Slow Axons Research Articles

Tissue-engineered grafts exploit axon-facilitated axon regeneration and pathway protection to enable recovery after 5-cm nerve defects in pigs.

Functional restoration following major peripheral nerve injury (PNI) is challenging, given slow axon growth rates and eventual regenerative pathway degradation in the absence of axons. We are developing tissue-engineered nerve grafts (TENGs) to simultaneously "bridge" missing nerve segments and "babysit" regenerative capacity by providing living axons to guide host axons and maintain the distal pathway. TENGs were biofabricated using porcine neurons and "stretch-grown" axon tracts. TENG neurons survived and elicited axon-facilitated axon regeneration to accelerate regrowth across both short (1 cm) and long (5 cm) segmental nerve defects in pigs. TENG axons also closely interacted with host Schwann cells to maintain proregenerative capacity. TENGs drove regeneration across 5-cm defects in both motor and mixed motor-sensory nerves, resulting in dense axon regeneration and electrophysiological recovery at levels similar to autograft repairs. This approach of accelerating axon regeneration while maintaining the pathway for long-distance regeneration may achieve recovery after currently unrepairable PNIs.

Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs.

Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3’UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.

Promoting and Orienting Axon Extension Using Scaffold-Free Dental Pulp Stem Cell Sheets.

Current treatments of facial nerve injury result in poor functional outcomes due to slow and inefficient axon regeneration and aberrant reinnervation. To address these clinical challenges, bioactive scaffold-free cell sheets were engineered using neurotrophic dental pulp stem/progenitor cells (DPCs) and their aligned extracellular matrix (ECM). DPCs endogenously supply high levels of neurotrophic factors (NTFs), growth factors capable of stimulating axonal regeneration, and an aligned ECM provides guidance cues to direct axon extension. Human DPCs were grown on a substrate comprising parallel microgrooves, inducing the cells to align and deposit a linearly aligned, collagenous ECM. The resulting cell sheets were robust and could be easily removed from the underlying substrate. DPC sheets produced NTFs at levels previously shown capable of promoting axon regeneration, and, moreover, inducing DPC alignment increased the expression of select NTFs relative to unaligned controls. Furthermore, the aligned DPC sheets were able to stimulate functional neuritogenic effects in neuron-like cells in vitro. Neuronally differentiated neuroblastoma SH-SY5Y cells produced neurites that were significantly more oriented and less branched when cultured on aligned cell sheets relative to unaligned sheets. These data demonstrate that the linearly aligned DPC sheets can biomechanically support axon regeneration and improve axonal guidance which, when applied to a facial nerve injury, will result in more accurate reinnervation. The aligned DPC sheets generated here could be used in combination with commercially available nerve conduits to enhance their bioactivity or be formed into stand-alone scaffold-free nerve conduits capable of facilitating improved facial nerve recovery.

Failures of nerve regeneration caused by aging or chronic denervation are rescued by restoring Schwann cell c-Jun.

After nerve injury, myelin and Remak Schwann cells reprogram to repair cells specialized for regeneration. Normally providing strong regenerative support, these cells fail in aging animals, and during chronic denervation that results from slow axon growth. This impairs axonal regeneration and causes significant clinical problems. In mice, we find that repair cells express reduced c-Jun protein as regenerative support provided by these cells declines during aging and chronic denervation. In both cases, genetically restoring Schwann cell c-Jun levels restores regeneration to control levels. We identify potential gene candidates mediating this effect and implicate Shh in the control of Schwann cell c-Jun levels. This establishes that a common mechanism, reduced c-Jun in Schwann cells, regulates success and failure of nerve repair both during aging and chronic denervation. This provides a molecular framework for addressing important clinical problems, suggesting molecular pathways that can be targeted to promote repair in the PNS.

Nerve excitability differences in slow and fast motor axons of the rat: more than just Ih.

