Axonal regeneration can overcome negative consequences of disease and injury of the nervous system. Higher vertebrates have evolved a complex set of traits that severely limit regeneration in the CNS. It is now known that limited regenerative capacity is mainly due to the inhibitors present in CNS myelin as well as injury induced changes in the cellular environment. While much is known about axon guidance in the developing nervous system, little is known about adult regenerative growth. Here we describe a limited known-target library screen where primary neurons are plated on non-permissive substrates and drug effects on neurite outgrowth are evaluated. This approach should allow for the identification of permissive and instructive regeneration signaling pathways. Preliminary data suggest that at least 10 classes of compounds are active in this assay. Further characterization of these signal transduction pathways as well as the molecular targets involved in this model of regeneration are under way.

In the granular layer of the cerebellar cortex, the Purkinje axons give off collaterals bending back at sharp angles and terminating in the vicinity of the layer of Purkinje cell bodies. These recurrent axons have been reported to make synapses on the somata and initial dendritic portions of neighboring Purkinje cells (Palay and Chan-Palay, 1974), although differences between species seem to exist. We report here, based on computer simulations, that sparse recurrent connections in a heterogeneous network of model Purkinje cells suffice to synchronize the firing of simple spikes. During fast, sustained activity (> 100 Hz), the Purkinje cell population exhibits spontaneous oscillations, indistinguishable in shape and frequency from those observed recently in the cerebellum of calretinin-calbindin knockout mice (Cheron et al., Soc. Neurosci. Abstr. 2001). The mechanism for synchronization requires: 1) a strong excitation of individual Purkinje cells, making them behave like fast oscillators, 2) fast inhibitor...

The trochlear motor axons project dorsally along midbrain-hindbrain boundary to exit the brain contralaterally. We have scrutinized the trajectory of the trochlear nerve with particular attention to the spatio-temporal pattern of Sema3F expression in chick embryos. Sema3F is expressed in the posterior limit of the midbrain along midbrain-hindbrain boundary throughout the dorsal projection of the trochlear motor axons and disappear after the decussation. On the other hand, neuropilin-2, a receptor for Sema3F, is expressed in the trochlear nucleus but predominantly in its anterior population. When Sema3F was expressed ectopically in the way of projection, all the trochlear motor axons were repelled or collapsed in vivo. These results suggest that neuropilin-2 positive trochlear axons from anterior trochlear nucleus are navigated by Sema3F along midbrain-hindbrain boundary and they are followed by the rest of the axons during the dorsal projection. After transformation of the midbrain into the hindbrain by tr...

Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice ( Mus musculus ) basal GC receptor ( Nr3c1 , also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions ( M. musculus ). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo , prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity.

Increased levels of inhibitors such as chondroitin sulfate proteoglycans (CSPGs) and those associated with disrupted myelin likely reduce axonal regrowth following spinal cord injury. Inhibition of the small guanine nucleotide triphosphatase (GTPase), RhoA, or its downstream effector, Rho kinase, increases axon extension on inhibitory myelin constituents in vitro, and may lead to increased regeneration following injury. In addition, recent evidence indicates that inhibition of Rho kinase increases retinal ganglion cell outgrowth on neural CSPGs in a choice assay, and increases axon extension from dorsal root ganglion (DRG) neurons on non-neural CSPGs. We tested whether the RhoA/Rho kinase signaling pathway contributes to neural CSPG-mediated inhibition of axon extension from DRG neurons, a cellular population affected by spinal cord injury. We grew chick DRG neurons and human neuroblastoma cells on patterned substrata consisting of alternating stripes of outgrowth promoting laminin and neural CSPGs, then t...

