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Neuronal morphology, axonal integrity, and axonal regeneration in situ are regulated by cytoskeletal phosphorylation in identified lamprey central neurons
Author(s) -
Hall Garth F.,
Yao Jun
Publication year - 2000
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/(sici)1097-0029(20000101)48:1<32::aid-jemt5>3.0.co;2-c
Subject(s) - axotomy , microbiology and biotechnology , axon , neurofilament , biology , colchicine , okadaic acid , phosphorylation , cytoskeleton , axoplasmic transport , regeneration (biology) , phosphatase , chemistry , biochemistry , cell , immunology , genetics , immunohistochemistry
The CNS of the sea lamprey (Petromyzon marinus) contains giant, individually identifiable neurons that can be microinjected intracellularly in the living animal. We have used the unique accessibility of this system to investigate the role played by serine/threonine kinases and phosphatases in regulating cytoskeletal stability in identified reticulospinal neurons (ABCs) in situ. Injection of broad spectrum kinase and phosphatase inhibitors induce marked changes in ABC gross morphology and in the extent and morphology of sprouts induced by axotomy. The kinase inhibitor K‐252a causes regenerating sprouts to be longer and narrower than those seen in control preparations, and significantly reduces the diameters of axon stumps; this latter effect is similar to the effect of microinjecting anti neurofilament (NF) antibodies. By contrast, the phosphatase inhibitor okadaic acid (OA) causes the selective disruption of axonal integrity, blocking axonal regeneration and causing axon stump retraction in axotomized ABCs. The microtubule (MT) disrupting drug colchicine has an effect similar but less marked than OA on ABC axonal morphology . Both OA and colchicine also induce the formation of large somatodendritic swellings in axotomized (but not intact) ABCs by 1–3 weeks post‐injection. Immunocytochemical analyses indicate that both colchicine and OA treatments result in the destabilization of MTs and the phosphorylation of NFs, while OA induces the accumulation of phosphorylated tau protein in some dendritic swellings. Control injections of inactive substances have none of these effects. These results suggest that OA does not have its primary effect on NF assembly at the doses used, but may block axonal regeneration by inducing a prolonged disruption of axonal MTs, possibly via an indirect mechanism involving the hyperphosphorylation of tau and other MAPs. K‐252a, on the other hand, may interfere with NF assembly and sidearm phosphorylation, thereby reducing NF transport into both axon stumps and sprouts and in turn reducing sprout diameter. The implications of these results for the respective roles of MTs, MAPs, and NFs in axonal regeneration in the vertebrate CNS are discussed. Microsc. Res. Tech. 48:32–46, 2000. © 2000 Wiley‐Liss, Inc.