The objective was to determine biophysical differences between fast and slow motor axons using threshold tracking and demonstrate confounds related to anesthetic. Nerve excitability of motor axons innervating the slow-twitch soleus (SOL) and fast-twitch tibialis anterior (TA) muscles was tested. The experiments were conducted with pentobarbital sodium (SP) anesthetic and compared with previous results that used ketamine-xylazine (KX). Nerve excitability indices measured with SP show definitive differences between TA and SOL motor axons that extend beyond previous reports. Nerve excitability indices sensitive to changes in Ih indicated an increase in SOL axons compared with TA axons [e.g., S3 t = 7.949 (df = 10), P < 0.001; hyperpolarizing threshold electrotonus (90-100 ms), t = 2.659 (df = 20); P = 0.01; hyperpolarizing I/V slope, t = 4.308 (df = 19); P < 0.001]. SOL axons also had a longer strength-duration time constant [t = 3.35 (df = 20); P = 0.003] and a longer and larger magnitude relative refractory period [RRP (ms) t = 3.53 (df = 12); P = 0.004; Refractoriness at 2 ms, t = 0.0055 (df = 9); P = 0.006]. Anesthetic choice affected many measures of peripheral nerve excitability with differences most apparent in tests of threshold electrotonus and recovery cycle. For example, recovery cycle with KX lacked a clear superexcitable and late subexcitable period. We conclude that KX had a confounding effect on nerve excitability results consistent with ischemic depolarization. Results using SP revealed the full extent of differences in nerve excitability measures between putative slow and fast motor axons of the rat. These results provide empirical evidence, beyond conduction velocity, that the biophysical properties of motor axons vary with the type of muscle fiber innervated. These differences suggest that fast axons may be predisposed to dysfunction during hyperpolarizing stresses, e.g., electrogenic sodium pumping following sustained impulse conduction.NEW & NOTEWORTHY Nerve excitability testing is a tool used to provide insight into the properties of ion channels in peripheral nerves. It is used clinically to assess pathophysiology of axons. Researchers customarily think of motor axons as homogeneous; however, we demonstrate there are clear differences between fast and slow axons in the rat. This is important for interpreting results with selective motor neuronopathy, like aging where fast axons are at high risk of degeneration.

Retinal ganglion cell function in recovered optic neuritis: Faster is not better

ObjectiveTo assess residual retinal ganglion cell (RGC) function in patients with recovered optic neuritis (ON) and multiple sclerosis (MS). MethodsAge-matched controls (C, n = 32) and MS patients (n = 17) with history of ON in one eye but normal visual acuity and color vision were tested with steady-state Pattern Electroretinogram (PERG). Light Emitting Diodes (LED)-generated bar gratings, robust signal averaging and Fourier analysis were used to assess response amplitude and latency. ResultsPERG amplitude was similar for C, ON and fellow eyes (FE) (P = 0.4), but PERG latency was shortened in ON by 3.2 ms (P = 0.002) and in FE by 2.0 ms (P = 0.02) and was correlated (P < 0.01) with both Retinal Nerve Fiber Layer (RNFL) and Ganglion Cell Inner Plexiform Layer (GCIPL) thicknesses. PERG latency shortening could be simulated in control subjects (n = 8) by dioptrically blurring the edges of gratings (high spatial frequencies), which reduced activity of parvocellular RGCs with smaller/slower axons. The blurred PERG latency was shorter than baseline by 2.9 ms (P = 0.01). ConclusionsPERG latency is shortened in both eyes of MS patients with recovered unilateral ON, suggesting relative dysfunction of RGCs with slower axons and sparing of RGCs with faster axons. SignificanceAssessment of PERG latency in MS and ON may help identifying and monitoring RGC dysfunction. PERG latency shortening in FE suggests primary retinopathy in MS.

Excitability and firing behavior of single slow motor axons transmitting natural repetitive firing of human motoneurons.

