Neurotrophin-3 and TrkC in the frog visual system: changes after axotomy

Neurotrophins are potent regulators of the survival of different neuronal populations in the CNS. Little is known of the immunodistribution of neurotrophin-3 (NT-3) and tyrosine kinase C (TrkC) receptor in the frog visual system, which can successfully regenerate and recover vision after injury. In this study we show that both NT-3 and TrkC are present in the frog retina and tectum, and that their distribution changes after optic nerve transection. Both NT-3 and TrkC are present in the ganglion cell layer, inner nuclear layer, nerve fiber layer and outer plexiform layer, and in Müller cells of control retinas. Quantification of identified RGCs shows that there are only small changes in the proportion, or intensity, of NT-3 immunostained cells surviving after axotomy and regeneration. Müller cell staining, however, is increased. TrkC staining in the retina does not change after axotomy. In the tectum, NT-3 immunoreactivity is present in the retinorecipient layer 9, and in radial processes of neurons and ependymoglia. TrkC is present in ependymoglia and in tectal neurons. After axotomy or colchicine treatment fewer NT-3-immunoreactive processes are present in layer 9 and there is decreased staining of tectal neurons. These data are consistent with the hypothesis that NT-3 is synthesized in the retina and anterogradely transported to the tectum. TrkC immunostaining, on the other hand, increases in tectal cells after optic nerve transection, suggesting that it may be regulated by the supply of NT-3 from the retina.     

Upregulation of BDNF mRNA and trkB mRNA in the nigrostriatal system and in the lesion site following unilateral transection of the medial forebrain bundle

We have performed unilateral transection of the medial forebrain bundle (MFB) and studied BDNF mRNA and trkB mRNA levels at different postlesion times in the nigrostriatal system by means of in situ hybridization. BDNF mRNA levels were transiently induced in the substantia nigra pars compacta at 1 day postaxotomy. The disposition of BDNF mRNA expressing cells at this postlesion time in substantia nigra mimicked that of the dopaminergic neurons expressing the mRNA for the dopamine transporter. TrkB mRNA levels remained unaltered in the ventral mesencephalon at the different postlesion times examined-1 to 14 days. In contrast, trkB mRNA levels were significantly induced in the striatum at the longer postlesion time examined-14 days-when all neurodegenerative events are completed. It is becoming apparent that nigral BDNF mRNA levels are anterogradely transported to its target tissue in striatum. However, following axotomy, the lesion site represents a second potential target for BDNF action. Consequently, we also analyzed the pattern of mRNA expression for BDNF and trkB at the lesion site where dopaminergic axons are disconnected. There, we found notable inductions of both BDNF mRNA and trkB mRNA levels at 4 days postaxotomy. BDNF mRNA expressing cells were confined at the site of axotomy, which coincided precisely to that showing induction of trkB mRNA. Altogether, our results anticipate promising trophic roles of BNDF in the injured nigrostriatal system.     

Changes in BDNF and neurotrophin receptor expression in degenerating and regenerating rat retinal ganglion cells

Purpose: Exogenously applied BDNF has been shown to rescue rat retinal ganglion cells (RGCs) from axotomy-induced apoptotic death, presumably via activation of its high affinity receptor TrkB. Since both TrkB and BDNF are endogenously expressed in RGCs, auto- or para-crine neurotrophic loops in the retina may be involved. In the present study, we investigated whether expression levels of BDNF, TrkA, TrkB, TrkC and p75 protein in RGCs are specifically regulated following axonal lesion and during regeneration of optic fibres in the adult rat. Methods: By double labelling retinal cryosections with Fluorogold and respective antibodies we determined the percentage of RGCs expressing the above-mentioned markers. In addition, mRNA levels of BDNF and TrkB were measured using quantitative RT-PCR. Results: Compared to controls the number of BDNF-positive RGCs increased twofold 2 days after axotomy and the percentage of RGCs expressing TrkB was elevated by 50 %. Correspondingly, mRNA levels of BDNF increased about twofold 2 days after axotomy. During regen-eration, the percentage of BDNF-immunoreactive RGCs was further elevated compared to axotomy alone. The number of TrkA-positive RGCs doubled after axotomy, whereas no significant change in TrkC expression was observed. P75 expression was not detected in adult rat RGCs. Conclusion: Our results suggest that intrinsic rescue mechanisms may contribute to short term neuronal survival and axonal regeneration of RGCs after axonal lesions.     

