W. and Marangos, EJ., Neurons switch from non-neuronal enolasc to neuron-specific enolase during differentiation, Brain Research, 190 (1980) 195-214. 19 Sternberger, L.A., The unlabeled antibody enzyme method. In L.A. Sternberger (Ed.), lmmunocytochemistry, Prentice-Hall, Englewood Cliffs, 1974, pp. 129-171.