Excitability of motor axons is critically important for realizing their main function, i.e., transmitting motoneuron firing to muscle fibers. The present study was designed to explore excitability recovery and firing behavior in single slow axons transmitting human motoneuron firing during voluntary muscle contractions. The abductor digiti minimi, flexor carpi ulnaris, and tibialis anterior were investigated during threshold stimulation of corresponding motor nerves. Motor unit (MU) firing index in response to testing volleys evoking M-responses was used as a physiological measure of axonal excitability and its changes throughout a target interspike interval (ISI) were explored. It was shown that axons displayed an early irresponsive period (within the first ~2-5 ms of a target ISI) that was followed by a responsive period (for the next 5-17 ms of the ISI), in which MUs fired axonal doublets, and a later irresponsive period. At the beginning of the responsive period, M-responses showed small latency delays. However, since at that ISI moment, MUs displayed excitability recovery with high firing index, slight latency changes may be considered as a functionally insignificant phenomenon. The duration of axonal doublet ISIs did not depend on motoneuron firing frequencies (range 4.3-14.6 imp/s). The question of whether or not traditionally described axonal recovery excitability cycle is realistic in natural motor control is discussed. In conclusion, the present approach, exploring, for the first time, excitability recovery in single slow axons during motoneuron natural activation, can provide further insight into axonal firing behavior in normal states and diseases.NEW & NOTEWORTHY Excitability of single slow axons was estimated by motor unit firing index in response to motor nerve stimulation, and its changes throughout a target interspike interval were explored during transmitting human motoneuron natural firing. It was found that axons exhibited early irresponsive, responsive, and later irresponsive periods. Findings question whether the traditionally described axonal excitability recovery cycle is realistic in natural motor control.

Axonal Degeneration in Retinal Ganglion Cells Is Associated with a Membrane Polarity-Sensitive Redox Process.

Axonal degeneration is a pathophysiological mechanism common to several neurodegenerative diseases. The slow Wallerian degeneration (WldS) mutation, which results in reduced axonal degeneration in the central and peripheral nervous systems, has provided insight into a redox-dependent mechanism by which axons undergo self-destruction. We studied early molecular events in axonal degeneration with single-axon laser axotomy and time-lapse imaging, monitoring the initial changes in transected axons of purified retinal ganglion cells (RGCs) from wild-type and WldS rat retinas using a polarity-sensitive annexin-based biosensor (annexin B12-Cys101,Cys260-N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine). Transected axons demonstrated a rapid and progressive change in membrane phospholipid polarity, manifested as phosphatidylserine externalization, which was significantly delayed and propagated more slowly in axotomized WldS RGCs compared with wild-type axons. Delivery of bis(3-propionic acid methyl ester)phenylphosphine borane complex, a cell-permeable intracellular disulfide-reducing drug, slowed the onset and velocity of phosphatidylserine externalization in wild-type axons significantly, replicating the WldS phenotype, whereas extracellular redox modulation reversed the WldS phenotype. These findings are consistent with an intra-axonal redox mechanism for axonal degeneration associated with the initiation and propagation of phosphatidylserine externalization after axotomy.SIGNIFICANCE STATEMENT Axonal degeneration is a neuronal process independent of somal apoptosis, the propagation of which is unclear. We combined single-cell laser axotomy with time-lapse imaging to study the dynamics of phosphatidylserine externalization immediately after axonal injury in purified retinal ganglion cells. The extension of phosphatidylserine externalization was slowed and delayed in Wallerian degeneration slow (WldS) axons and this phenotype could be reproduced by intra-axonal disulfide reduction in wild-type axons and reversed by extra-axonal reduction in WldS axons. These results are consistent with a redox mechanism for propagation of membrane polarity asymmetry in axonal degeneration.