Changes in BDNF and neurotrophin receptor expression in degenerating and regenerating rat retinal ganglion cells

Purpose: Exogenously applied BDNF has been shown to rescue rat retinal ganglion cells (RGCs) from axotomy-induced apoptotic death, presumably via activation of its high affinity receptor TrkB. Since both TrkB and BDNF are endogenously expressed in RGCs, auto- or para-crine neurotrophic loops in the retina may be involved. In the present study, we investigated whether expression levels of BDNF, TrkA, TrkB, TrkC and p75 protein in RGCs are specifically regulated following axonal lesion and during regeneration of optic fibres in the adult rat. Methods: By double labelling retinal cryosections with Fluorogold and respective antibodies we determined the percentage of RGCs expressing the above-mentioned markers. In addition, mRNA levels of BDNF and TrkB were measured using quantitative RT-PCR. Results: Compared to controls the number of BDNF-positive RGCs increased twofold 2 days after axotomy and the percentage of RGCs expressing TrkB was elevated by 50 %. Correspondingly, mRNA levels of BDNF increased about twofold 2 days after axotomy. During regen-eration, the percentage of BDNF-immunoreactive RGCs was further elevated compared to axotomy alone. The number of TrkA-positive RGCs doubled after axotomy, whereas no significant change in TrkC expression was observed. P75 expression was not detected in adult rat RGCs. Conclusion: Our results suggest that intrinsic rescue mechanisms may contribute to short term neuronal survival and axonal regeneration of RGCs after axonal lesions.     

Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy

Numerous experimental therapies to promote axonal regeneration have shown promise in animal models of acute spinal cord injury, but their effectiveness is often found to diminish with a delay in administration. We evaluated whether brain-derived neurotrophic factor (BDNF) application to the spinal cord injury site 2 months after cervical axotomy could promote a regenerative response in chronically axotomized rubrospinal neurons. BDNF was applied to the spinal cord in three different concentrations 2 months after cervical axotomy of the rubrospinal tract. The red nucleus was examined for reversal of neuronal atrophy, GAP43 and Talpha1 tubulin mRNA expression, and trkB receptor immunoreactivity. A peripheral nerve transplant paradigm was used to measure axonal regeneration into peripheral nerve transplants. Rubrospinal axons were anterogradely traced and trkB receptor immunohistochemistry performed on the injured spinal cord. We found that BDNF treatment did not reverse rubrospinal neuronal atrophy, nor promote GAP-43 and Talpha1 tubulin mRNA expression, nor promote axonal regeneration into peripheral nerve transplants. TrkB receptor immunohistochemistry demonstrated immunoreactivity on the neuronal cell bodies, but not on anterogradely labeled rubrospinal axons at the injury site. These findings suggest that the poor response of rubrospinal neurons to BDNF applied to the spinal cord injury site 2 months after cervical axotomy is not related to the dose of BDNF administered, but rather to the loss of trkB receptors on the injured axons over time. Such obstacles to axonal regeneration will be important to identify in the development of therapeutic strategies for chronically injured individuals.     

Distribution of substance P-like immunoreactive retinal ganglion cells and their pattern of termination in the optic tectum of chick (Gallus gallus)

