Distribution of neurotrophin-3 during the ontogeny and regeneration of the lizard (Gallotia galloti) visual system

We have previously described the spontaneous regeneration of retinal ganglion cell axons after optic nerve (ON) transection in the adult Gallotia galloti. As neurotrophin-3 (NT-3) is involved in neuronal differentiation, survival and synaptic plasticity, we performed a comparative immunohistochemical study of NT-3 during the ontogeny and regeneration (after 0.5, 1, 3, 6, 9, and 12 months postlesion) of the lizard visual system to reveal its distribution and changes during these events. For characterization of NT-3(+) cells, we performed double labelings using the neuronal markers HuC-D, Pax6 and parvalbumin (Parv), the microglial marker tomato lectin or Lycopersicon esculentum agglutinin (LEA), and the astroglial markers vimentin (Vim) and glial fibrillary acidic protein (GFAP). Subpopulations of retinal and tectal neurons were NT-3(+) from early embryonic stages to adulthood. Nerve fibers within the retinal nerve fiber layer, both plexiform layers and the retinorecipient layers in the optic tectum (OT) were also stained. In addition, NT-3(+)/GFAP(+) and NT-3(+)/Vim(+) astrocytes were detected in the ON, chiasm and optic tract in postnatal and adult lizards. At 1 month postlesion, abundant NT-3(+)/GFAP(+) astrocytes and NT-3(-)/LEA(+) microglia/macrophages were stained in the lesioned ON, whereas NT-3 became downregulated in the experimental retina and OT. Interestingly, at 9 and 12 months postlesion, the staining in the experimental retina resembled that in control animals, whereas bundles of putative regrown fibers showed a disorganized staining pattern in the OT. Altogether, we demonstrate that NT-3 is widely distributed in the lizard visual system and its changes after ON transection might be permissive for the successful axonal regrowth.     

The outgrowth response of the axons of developing and regenerating rat retinal ganglion cells in vitro to neurotrophin treatment

BDNF and NT-4 (but not NT-3 or CNTF) significantly enhanced the outgrowth of early embryonic and adult regenerating RGC axons when provided with a supportive substrate in vitro. BDNF and NT-4 treatment transiently increased RGC axon outgrowth from E15 rat retinas but not from retinas at older embryonic ages. The transient effect of BDNF and NT-4 and the inability of the neurotrophins to promote outgrowth from older embryonic retinal explants suggests a time frame of neurotrophin action and that other chemical factors (target-derived or otherwise) may be necessary for the continued maintenance of developing RGC axons. BDNF and NT-4 also enhanced the outgrowth of regenerating axons from adult retinal explants, but appeared to have a more subtle effect on axon outgrowth, in that the growth-promoting effects of BDNF and NT-4 appeared continuous throughout the incubation period. The suppression of RGC axon outgrowth from embryonic and adult retinae cultured in trkB-IgG-containing medium suggests that the response of developing and regenerating axons, to BDNF and NT-4 are likely to occur through trkB signalling.     

Regeneration of retinal axons in the lizard Gallotia galloti is not linked to generation of new retinal ganglion cells

Using anterograde tracing with HRP and antibodies (ABs) against neurofilaments, we show that regrowth of retinal ganglion cell (RGC) axons in the lizard Gallotia galloti commences only 2 months after optic nerve transection (ONS) and continues over at least 9 months. This is unusually long when compared to RGC axon regeneration in fish or amphibians. Following ONS, lizard RGCs up-regulate the immediate early gene C-JUN for 9 months or longer, indicating their reactive state. In keeping with the in vivo data, axon outgrowth from lizard retinal explants is increased above control levels from 6 weeks, reaches its maximum as late as 3 months, and remains elevated for at least 1 year after ONS. By means of BrdU incorporation assays and antiproliferating cell nuclear antigen immunohistochemistry, we show that the late axon outgrowth is not derived from new RGCs that might have arisen in reaction to ONS: no labeled cells were detected in lizard retinas at 0.5, 1, 1.5, 3, 6, and 12 months after ONS. Conversely, numbers of RGCs undergoing apoptosis were too low to be detectable in TUNEL assays at any time after ONS. These results demonstrate that retinal axon regeneration in G. galloti is due to axon regrowth from the resident population of RGCs, which remain in a reactive state over an extended time interval. Neurogenesis does not appear to be involved in RGC axon regrowth in G. galloti.     