L1 has been shown to be one of the major cell adhesion molecules involved in axon pathfinding. Previous studies have demonstrated L1 expression on retinal axons and on a population of neurons in the ventral diencephalon during the major axon in growth period at the chiasm. However, the function of L1 in development of the optic chiasm is unclear. In the present study, we have used DiI to trace the course and decussation patterns of axons in mice mutant for L1. We find a number of aberrant projections in the chiasm at E13 when retinal axons first reach the chiasm. L1 mutants had a greater degree of disorganisation as the axons pass into the chiasm. Aberrant projections could be observed, including to the hypothalamus and opposite optic nerve. There is also an apparent increase in the complexity of the growth cone morphology in L1 mutant mice. In embryos at E14 and E15, we show that the ipsilateral projection is reduced. These results demonstrate L1 may be involved in axon guidance at the chiasm and in estab...

Increased mTOR activity has been shown to enhance regeneration of injured axons by increasing neuronal protein synthesis, while PTEN signaling can block mTOR activity to attenuate protein synthesis. MicroRNAs (miRs) have been implicated in regulation of PTEN and mTOR expression, and previous work in spinal cord showed an increase in miR-199a-3p after spinal cord injury (SCI) and increase in miR-21 in SCI animals that had undergone exercise. Pten mRNA is a target for miR-21 and miR-199a-3p is predicted to target mTor mRNA. Here, we show that miR-21 and miR-199a-3p are expressed in adult dorsal root ganglion (DRG) neurons, and we used culture preparations to test functions of the rat miRs in adult DRG and embryonic cortical neurons. miR-21 increases and miR-199a-3p decreases in DRG neurons after in vivo axotomy. In both the adult DRG and embryonic cortical neurons, miR-21 promotes and miR-199a-3p attenuates neurite growth. miR-21 directly bound to Pten mRNA and miR-21 overexpression decreased Pten mRNA level...

Growth cones of cortical axons pause for many hours in preparation for axon branching. They become large and complex compared with small advancing growth cones. We wanted to investigate whether calcium transients regulate the advance of mammalian CNS growth cones. We found that spontaneous calcium transients in developing cortical neurons have characteristic patterns, frequencies, and amplitudes. Importantly, neurons with large paused growth cones exhibit high-frequency spontaneous calcium transients, which are rare in those with small advancing growth cones. The incidence, frequencies, and amplitudes of calcium transients are inversely related to rates of axon outgrowth. The transients are mediated primarily by L-type voltage-gated calcium channels, and silencing them with channel blockers promotes axon outgrowth. Thus calcium transients regulate growth cone advance by direct effects on the growth cone.

The role of microglia in degenerative and regenerative processes after damage of the nervous system remains ambiguous, partially due to the paucity of appropriate investigative methods. Here, we show that treatment with the pharmacological colony stimulating factor 1 receptor inhibitor PLX5622 specifically eliminated microglia in murine retinae and optic nerves with high efficiency. Interestingly, time course and extent of retinal ganglion cell (RGC) degeneration after optic nerve crush remained unaffected upon microglia depletion, although remnants of prelabeled apoptotic RGCs were not cleared from the retina in these animals. In addition, microglia depletion neither affected the induction of regeneration associated genes upon optic nerve injury nor the increased regenerative potential of RGCs upon lens injury (LI). However, although the repopulation of the optic nerve lesion site by astrocytes was significantly delayed upon microglia depletion, spontaneous and LI-induced axon regeneration were unaffected...

Many genes involved in developmental axon outgrowth are poorly expressed in mature neurons, and successful axon regeneration has been linked to re-expression of at least some of these genes in adults. We have now used large-scale cDNA microarrays to obtain a more global profile of genes associated with successful regeneration in adults. Dorsal root ganglion (DRG) neurons were isolated from embryonic and adult mice, with or without a peripheral nerve injury to stimulate regeneration. RNA was amplified, fluorescently labeled, and hybridized with microarrays containing more than 25,000 cDNA's. The results confirm that nerve injury stimulates re-expression of several developmentally-expressed genes, but not all genes associated with developmental growth are re-activated by adult axotomy. In some cases, developmental outgrowth and adult regeneration are distinguished by expression of different members of a gene family. For example, the gene for SCG10, a member of the stathmin gene family, is preferentially expr...