Substance P-like immunoreactive (SP-LI) neurons were identified within the inner nuclear layer and ganglion cell layer of the chick retina. The SP-LI cells in the inner nuclear layer consisted of several subtypes of neurons, differing in soma size and dendritic arborization. In the ganglion cell layer a population of moderately labelled SP-LI neurons was also present. About 6-9 microns in diameter and spaced 50-80 microns apart, they formed a regular array across the entire retina, with a density of about 400 cells/mm2 in the superior temporal retina, declining to less than 100 cells/mm2 in the peripheral retina. The total number of SP-LI cells in the ganglion cell layer was approximately 75,000. Individual axons could be followed toward the optic nerve head. Lesions near the optic nerve head resulted in axotomy of ganglion cells within a limited portion of the retina. Two days of postaxotomy there were numerous SP-LI swellings in the proximal segments of axotomized axons. SP-LI neurons in the axotomized zone were larger, more numerous, and showed increased staining of their processes. Fourteen days following a retinal lesion, there was depletion of all SP-LI cells in the ganglion cell layer within the axotomized zone, but the SP-LI neurons in the inner nuclear layer were not noticeably affected. Following a localized injection of rhodamine-coupled latex beads into the optic tectum, a population of retinal ganglion cells (RGCs) in the contralateral retina was retrogradely labelled. Many of these cells also exhibited SP-like immunoreactivity. Examination of the optic tectum indicated the presence of SP-LI fibres in laminae 2-13 (nomenclature of Cajal: Histologie du Systeme Nerveux. Vol. 2. Paris: Maloine, '11), with immunoreactive terminal regions present mainly in laminae 2-4, 7, and 9-13. SP-LI cell bodies were found predominantly in laminae 10-12 and 13. Fourteen days following a retinal lesion, SP-LI processes and terminals were depleted from laminae 2 and 3. Immunoreactive cells and processes in the remaining laminae of the optic tectum were not noticeably altered. The present report confirms the existence of SP-LI retinal ganglion cells in the chick retina and demonstrates their contribution to lamina specific SP-LI arborization in the optic tectum.     

Basic fibroblast growth factor applied to the optic nerve after injury increases long-term cell survival in the frog retina

The neuroprotective effects of basic fibroblast growth factor (bFGF) on the long-term survival of axotomized retinal ganglion cells (RGCs) were studied in the frog Rana pipiens. Cell loss was quantified in different regions of the ganglion cell layer using Nissl staining and tetramethylrhodamine dextran amine backfilling. All regions of the retina showed a significant decrease (32-66%) in RGC numbers between 4 and 16 weeks after axotomy. Some cells showed morphological and biochemical signs of apoptosis. A single application of bFGF to the optic nerve stump at the time of axotomy protected many of the cells 6 weeks after the injury, but this effect was lost by 12 weeks. A second application of bFGF, 6 weeks after the injury, rescued many RGCs at 12 weeks. In contrast, single or double injections of bFGF into the eyeball had no effect on RGC survival. Axotomized RGCs were significantly enlarged and elongated after axotomy, and these morphological changes were increased by bFGF treatment. In the normal retina and optic nerve, immunocytochemical staining showed bFGF-like immunoreactivity (-LI) in the pigment epithelial layer, in the outer segments of photoreceptors, and in occasional RGCs. Strong bFGF-LI was present in Müller cells and in optic nerve astrocytes and oligodendrocytes. FGF receptor-LI was present in photoreceptors, outer plexiform layer, retinal ganglion cell axons, and Müller cells. FGF receptor-LI was also observed in optic nerve glia.     

The Distribution of Brain-derived Neurotrophic Factor and its Receptor trkB in Parvlbumin-containing Neurons of the Rat Visual Cortex

We analysed the distribution of brain-derived neurotrophic factor (BDNF) and its receptor trkB in the adult rat visual cortex, paying particular attention to a GABAergic neuronal subpopulation—the parvalburnin-positive cells. We found expression of trkB in the cell body and apical dendrite of pyramidal neurons and in the cell body of non-pyramidal neurons. Double labelling experiments revealed extensive colocalization of parvalbumin and trkB immunoreactivity in non-pyramidal neurons. Interestingly, the trkB-positive pyramidal neurons appeared surrounded by parvalbumin-labelled boutons. The use of double immunohistochemistry and in situ hybridization histochemistry showed that parvalbumin-positive neurons express trkB mRNA. BDNF rnRNA was found in several cells. Coexpression of BDNF mRNA and parvalbumin immunoreactivity was extremely rare. These data strongly suggest that BDNF synthesized by cortical neurons acts as a postsynaptically derived factor for parvalbumin-positive neurons in the adult rat visual cortex.     