Activation of oligodendroglial Stat3 is required for efficient remyelination

Multiple sclerosis is the most prevalent demyelinating disease of the central nervous system (CNS) and is histologically characterized by perivascular demyelination as well as neurodegeneration. While the degree of axonal damage is correlated with clinical disability, it is believed that remyelination can protect axons from degeneration and slow disease progression. Therefore, understanding the intricacies associated with myelination and remyelination may lead to therapeutics that can enhance the remyelination process and slow axon degeneration and loss of function. Ciliary neurotrophic factor (CNTF) family cytokines such as leukemia inhibitory factor (LIF) and interleukin 11 (IL-11) are known to promote oligodendrocyte maturation and remyelination in experimental models of demyelination. Because CNTF family member binding to the gp130 receptor results in activation of the JAK2/Stat3 pathway we investigated the necessity of oligodendroglial Stat3 in transducing the signal required for myelination and remyelination. We found that Stat3 activation in the CNS coincides with myelination during development. Stimulation of oligodendrocyte precursor cells (OPCs) with CNTF or LIF promoted OPC survival and final differentiation, which was completely abolished by pharmacologic blockade of Stat3 activation with JAK2 inhibitor. Similarly, genetic ablation of Stat3 in oligodendrocyte lineage cells prevented CNTF-induced OPC differentiation in culture. In vivo, while oligodendroglial Stat3 signaling appears to be dispensable for developmental CNS myelination, it is required for oligodendrocyte regeneration and efficient remyelination after toxin-induced focal demyelination in the adult brain. Our data suggest a critical function for oligodendroglial Stat3 signaling in myelin repair.

Effects of 4-methylbenzylidene camphor (4-MBC) on neuronal and muscular development in zebrafish (Danio rerio) embryos

The negative effects of overexposure to ultraviolet (UV) radiation in humans, including sunburn and light-induced cellular injury, are of increasing public concern. 4-Methylbenzylidene camphor (4-MBC), an organic chemical UV filter, is an active ingredient in sunscreen products. To date, little information is available about its neurotoxicity during early vertebrate development. Zebrafish embryos were exposed to various concentrations of 4-MBC in embryo medium for 3days. In this study, a high concentration of 4-MBC, which is not being expected at the current environmental concentrations in the environment, was used for the purpose of phenotypic screening. Embryos exposed to 15μM of 4-MBC displayed abnormal axial curvature and exhibited impaired motility. Exposure effects were found to be greatest during the segmentation period, when somite formation and innervation occur. Immunostaining of the muscle and axon markers F59, znp1, and zn5 revealed that 4-MBC exposure leads to a disorganized pattern of slow muscle fibers and axon pathfinding errors during the innervation of both primary and secondary motor neurons. Our results also showed reduction in AChE activity upon 4-MBC exposure both in vivo in the embryos (15μM) and in vitro in mammalian Neuro-2A cells (0.1μM), providing a possible mechanism for 4-MBC-induced muscular and neuronal defects. Taken together, our results have shown that 4-MBC is a teratogen and influences muscular and neuronal development, which may result in developmental defects.

Dynamics of signal propagation and collision in axons.

Long-range communication in the nervous system is usually carried out with the propagation of action potentials along the axon of nerve cells. While typically thought of as being unidirectional, it is not uncommon for axonal propagation of action potentials to happen in both directions. This is the case because action potentials can be initiated at multiple "ectopic" positions along the axon. Two ectopic action potentials generated at distinct sites, and traveling toward each other, will collide. As neuronal information is encoded in the frequency of action potentials, action potential collision and annihilation may affect the way in which neuronal information is received, processed, and transmitted. We investigate action potential propagation and collision using an axonal multicompartment model based on the Hodgkin-Huxley equations. We characterize propagation speed, refractory period, excitability, and action potential collision for slow (type I) and fast (type II) axons. In addition, our studies include experimental measurements of action potential propagation in axons of two biological systems. Both computational and experimental results unequivocally indicate that colliding action potentials do not pass each other; they are reciprocally annihilated.

Local axonal protection by WldS as revealed by conditional regulation of protein stability.

The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4-5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD(+), the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment.

IH activity is increased in populations of slow versus fast motor axons of the rat.

