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To gain insight into the possible molecular mechanisms underlying axonal regeneration of neurons of the adult central nervous system (CNS), we have investigated, by in situ hybridization and by immunocytochemistry, the localization and sites of synthesis of the neurite outgrowth-promoting cell surface molecules L1, N-CAM and its highly sialylated form, N-CAM-PSA, in and around peripheral nerve grafts implanted into the thalamus of adult rats. Normal unoperated adult rat thalamus contains N-CAM and L1 but no N-CAM-PSA immunoreactive axons. Between 7 days and 13 weeks after graft implantation, L1, N-CAM and N-CAM-PSA were all present at the surface of axonal sprouts in the brain parenchyma close to grafts and in the central parts of Schwann cell columns within grafts. Schwann cell membranes were L1 and N-CAM positive at all postgraft survival times, more strongly at 2-4 weeks than other times, but were associated with N-CAM-PSA reaction product only where they abutted N-CAM-PSA positive axons. Schwann cell membranes apposed to basal laminae (which were avoided by regenerating CNS axons) were L1, N-CAM and N-CAM-PSA negative. Between 3 days and 8 weeks after grafting, N-CAM and L1 mRNA were generally weakly upregulated in neurons of the ipsilateral thalamus, but, most conspicuously, L1 mRNA was strongly upregulated in the neurons of the thalamic reticular nucleus; these neurons are known to regenerate axons very effectively into peripheral nerve grafts and are the probable source of most of the axons which enter thalamic grafts. N-CAM and L1 mRNA were also strongly upregulated in presumptive Schwann cells in the graft. These results show that regenerating CNS axons (re)express N-CAM-PSA and upregulate L1 and N-CAM, suggesting that all of these molecules may play a role in cellular interactions during the regeneration of CNS axons. Furthermore L1 synthesis appears to be particularly well correlated with the ability of CNS neurons to regenerate axons into peripheral nerve grafts.

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We have used in situ hybridization with a digoxigenin-labelled probe for tenascin-C mRNA and immunocytochemistry with antibodies against tenascin-C, glial fibrillary acidic protein, OX-42 and the 200 kDa neurofilament protein to study the expression, distribution and cellular relationships of tenascin-C mRNA and protein in the developing (postnatal) and adult spinal cord of rat, and the effects thereon of dorsal root, ventral root and sciatic nerve injuries. The most interesting finding was that on postnatal day 7 (P7), P14 and in the adult, but not on P0 or P3, a group of neurons in the lumbar ventral horn expressed the tenascin-C mRNA gene. They represented about 5% of ventral horn neurons in the adult and were among the smaller such neurons. Since 40-60% of such cells were lost at P13 following sciatic nerve crush on P0, some were almost certainly motor neurons. In addition, we found that at P0 and P3, mRNA-containing glial cells were widespread in grey and white matter but sparse in the developing dorsal columns; tenascin-C immunofluorescence showed a similar distribution. By P7 there were fewer mRNA-containing cells in the ventral horns and in the area of the dorsal columns containing the developing corticospinal tract where immunofluorescence was also weak. At P14 there were no glial-like mRNA-containing cells in the grey matter; such cells were confined to the periphery of the lateral and ventral white columns but were present throughout the dorsal columns where tenascin-C immunofluorescence was also strong. No glial-like mRNA-containing cells were present in the adult lumbar spinal cord and tenascin-C immunofluorescence was confirmed to irregular patches in the ventral horn, especially around immunonegative cell bodies of small neurons, a zone around the central canal, and a thin zone adjacent to the glia limitans. Thus the expression of tenascin-C is differentially developmentally regulated in the grey matter and in different parts of the white matter. Three days after injury of dorsal roots L4-6, many cells containing tenascin-C mRNA, some identified as glial fibrillary acidic protein-positive astrocytes, were present in the ipsilateral dorsal column, but were rare after longer survivals. Immunoreactivity, however, was elevated in the ipsilateral dorsal column at 3 days, remained high for several months and disappeared at 6.5 months. Dorsal root injury had no effect on tenascin-C mRNA or protein in the grey matter. Sciatic nerve or ventral root injury had no effect on these molecules in any part of the spinal cord.