Up-regulation of brain-derived neurotrophic factor by application of fibroblast growth factor-2 to the cut optic nerve is important for long-term survival of retinal ganglion cells

Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes the survival of retinal ganglion cells (RGCs) in the frog Rana pipiens and results in a rapid up-regulation of brain-derived neurotrophic factor (BDNF) and TrkB synthesis by the RGCs. Here we investigate whether this up-regulation is maintained over the long term and whether it is required for FGF-2's survival effect. At 6 weeks after axotomy and FGF-2 treatment, we found more RGCs immunopositive for BDNF protein and higher intensity of BDNF and TrkB immunostaining, accompanied by increases in BDNF and TrkB mRNA in RGCs. Application of fluorescently labeled siRNA targeted against BDNF to the cut RGC axons showed that it was transported to the cell bodies. Axonal siRNA treatment eliminated the increases in BDNF immunostaining and mRNA that were induced by FGF-2 and had no effect on TrkB mRNA. This reduction in BDNF synthesis by siRNA greatly reduced the long-term survival effect of FGF-2 on RGCs. This, taken together with previous results, suggests that, although FGF-2 may initially activate survival pathways via ERK signaling, its main long-term survival effects are mediated via its up-regulation of BDNF synthesis by the RGCs.     

BDNF and TrkB expression in intrastriatal ventral mesencephalic grafts in a rat model of Parkinson's disease

Summary. We investigated the expression of BDNF and its high affinity receptor trkB in fetal dopaminergic grafts in a rat model of Parkinson's disease. Grafts were allowed to differentiate for 7, 14, 28, or 56 days, respectively and were analyzed immunocytochemically thereafter with antibodies directed against tyrosine hydroxylase, BDNF and trkB. At all time points investigated, grafts contained tyrosine hydroxylase immunoreactive neurons. Immature grafts (7 days) displayed no immunoreactivity for BDNF which was restricted to glial cells at the graft-host interface. After longer differentiation periods BDNF-immunoreactivity was detectable in neurons and astrocytes within the grafts. No trkB immunoreactivity was found in immature grafts but a strong signal for trkB emerged in grafted neurons older than 14 days whereas glial cells remained unlabeled at all time points investigated. Expression of BDNF and trkB in grafted neurons and of BDNF in sourrounding glial cells suggests an autocrine or paracrine action of BDNF on dopaminergic neurons possibly mediated by activated glia. Accepted December 15, 1997; received October 28, 1997     

Brain-derived neurotrophic factor modulates GAP-43 but not tα1 expression in injured retinal ganglion cells of adult rats

The administration of neurotrophins affects neuronal survival and growth, but less is known about their ability to modify the expression of growth associated genes following injury to CNS neurons. Here we characterize the effect of brain-derived neurotrophic factor (BDNF) on mRNA levels for Tα1 α-tubulin, and for GAP-43, two genes whose expression levels in retinal ganglion cells (RGC) tend to correlate with growth. We first determined that most adult rat RGCs can retrogradely transport BDNF by injecting ¹²⁵I-BDNF into RGC target sites in vivo. We then used quantitative in situ hybridization to characterize the effect of axotomy, or axotomy and BDNF administration on mRNA levels for GAP-43 and Tα1. Axotomy alone resulted in a general decrease in Tα1 α-tubulin mRNA levels by 2 weeks, and elicited an increase in GAP-43 mRNA levels in an average of 30% of surviving RGCs. The intravitreal administration of a single dose of BDNF (5 μg) to axotomized RGCs on the day of injury did not affect Tα1 α-tubulin mRNA levels, but was followed by a moderate (approximately 80%), and short-lasting enhancement of GAP-43 mRNA levels in most RGCs during the first week after axotomy. No significant increase in GAP-43 mRNA levels was observed when BDNF was injected into the uninjured eye. We conclude that BDNF specifically enhances GAP-43 but not Tα1 mRNA levels in injured RGCs. Because BDNF is known to stimulate branch length of injured RGCs, we suggest that changes in the expression of GAP-43, but not Tα1 tubulin, correlate with branching of injured neurons as opposed to long distance regrowth. J. Neurosci. Res. 47:561–572, 1997. © 1997 Wiley-Liss, Inc.     