Much is known about the electrophysiological variation in motoneuron somata across different motor units. However, comparatively less is known about electrophysiological variation in motor axons and how this could impact function or electrodiagnosis in healthy or diseased states. We performed nerve excitability testing on two groups of motor axons in Sprague–Dawley rats that are known to differ significantly in their chronic daily activity patterns and in the relative proportion of motor unit types: one group innervating the soleus (“slow motor axons”) and the other group innervating the tibialis anterior (“fast motor axons”) muscles. We found that slow motor axons have significantly larger accommodation compared to fast motor axons upon application of a 100 ms hyperpolarizing conditioning stimulus that is 40% of axon threshold (Z = 3.24, p = 0.001) or 20% of axon threshold (Z = 2.67, p = 0.008). Slow motor axons had larger accommodation to hyperpolarizing currents in the current-threshold measurement (-80% Z = 3.07, p = 0.002; -90% Z = 2.98, p = 0.003). In addition, we found that slow motor axons have a significantly smaller rheobase than fast motor axons (Z = -1.99, p = 0.047) accompanied by a lower threshold in stimulus-response curves. The results provide evidence that slow motor axons have greater activity of the hyperpolarization-activated inwardly rectifying cation conductance (IH) than fast motor axons. It is possible that this difference between fast and slow axons is caused by an adaptation to their chronic differences in daily activity patterns, and that this adaptation might have a functional effect on the motor unit. Moreover, these findings indicate that slow and fast motor axons may react differently to pathological conditions.

Correction: A Slow Axon Antidromic Blockade Hypothesis for Tremor Reduction via Deep Brain Stimulation

Correction: A Slow Axon Antidromic Blockade Hypothesis for Tremor Reduction via Deep Brain Stimulation

Variable functional recovery and minor cell loss in the ganglion cell layer of the lizard Gallotia galloti after optic nerve axotomy

The lizard Gallotia galloti shows spontaneous and slow axon regrowth through a permissive glial scar after optic nerve axotomy. Although much of the expression pattern of glial, neuronal and extracellular matrix markers have been analyzed by our group, an estimation of the cell loss in the ganglion cell layer (GCL) and the degree of visual function recovery remained unresolved. Thus, we performed a series of tests indicative of effective visual function (pupillary light reflex, accommodation, visually elicited behavior) in 18 lizards at 3, 6, 9 and 12 months post-axotomy which were then processed for immunohistochemistry for the neuronal markers SMI-31 (neurofilaments), Tuj1 (beta-III tubulin) and SV2 (synaptic vesicles) at the last timepoint. Separately, cell loss in the GCL was estimated by comparative quantitation of DAPI+ nuclei in control and 12 months experimental lizards. Additionally, 15 lizards were processed for electron microscopy to monitor relevant ultrastructural changes in the GCL, optic nerve and optic tract throughout regeneration. Hypertrophy of RGCs was persistent, morphology of the regenerated nerves varied from narrow to neuroma-like features and larger regenerated axons underwent remyelination by 9 months. The estimated cell loss in the GCL was 27% and two-third of the animals recovered the pupillary light reflex which involves the pretectum. Strikingly, visually elicited behavior involving the tectum was only restored in two specimens, presumably due to the higher complexity of this pathway. These preliminary results indicate that limited functional regeneration occurs spontaneously in the severely injured visual system of the lacertid G. galloti.

A Slow Axon Antidromic Blockade Hypothesis for Tremor Reduction via Deep Brain Stimulation

Parkinsonian and essential tremor can often be effectively treated by deep brain stimulation. We propose a novel explanation for the mechanism by which this technique ameliorates tremor: a reduction of the delay in the relevant motor control loops via preferential antidromic blockade of slow axons. The antidromic blockade is preferential because the pulses more rapidly clear fast axons, and the distribution of axonal diameters, and therefore velocities, in the involved tracts, is sufficiently long-tailed to make this effect quite significant. The preferential blockade of slow axons, combined with gain adaptation, results in a reduction of the mean delay in the motor control loop, which serves to stabilize the feedback system, thus ameliorating tremor. This theory, without any tuning, accounts for several previously perplexing phenomena, and makes a variety of novel predictions.