Enriched Environment Protects the Optic Nerve from Early Diabetes-Induced Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Axoglial alterations of the distal (close to the chiasm) optic nerve (ON) could be the first structural change of the visual pathway in streptozotocin (STZ)-induced diabetes in rats. We analyzed the effect of environmental enrichment on axoglial alterations of the ON provoked by experimental diabetes. For this purpose, three days after vehicle or STZ injection, animals were housed in enriched environment (EE) or remained in a standard environment (SE) for 6 weeks. Anterograde transport, retinal morphology, optic nerve axons (toluidine blue staining and phosphorylated neurofilament heavy immunoreactivity), microglia/macrophages (ionized calcium binding adaptor molecule 1 (Iba-1) immunoreactivity), astrocyte reactivity (glial fibrillary acid protein-immunostaining), myelin (myelin basic protein immunoreactivity), ultrastructure, and brain derived neurotrophic factor (BDNF) levels were assessed in non-diabetic and diabetic animals housed in SE or EE. No differences in retinal morphology or retinal ganglion cell number were observed among groups. EE housing which did not affect the STZ-induced weight loss and hyperglycemia, prevented a decrease in the anterograde transport from the retina to the superior colliculus, ON axon number, and phosphorylated neurofilament heavy immunoreactivity. Moreover, EE housing prevented an increase in Iba-1 immunoreactivity, and astrocyte reactivity, as well as ultrastructural myelin alterations in the ON distal portion at early stages of diabetes. In addition, EE housing avoided a decrease in BDNF levels induced by experimental diabetes. These results suggest that EE induced neuroprotection in the diabetic visual pathway.     

Differential effects of intravitreal optic nerve and sciatic nerve grafts on the survival of retinal ganglion cells and the regeneration of their axons

We have investigated the effects of intravitreal sciatic nerve (SN) and/or optic nerve (ON) grafts on the survival and the axonal regeneration of retinal ganglion cells (RGCs). Following transection of the ON, approximately 40% RGCs survived at 7 days post-axotomy (dpa). Results showed that the intravitreal ON graft significantly promoted the survival of RGCs at 7 dpa (39,063 vs 28,246). Intravitreal SN graft, however, did not rescue axotomized RGCs at 5, 7 or 14 dpa. Axotomized RGCs could be induced to regenerate axons along a segment of SN graft attached to the proximal stump of ON. On average, 608 axotomized RGCs were induced to regenerate axons along the attached SN graft. The presence of intravitreal SN graft promoted about 100% increase in the number of regenerating RGCs (1,227) relative to the control groups. The intravitreal ON graft, surprisingly, also induced about 100% more regenerating RGCs (1220) than in the control group. When SN and ON grafts were co-transplanted into the vitreous, about 200% more regenerating RGCs (1916) were observed than in the control group. These findings illustrated that the intravitreal ON graft rescued axotomized RGCs and enhanced the regeneration of retinal axons. This is the first report to show that ON promotes RGC axonal regeneration. The intravitreal SN graft did not rescue RGCs but promoted axonal regeneration. The differential effects of intravitreal ON and SN grafts on the survival and the RGC regeneration suggest that these might be two independently operating events.     

Glial cell line–derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: Comparison to and combination with brain‐derived neurotrophic factor (BDNF)

Adult rat retinal ganglion cells (RGC) undergo degeneration after optic nerve transection. Studies have shown that exogenously applied neurotrophic factors such as brain‐derived neurotrophic factor (BDNF) can attenuate axotomy‐induced as well as developmental RGC death. Here, we examined whether glial cell line–derived neurotrophic factor (GDNF), a known neurotrophic factor for dopaminergic neurons and motor neurons, could provide neurotrophic support to RGC in adult rats. We determined whether RGC could retrogradely transport GDNF from their target tissue. After injection into the superior colliculus of adult rats, ¹²⁵I‐GDNF was retrogradely transported to contralateral eyes but not to ipsilateral eyes. The transport of ¹²⁵I‐GDNF could be blocked by coinjection of excess unlabeled GDNF, indicating that it was receptor mediated. We tested whether intravitreally applied GDNF could prevent axotomy‐induced RGC degeneration. The RGC were prelabeled with Fluorogold (FG) and axotomized by intraorbital optic nerve transection. GDNF, BDNF (positive control), cytochrome c (negative control), or a GDNF/BDNF combination was injected intravitreally on days 0 and 7. On day 14, FG‐labeled RGC were counted from whole‐mount retinas. We found that, similar to BDNF, GDNF could significantly attenuate the degeneration of RGC in a dose‐dependent fashion. Furthermore, the combination treatment of GDNF and BDNF showed better protection than either factor used individually. Our data indicate that GDNF is a neurotrophic factor for the adult rat RGC. GDNF, like BDNF, may be useful for the treatment of human RGC degenerative diseases. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 382–390, 1999     

Tenascin-R and axon growth-promoting molecules are up-regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway.     

