Astrocytes are specialized and heterogeneous cells that contribute to central nervous system function and homeostasis. However, the mechanisms that create and maintain differences among astrocytes and allow them to fulfill particular physiological roles remain poorly defined. We reveal that neurons actively determine the features of astrocytes in the healthy adult brain and define a role for neuron-derived sonic hedgehog (Shh) in regulating the molecular and functional profile of astrocytes. Thus, the molecular and physiological program of astrocytes is not hardwired during development but, rather, depends on cues from neurons that drive and sustain their specialized properties.

Brain cells are immersed in a complex structure forming the extracellular matrix. The composition of the matrix gradually matures during postnatal development, as the brain circuitry reaches its adult form. The fully developed extracellular environment stabilizes neuronal connectivity and decreases cortical plasticity as highlighted by the demonstration that treatments degrading the matrix are able to restore synaptic plasticity in the adult brain. The mechanisms through which the matrix inhibits cortical plasticity are not fully clarified. Here we show that a prominent component of the matrix, chondroitin sulfate proteoglycans (CSPGs), restrains morphological changes of dendritic spines in the visual cortex of adult mice. By means of in vivo and in vitro two-photon imaging and electrophysiology, we find that after enzymatic digestion of CSPGs, cortical spines become more motile and express a larger degree of structural and functional plasticity.

The processes activated at the time of axotomy and leading to the formation of a new growth cone are the first step in regeneration, but are still poorly characterized. We investigated this event in an in vitro model of axotomy performed on dorsal root ganglia and retinal explants. We observed that the dorsal root ganglion axons and retinal ganglion cell axons, which had grown out on a poly D-lysine/laminin substrate at the time of culture preparation greatly differed in their regenerative response after a subsequent in vitro lesion made far from the cell body. The majority of axons of adult dorsal root ganglia but only a small percentage of axons of adult retinal ganglion cells regenerated new growth cones within four hours after in vitro axotomy, though both kinds of axons were growing before the lesion. The depletion of extracellular calcium and the inhibition of extracellular-signal regulated kinase 1,2 (ERK) and protein kinase A (PKA) at the time of injury significantly impaired the capacity of dorsal root ganglia axons to re-initiate growth cones without affecting growth cone motility. Pharmacological treatments directed at increasing the level of cAMP promoted growth cone regeneration in adult retinal ganglion cell axons in spite of the low regenerative potential exhibited in normal conditions. Understanding the cellular mechanisms activated at the time of lesion and leading to the formation of a new growth cone is necessary for devising treatments aimed at enhancing the regenerative response of injured axons.

Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative abilityin vivocorrelates with the potential to form a new growth cone after axotomyin vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomyin vitroleads to a fourfold to sixfold increase in3H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin andN-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery bothin vitroandin vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.

In young animals, monocular deprivation leads to an ocular dominance shift, whereas in adults after the critical period there is no such shift. Chondroitin sulphate proteoglycans (CSPGs) are components of the extracellular matrix (ECM) inhibitory for axonal sprouting. We tested whether the developmental maturation of the ECM is inhibitory for experience-dependent plasticity in the visual cortex. The organization of CSPGs into perineuronal nets coincided with the end of the critical period and was delayed by dark rearing. After CSPG degradation with chondroitinase-ABC in adult rats, monocular deprivation caused an ocular dominance shift toward the nondeprived eye. The mature ECM is thus inhibitory for experience-dependent plasticity, and degradation of CSPGs reactivates cortical plasticity.

Since the first studies on axonal regeneration, the optic nerve (ON) of higher vertebrates has been considered a good experimental system to investigate the failure of mature CNS neurons to re-grow after axotomy. The optic nerve is composed of a single population of fibers the RGC axons and, being separated from the rest of the brain, it is easily accessible to surgical manipulations. All the fibers can be transected without massive damage to the surrounding tissue, so their reaction to axotomy is not perturbed by extended inflammation processes. Another advantage of the system is the accessibility of RGCs. Being in the more internal retinal layer, RGCs are directly exposed to the humor vitreus, the liquid filling the posterior chamber of the eye. Pharmaceutical treatments are easily injected into the eye and, diffusing in the vitreus, can reach all the RGCs. Last but not least, functional recovery can be easily monitored in the optic nerve; measurement of electrical activity in response to visual stimuli in CNS regions that receive inputs from the retina such as superior colliculus or visual cortex allows evaluation of the re-growth of ON fibers and the restoration of connections. All the experiments carried out so far indicate that the failure of regeneration in the ON, as in the majority of the CNS districts, is a multi-factorial phenomenon, involving three classes of negative events. 1) RGCs die after axotomy: in the adult rat, their number is reduced to a very small percentage in a few weeks after the lesion. 2) The majority of mature axotomised RGCs are not programmed to re-start the process of axonal elongation that they displayed in immature stages. 3) The optic nerve environment contains molecules many of them upregulated after the lesion that are inhibitory for axonal growth. This review, focused on experiments performed in the mammalian optic nerve, traces attempts made to overcome each of these three obstacles, and maps progress towards a combined therapeutic strategy.