Intra-axonal calcium changes after axotomy in wild-type and slow Wallerian degeneration axons

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt–Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (WldS) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4–24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from WldS rats. The step gradient of calcium distribution in WldS is absent in around 20% of the intact nerves beneath SLCs but 4–24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between WldS and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in WldS neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that WldS neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

Corticospinal reorganization after spinal cord injury

The corticospinal tract (CST) is a major descending pathway contributing to the control of voluntary movement in mammals. During the last decades anatomical and electrophysiological studies have demonstrated significant reorganization in the CST after spinal cord injury (SCI) in animals and humans. In animal models of SCI, anatomical evidence showed corticospinal sprouts rostral and caudal to the lesion and their integration into intraspinal axonal circuits. Electrophysiological data suggested that indirect connections from the primary motor cortex to forelimb motoneurons, via brainstem nuclei and spinal cord interneurons, or direct connections from slow uninjured corticospinal axons, might contribute to the control of movement after a CST injury. In humans with SCI, post mortem spinal cord tissue revealed anatomical changes in the CST some of which were similar but others markedly different from those found in animal models of SCI. Human electrophysiological studies have provided ample evidence for corticospinal reorganization after SCI that may contribute to functional recovery. Together these studies have revealed a large plastic capacity of the CST after SCI. There is also a limited understanding of the relationship between anatomical and electrophysiological changes in the CST and control of movement after SCI. Increasing our knowledge of the role of CST plasticity in functional restoration after SCI may support the development of more effective repair strategies.

Corticospinal tract regeneration after spinal cord injury in receptor protein tyrosine phosphatase sigma deficient mice

Receptor protein tyrosine phosphatase sigma (RPTPsigma) plays a role in inhibiting axon growth during development. It has also been shown to slow axon regeneration after peripheral nerve injury and inhibit axon regeneration in the optic nerve. Here, we assessed the ability of the corticospinal tract (CST) axons to regenerate after spinal hemisection and contusion injury in RPTPsigma deficient (RPTPsigma(-/-)) mice. We show that damaged CST fibers in RPTPsigma(-/-) mice regenerate and appear to extend for long distances after a dorsal hemisection or contusion injury of the thoracic spinal cord. In contrast, no long distance axon regeneration of CST fibers is seen after similar lesions in wild-type mice. In vitro experiments indicate that cerebellar granule neurons from RPTPsigma(-/-) mice have reduced sensitivity to the inhibitory effects of chondroitin sulfate proteoglycan (CSPG) substrate, but not myelin, which may contribute to the growth of CST axons across the CSPG-rich glial scar. Our data suggest that RPTPsigma may function to prevent axonal growth after injury in the adult mammalian spinal cord and could be a target for promoting long distance regeneration after spinal cord injury.

Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat.

The role of endogenous GABA and ATP in regulating transmitter release from primary afferent terminals in the superficial dorsal horn of the spinal cord is still controversial. ATP is co-released with GABA from some inhibitory dorsal horn neurons raising the possibility that ATP could act in concert with GABA to regulate transmitter release from primary afferent terminals if receptors to both transmitters are expressed there. Using electrophysiology together with immunocytochemistry, we have investigated the expression of ATP-gated P2X and GABAA receptors by identified subpopulations of dorsal root ganglion (DRG) neurons known to project primarily to the superficial dorsal horn. Expression of the heat-sensitive vanilloid receptor 1 (VR1) and sensitivity to capsaicin were used to characterize DRG neurons sensitive to noxious heat. Both P2X and GABAA receptors were expressed on the majority of DRG neurons examined. Recording compound action potentials (CAPs) from dorsal roots in the presence of muscimol, alpha,beta-methylene-ATP (alpha,beta-meATP) or capsaicin resulted in depression of CAP in the slow and medium conducting fibres, indicating cognate receptor expression on the small diameter axons. Dorsal root-evoked dorsal root potentials (DR-DRPs), reflecting depolarization of primary afferent terminals by endogenously released substances, were depressed by the GABAA receptor antagonist SR95531 and alpha,beta-meATP. These results suggest that GABAA and P2X receptors are expressed on DRG cell bodies and slow fibre axons, many of which are heat-nociceptive. These fibres project to the superficial lamina of the dorsal horn where the receptors may function to modulate transmitter release near their central terminals.