In adult mammalian CNS, axons mostly fail to regenerate after injury, while in the PNS they often succeed in reaching their previous targets. Crucial differences are present in the local tissue microenvironment of CNS and PNS. To investigate the substrate properties of nervous tissue for neuronal adhesion and fiber growth, we used frozen sections of rat CNS and PNS as culture substrates for neuroblastoma cells and for sympathetic and dorsal root ganglia. The results showed that CNS white matter from adult rat spinal cord, cerebellum, forebrain, or optic nerve did not allow cell adhesion and axonal elongation. In contrast, gray matter areas, sciatic nerve, and also trout CNS white and gray matter were permissive substrates. To delineate the tissue components of white matter involved in this nonpermissive substrate effect, newborn rats were injected for 13 d with the antimitotic agent 5-azacytidine. This treatment strongly reduced the oligodendrocyte population and the myelin content of the spinal cord. The immunoreactivity for specific oligodendrocyte and astrocyte markers confirmed the selective suppression of oligodendroglia in these rats. Neuroblastoma cells plated on spinal cord sections taken from these animals were no longer exclusively localized on the gray matter but were also found on regions normally rich in myelin. A significant reduction of the white matter nonpermissive substrate effect was also obtained by the monoclonal antibody IN-1 directed against 2 defined myelin proteins with inhibitory substrate properties (Caroni and Schwab, 1988b). Our results, therefore, show that, in the adult mammalian CNS, cell adhesion and axonal elongation are prevented by white matter components, which are, at least in part, associated with oligodendrocytes and myelin.

B-50/GAP-43 is a nervous tissue-specific protein, the expression of which is associated with axon growth and regeneration. Its overexpression in transgenic mice produces spontaneous axonal sprouting and enhances induced remodeling in several neuron populations (Aigner et al., 1995;Holtmaat et al., 1995). We examined the capacity of this protein to increase the regenerative potential of injured adult central axons, by inducing targeted B-50/GAP-43 overexpression in Purkinje cells, which normally show poor regenerative capabilities. Thus, transgenic mice were produced in which B-50/GAP-43 overexpression was driven by the Purkinje cell-specific L7 promoter. Uninjured transgenic Purkinje cells displayed normal morphology, indicating that transgene expression does not modify the normal phenotype of these neurons. By contrast, after axotomy numerous transgenic Purkinje cells exhibited profuse sprouting along the axon and at its severed end. Nevertheless, despite these growth phenomena, which never occurred in wild-type mice, the severed transgenic axons were not able to regenerate, either spontaneously or into embryonic neural or Schwann cell grafts placed into the lesion site. Finally, although only a moderate Purkinje cell loss occurred in wild-type cerebella after axotomy, a considerable number of injured transgenic neurons degenerated, but they could be partially rescued by the different transplants placed into the lesion site. Thus, B-50/GAP-43 overexpression substantially modifies Purkinje cell response to axotomy, by inducing growth processes and decreasing their resistance to injury. However, the presence of this protein is not sufficient to enable these neurons to accomplish a full program of axon regeneration.

In the adult central nervous system (CNS) of higher vertebrates lesioned axons seemed unable to regenerate and reach their former target regions due to influences of the CNS microenvironment. Evidence from in vitro and biochemical experiments has demonstrated the presence of inhibitory substrate components in CNS tissue, in particular in white matter. These CNS components, which strongly inhibit neurite growth, were identified as minor membrane proteins ofdefined molecular mass (35 and 250 kDa) in oligodendrocyte membranes and CNS myelin. Oligodendrocyte development and myelin formation can be prevented by x-irradiation ofnewborn rats. Here we show that in myelin-free spinal cords corticospinal tract fibers transected at 2 weeks of age show reelongation ofmany millimeters within 2-3 weeks after the lesion. In normally myelinated controls, regenerative sprouts grew less than 1.7 mm caudal to the lesion.

The highly specific connection patterns of the mature CNS are shaped through finely regulated processes of axon growth and retraction. To investigate the relative contribution of cell-autonomous mechanisms and extrinsic cues in these events, we examined the development of Purkinje axon intracortical plexus in the rat cerebellum. During the first postnatal week, several new processes sprout from focal swellings along the initial portion of the Purkinje neurite and spread in the granular layer. Intense structural plasticity occurs during the following week, with pruning of collateral branches and remodeling of terminal arbors. The mature distribution of the Purkinje infraganglionic plexus, confined within the most superficial portion of the granular layer, is attained at approximately postnatal day 15. A similar neuritic branching pattern is also developed by Purkinje cells grown in cultures of dissociated cerebellar cells or transplanted to extracerebellar CNS regions, suggesting that cell-autonomous mechanisms contribute to determining the Purkinje axon phenotype. The structural remodeling of Purkinje intracortical plexus is concomitant with the development of cerebellar myelin. To ask whether myelin-associated factors contribute to the morphological maturation of Purkinje neurites, we prevented normal myelinogenesis by killing oligodendrocyte precursors with 5'-azacytidine or by applying neutralizing antibodies against the myelin-associated neurite growth inhibitor Nogo-A. In both conditions, Purkinje axons retained exuberant branches, and the terminal plexus spanned the entire extent of the granular layer. Thus, the formation of Purkinje axon collaterals is, in part, controlled by intrinsic determinants, but their growth and distribution are regulated by environmental signals, among which are myelin-derived cues.

The neuronal loss observed in AIDS patients may be partly due to the neurotoxicity of HIV coat protein gp120, whose mechanism of action has been suggested to involve an interaction with voltage-dependent Ca2+ channels and NMDA receptors (Lipton, Trends Neurosci 15:75-79, 1992). In the present investigation we analyzed the acute neurotoxicity of gp120 on a purified neuronal population (rat cerebellar granule cell cultures) amply used for studies on glutamate toxicity. Cultures of 7-8 days were exposed for 15 min to a buffered Locke's solution containing the substances under study, washed, and cultured for another 24 hr in their original medium. The cells were stained with the nuclear dyes propidium iodide (for dead cells) and Hoechst 33258 (for total cells) and counted. Average cell death in controls was 8%. gp120 (1 pM-10 nM) caused an increase of cell death of about 80%. The effect was totally antagonized by NMDA antagonists (1 mM APV and 10 microM MK-801), by 1 microM nifedipine, and by anti-gp120 antibodies. At a concentration of 100 microM glutamate caused an average 130% increase of cell death, which was totally antagonized by APV. The effect of gp120 or glutamate did not appear to be mediated by the secretion of neurotoxins by nonneuronal cells present in a low proportion in the cultures nor to be due to the inactivation of (or competition with) neurotrophic factors present in the medium. The simultaneous administration of gp120 and glutamate (in various combinations of concentrations) had an effect that was less than additive.(ABSTRACT TRUNCATED AT 250 WORDS)