Reelin expression in the retina and optic tectum of developing common brown trout

Reelin (RELN) is an extracellular matrix protein largely related with laminar organization in several brain areas. The development of RELN immunoreactivity in the retina and the optic tectum of the brown trout are analyzed with a monoclonal (142) antibody against RELN whose suitability has been ascertained by western blot. In the retina of embryos and alevins, RELN immunoreactivity is detected in cells of the ganglion cell layer (GCL) and inner nuclear layer (INL), and in the inner plexiform layer (IPL), where it appears as "diffuse" material confined to the ON-sublayer. In juveniles, RELN expression becomes restricted to a stripe of cells in the INL. RELN-immunoreactive (RELN-ir) cells are absent from the outer nuclear layer (ONL) at any developmental stage. The developmental pattern of RELN expression in the trout retina shows many similarities with that of amniotes: (a) RELN expression parallels the vitreal to scleral progression of differentiation of the retina and, within each cell layer, RELN immunoreactivity appears confined to a subpopulation of postmitotic cells; (b) at early stages RELN expression is exclusively observed in the central retina and as maturation progresses from the center to the periphery, more RELN-ir cells are observed following the same spatial pattern. Differences with amniotes are noted regarding the absence of RELN expression in the GCL and INL in adulthood, and in the ONL at any developmental stage. In the optic tectum (OT) of trout, as in amniotes, RELN immunoreactivity increases within specific cell layers as lamination proceeds, and decreases when it is complete, except in the stratum opticum (SO), where RELN-ir cells are observed throughout life. Time-course expression of RELN in the OT suggests a role in the early modeling of synaptic contacts and the accommodation of new retinal arriving axons throughout life.     

Differential taurine effect on outgrowth from goldfish retinal ganglion cells after optic crush or axotomy. Influence of the optic tectum

The interaction between innervated tissues, targets and nerves is crucial in the maintenance of physiological conditions, and the disturbance of this harmony causes the production of morphological and biochemical changes. After lesion of the optic nerve, several modifications take place in the retina, the optic tectum and the optic nerve. The influence of the tectum on the outgrowth from the goldfish retina and the possible role of taurine was studied. Ganglion retinal cells were identified by retrolabeling with Dil. Crushing the optic nerve 10 days prior to plating retinal cells, as compared with optic axotomy, did not affect the survival of cultured retinal cells, as well as the length of the neurites. However, the number of neurites per cell and the branching of the longest fiber were higher after axotomy than after crushing. The addition of taurine to the medium did not modify this response at 5 days in culture. At early periods in culture, the stimulatory effect on isolated ganglion cell outgrowth produced by taurine was enhanced after axotomy respecting crushing of the optic nerve, but was not affected in retinal explants. The addition of medium from cultured optic tectum several days post-crush of the optic nerve to retinal explants from intact retinas or coming from post-crush retina modified the outgrowth, being inhibitory or stimulatory in a time-dependent manner. The co-culture of optic tectum and retina also affected the outgrowth from the retina with a byphasic shape. The results support the differential response of the retina facing partial or complete interruption with the target and limit the effect of taurine to early periods in culture. In addition, the production of inhibitory factors from the tectum, plus the stimulatory ones, are strongly supported by this work.     

Anterograde axonal transport of BDNF and NT-3 by retinal ganglion cells: roles of neurotrophin receptors

Retinal ganglion cells (RGCs) transport exogenous neurotrophins anterogradely to the midbrain tectum/superior colliculus with significant downstream effects. We determined contributions of neurotrophin receptors for anterograde transport of intraocularly injected radiolabeled neurotrophins. In adult rodents, anterograde transport of brain-derived neurotrophic factor (BDNF) was receptor-mediated, and transport of exogenous BDNF and neurotrophin-3 (NT-3) was more efficient, per RGC, in rodents than chicks. RT-PCR and Western blot analysis of purified murine RGCs showed that adult RGCs express the p75 receptor. Anterograde transport of BDNF or NT-3 was not diminished in p75 knock-out mice (with unaltered final numbers of RGCs), but BDNF transport was substantially reduced by co-injected trkB antibodies. In chick embryos, however, p75 antisense or co-injected p75 antibodies significantly attenuated anterograde transport of NT-3 by RGCs. Thus, neither BDNF nor NT-3 utilizes p75 for anterograde transport in adult rodent RGCs, while anterograde NT-3 transport requires the p75 receptor in embryonic chicken RGCs.     