Increased expression and activation of poly(ADP-ribose) polymerase (PARP) contribute to retinal ganglion cell death following rat optic nerve transection

Excessive activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) by free-radical damaged DNA mediates necrotic cell death in injury models of cerebral ischemia-reperfusion and excitotoxicity. We recently reported that secondary retinal ganglion cell (RGC) death following rat optic nerve (ON) transection is mainly apoptotic and can significantly but not entirely be blocked by caspase inhibition. In the present study, we demonstrate transient, RGC-specific PARP activation and increased retinal PARP expression early after ON axotomy. In addition, intravitreal injections of 3-aminobenzamide blocked PARP activation in RGCs and resulted in an increased number of surviving RGCs when compared to control animals 14 days after ON transection. These data indicate that secondary degeneration of a subset of axotomized RGCs results from a necrotic-type cell death mediated by PARP activation and increased PARP expression. Furthermore, PARP inhibition may constitute a relevant strategy for clinical treatment of traumatic brain injury.     

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: comparison to and combination with brain-derived neurotrophic factor (BDNF)

Adult rat retinal ganglion cells (RGC) undergo degeneration after optic nerve transection. Studies have shown that exogenously applied neurotrophic factors such as brain-derived neurotrophic factor (BDNF) can attenuate axotomy-induced as well as developmental RGC death. Here, we examined whether glial cell line-derived neurotrophic factor (GDNF), a known neurotrophic factor for dopaminergic neurons and motor neurons, could provide neurotrophic support to RGC in adult rats. We determined whether RGC could retrogradely transport GDNF from their target tissue. After injection into the superior colliculus of adult rats, 125I-GDNF was retrogradely transported to contralateral eyes but not to ipsilateral eyes. The transport of 125I-GDNF could be blocked by coinjection of excess unlabeled GDNF, indicating that it was receptor mediated. We tested whether intravitreally applied GDNF could prevent axotomy-induced RGC degeneration. The RGC were prelabeled with Fluorogold (FG) and axotomized by intraorbital optic nerve transection. GDNF, BDNF (positive control), cytochrome c (negative control), or a GDNF/BDNF combination was injected intravitreally on days 0 and 7. On day 14, FG-labeled RGC were counted from whole-mount retinas. We found that, similar to BDNF, GDNF could significantly attenuate the degeneration of RGC in a dose-dependent fashion. Furthermore, the combination treatment of GDNF and BDNF showed better protection than either factor used individually. Our data indicate that GDNF is a neurotrophic factor for the adult rat RGC. GDNF, like BDNF, may be useful for the treatment of human RGC degenerative diseases.     

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.     

Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons

Trigeminal sensory axons project to several epithelial targets, including those of the maxillary and mandibular processes. Previous studies identified a chemoattractant activity, termed Maxillary Factor, secreted by these processes, which can attract developing trigeminal axons in vitro. We report that Maxillary Factor activity is composed of two neurotrophins, neurotrophin-3 (NT-3) and Brain-Derived Neurotrophic Factor (BDNF), which are produced by both target epithelium and pathway mesenchyme and which are therefore more likely to have a trophic effect on the neurons or their axons than to provide directional information, at least at initial stages of trigeminal axon growth. Consistent with this, the initial trajectories of trigeminal sensory axons are largely or completely normal in mice deficient in both BDNF and NT-3, indicating that other cues must be sufficient for the initial stages of trigeminal axon guidance.     

Ontogeny of the conus papillaris of the lizard Gallotia galloti and cellular response following transection of the optic nerve: an immunohistochemical and ultrastructural study