BACKGROUND:The neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5 and NT-6, are a family of proteins that play fundamental roles in the differentiation, survival and maintenance of peripheral and central neurons. Much research has focused on the role of neurotrophins as target-derived, retrogradely transported trophic molecules. Although there is recent evidence that BDNF and NT-3 can be transported in an anterograde direction along peripheral and central axons, there is as yet no conclusive evidence that these anterograde factors have direct post-synaptic actions. RESULTS:We report that BDNF travels in an anterograde direction along the optic nerve. The anterogradely transported BDNF had rapid effects on retinal target neurons in the superior colliculus and lateral geniculate nucleus of the brain. When endogenous BDNF within the developing superior colliculus was neutralised, the rate of programmed neuronal death increased. Conversely, provision of an afferent supply of BDNF prevented the degeneration of geniculate neurons after removal of their cortical target. CONCLUSIONS:BDNF released from retinal ganglion cells acts as a survival factor for post-synaptic neurons in retinal target fields.

Retinal ganglion cells of transgenic mice overexpressing the anti-apoptotic protein Bcl-2 in neurons show a dramatic increase of survival rate after axotomy. We used this experimental system to test the regenerative potentials of central neurons after reduction of nonpermissive environmental factors. Survival of retinal ganglion cells 1 month after intracranial crush of the optic nerve was found to be 100% in adult bcl-2 mice and 44% in matched wild-type (wt) mice. In the optic nerve, and particularly at the crush site, fibers regrowing spontaneously or simply sprouting were absent in both wt and bcl-2 mice. We attempted to stimulate regeneration implanting in the crushed nerves hybridoma cells secreting antibodies that neutralize central myelin proteins, shown to inhibit regeneration (IN-1 antibodies) (Caroni and Schwab, 1988). Again, we found that regeneration of fibers beyond the site of crush was virtually absent in the optic nerves of both wt and bcl-2 mice. However, in bcl-2 animals treated with IN-1 antibodies, fibers showed sprouting in the proximity of the hybridoma implant. These results suggest that neurons overexpressing bcl-2 are capable of surviving axotomy and sprout when faced with an environment in which inhibition of regeneration has been reduced. Nevertheless, extensive regeneration does not occur, possibly because other factors act by preventing it.

Multicellular organisms face the necessity of removing superfluous or injured cells during normal development, tissue turn-over and in response to damaging conditions. These finalised killings occur throughout a process, commonly called programmed cell death (PCD), which is placed under strict cellular control. PCD is regulated by the products of the expression of a number of genes. This fact raises the intriguing possibility of inhibiting such degenerative processes by operating on some of the controlling genes. Central neurons of transgenic mice overexpressing bcl-2, a powerful inhibitor of PCD, are remarkably resistant to degeneration induced by noxious stimuli. We have explored the fate of retinal ganglion cells and of their axons, when such transgenic animals have been challenged by a lesion of the optic nerve. These results have direct bearing on the possibility of attaining functional restoration of the injured pathway.

Approximately half of the retinal ganglion cells (RGCs) present in the rodent retina at birth normally die during early development. Overexpression of the proto-oncogene bcl-2 recently has been shown to rescue some neuronal populations from natural cell death and from degeneration induced by axotomy of nerves within the peripheral nervous system. Here we study in vivo the role of the overexpression of bcl-2 in the natural cell death of RGCs and in the degenerative process induced in these cells by transection of the optic nerve. We find that in newborn bcl-2 transgenic mice, the number of RGCs undergoing natural cell death is considerably lower than in wild-type pups. Consistently, a vast majority (90%) of the ganglion cells found in the retina of neonatal transgenics are maintained in adulthood, whereas only 40% survive in wild-type mice. After transection of the optic nerve, the number of degenerating ganglion cells, determined by counting pyknotic nuclei or nuclei with fragmented DNA, is substantially reduced in transgenic mice. In wild-type animals, almost 50% of ganglion cells degenerate in the 24 hr after the lesion, whereas almost the entire ganglion cell population survives axotomy in transgenic mice. Therefore, overexpression of bcl-2 is effective in preventing degeneration of this neuronal population, raising the possibility that ganglion cells are dependent on the endogenous expression of bcl-2 for survival. The remarkable rescue capacity of bcl-2 overexpression in these neurons makes it an interesting model for studying natural cell death and responses to injury in the CNS.