Target-derived BDNF (brain-derived neurotrophic factor) is essential for the survival of developing neurons in the isthmo-optic nucleus

Neurons in the peripheral nervous system depend on single neurotrophic factors, whereas those in the brain are thought to utilize many different trophic factors. This study examined whether some neurons in the brain critically depend on a single trophic factor during development. Neurons in the isthmo-optic nucleus (ION) of chick embryos respond to exogenous brain-derived neurotrophic factor (BDNF). Relatively high concentrations of endogenous BDNF were present in the ION of 14-18-day-old chick embryos. ION target cells in the retina were immunolabeled for BDNF but showed surprisingly low levels of BDNF mRNA. These data suggest that ION target cells derive some BDNF from other retinal sources. No BDNF mRNA was detected in the ION itself. ION neurons had a very efficient retrograde transport system for BDNF and exogenous BDNF arrived in the ION intact. When the ION was deprived of endogenous trkB ligands by injection of trkB fusion proteins in the eye, cell death of ION neurons was enhanced, and this effect was mimicked by BDNF-specific blocking antibodies in the eye. TrkB fusion proteins in the retina induced cell death of ION neurons prior to visible effects on ION target cells in the retina. Immunolabel for endogenous BDNF was sparse in pyknotic ION neurons, suggesting that ION neurons with low BDNF content were eliminated by apoptosis. These data show that BDNF is an essential target-derived trophic factor for developing ION neurons and thereby validate the neurotrophic hypothesis for at least one neuronal population in the brain.     

BDNF reduces miniature inhibitory postsynaptic currents by rapid downregulation of GABA(A) receptor surface expression

Changes in neurotransmitter receptor density at the synapse have been proposed as a mechanism underlying synaptic plasticity. Neurotrophic factors are known to influence synaptic strength rapidly. In the present study, we found that brain-derived neurotrophic factor (BDNF) acts postsynaptically to reduce gamma-aminobutyric acid (GABA)-ergic function. Using primary cultures of rat hippocampal neurons, we investigated the effects of BDNF on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and on the localization of GABAA receptors. Application of BDNF (100 ng/mL) led within minutes to a marked reduction (33.5%) of mIPSC amplitudes in 50% of neurons, recorded in the whole-cell patch-clamp mode, leaving frequency and decay kinetics unaffected. This effect was blocked by the protein kinase inhibitor K252a, which binds with high affinity to trkB receptors. Immunofluorescence staining with an antibody against trkB revealed that about 70% of cultured hippocampal pyramidal cells express trkB. In dual labelling experiments, use of neurobiotin injections to label the recorded cells revealed that all cells responsive to BDNF were immunopositive for trkB. Treatment of cultures with BDNF reduced the immunoreactivity for the GABAA receptor subunits-alpha2, -beta2,3 and -gamma2 in the majority of neurons. This effect was detectable after 15 min and lasted at least 12 h. Neurotrophin-4 (NT-4), but not neurotrophin-3 (NT-3), also reduced GABAA receptor immunoreactivity, supporting the proposal that this effect is mediated by trkB. Altogether the results suggest that exposure to BDNF induces a rapid reduction in postsynaptic GABAA receptor number that is responsible for the decline in GABAergic mIPSC amplitudes.     

FGF-2 modulates expression and distribution of GAP-43 in frog retinal ganglion cells after optic nerve injury

Basic fibroblast growth factor (bFGF or FGF-2) has been implicated as a trophic factor that promotes survival and neurite outgrowth of neurons. We found previously that application of FGF-2 to the proximal stump of the injured axon increases retinal ganglion cell (RGC) survival. We determine here the effect of FGF-2 on expression of the axonal growth-associated phosphoprotein (GAP)-43 in retinal ganglion cells and tectum of Rana pipiens during regeneration of the optic nerve. In control retinas, GAP-43 protein was found in the optic fiber layer and in optic nerve; mRNA levels were low. After axotomy, mRNA levels increased sevenfold and GAP-43 protein was significantly increased. GAP-43 was localized in retinal axons and in a subset of RGC cell bodies and dendrites. This upregulation of GAP-43 was sustained through the period in which retinal axons reconnect with their target in the tectum. FGF-2 application to the injured nerve, but not to the eyeball, increased GAP-43 mRNA in the retina but decreased GAP-43 protein levels and decreased the number of immunopositive cell bodies. In the tectum, no treatment affected GAP-43 mRNA but FGF-2 application to the axotomized optic nerve increased GAP-43 protein in regenerating retinal projections. We conclude that FGF-2 upregulates the synthesis and alters the distribution of the axonal growth-promoting protein GAP-43, suggesting that it may enhance axonal regrowth.