Spontaneous regrowth of the axons of retinal ganglion cells (RGC) occurs after unilateral optic nerve transection (ONT) in the lizard Gallotia galloti. We have performed an immunohistochemical and ultrastructural study of the conus papillaris (CP) of this lizard during ontogeny and after ONT in order to characterize its cell subpopulations, innervation and putative blood-brain barrier (BBB) and to evaluate changes occurring throughout regeneration. Proliferating PCNA⁺ cells were abundant between embryonic stage 33 (E33) and hatching. From E33, we observed Pax2⁺/GS⁺ glial cells in the primitive CP, which became increasingly pigmented and vascularised from E35. Conal astrocytes coexpressing Pax2 with vimentin and/or GFAP were identified from E37-E38. GluT-1⁺/LEA⁺/Pax2^- endothelial cells (ECs) formed a continuous endothelium with tight junctions and luminal and abluminal microfolds. In adults, the peripheral blood vessels showed a thinner calibre, stronger GluT-1 staining and more abundant microfolds than those of the central CP indicating the higher specialization involved during transport within the former. Occasional pericytes, abundant Pax2⁺ pigment cells, LEA⁺ microglia/macrophages, unmyelinated Tuj1⁺ nerve fibres and SV2⁺ synaptic vesicles were also observed in the perivascular zone. After ONT, the expression of GluT-1 and p75^NTR persisted in ECs, suggesting the preservation/early recovery of the BBB. Relevant ultrastructural alterations were observed at 0.5?months postlesion, although, by 3?months, the CP had recovered the ultrastructure of controls indicating tissue recovery. Abnormal newly formed blood vessels had developed in the CP-optic nerve junction. Thus, the CP is a central nervous system structure whose regenerating capacity might be key for the nutritional support of regenerating RGCs in G. galloti.     

Neuronal Injury External to the Retina Rapidly Activates Retinal Glia,Followed by Elevation of Markers for Cell Cycle Re-Entry and Death in Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.     

Peculiar and typical oligodendrocytes are involved in an uneven myelination pattern during the ontogeny of the lizard visual pathway

We studied the myelination of the visual pathway during the ontogeny of the lizard Gallotia galloti using immunohistochemical methods to stain the myelin basic protein (MBP) and proteolipid protein (PLP/DM20), and electron microscopy. The staining pattern for the PLP/DM20 and MBP overlapped during the lizard ontogeny and was first observed at E39 in cell bodies and fibers located in the temporal optic nerve, optic chiasm, middle optic tract, and in the stratum album centrale of the optic tectum (OT). The expression of these proteins extended to the nerve fiber layer (NFL) of the temporal retina and to the outer strata of the OT at E40. From hatching onwards, the labeling became stronger and extended to the entire visual pathway. Our ultrastructural data in postnatal and adult animals revealed the presence of both myelinated and unmyelinated retinal ganglion cell axons in all visual areas, with a tendency for the larger axons to show the thicker myelin sheaths. Moreover, two kinds of oligodendrocytes were described: peculiar oligodendrocytes displaying loose myelin sheaths were only observed in the NFL, whereas typical medium electron-dense oligodendrocytes displaying compact myelin sheaths were observed in the rest of the visual areas. The weakest expression of the PLP/DM20 in the NFL of the retina appears to be linked to the loose appearance of its myelin sheaths. We conclude that typical and peculiar oligodendrocytes are involved in an uneven myelination process, which follows a temporo-nasal and rostro-caudal gradient in the retina and ON, and a ventro-dorsal gradient in the OT.     

Developing vestibular ganglion neurons switch trophic sensitivity from BDNF to GDNF after target innervation

Recent evidence showing a distinctive cell loss in vestibular and cochlear ganglia of brain-derived neurotrophic factor (BDNF) versus neurotrophin-3 (NT-3) null mutant mice demonstrates that these neurotrophins play a critical role in inner ear development. In this study, biological functions of BDNF and NT-3 in the chick vestibular and cochlear ganglion development was assessed in vitro and compared to those of other neurotrophic factors. The embryonic day (E)8-12 vestibular ganglion neurons showed an extensive outgrowth in response to BDNF with less outgrowth to NT-3. In contrast, NT-3 had stronger neurotrophic effects on the E12 cochlear ganglion neurons compared to BDNF. These results support previous evidence that neurotrophins play important roles in the vestibular and cochlear ganglion neuron development. However, the responsiveness to the neurotrophins declined and became undetectable by E16. Unexpectedly, glial cell line-derived neurotrophic factor (GDNF) promoted neurite outgrowth from vestibular ganglia at E12-16, later than the stages at which BDNF had neurotrophic effects. The time of switching sensitivity of the vestibular ganglion neurons from BDNF to GDNF correlated with the time of completion of synaptogenesis on their peripheral and central targets. Furthermore, a factor released from E12 inner ears exerted neurotrophic effects on late-stage vestibular ganglion neurons that were not responsive to the E4 otocyst-derived factor. These results raise the possibility that the vestibular ganglion neurons become responsive to GDNF upon target innervation and that the changes in sensitivity are regulated by changes in available factors released from their peripheral targets, the inner ear epithelia.     

Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats

To investigate the short-and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent market diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or in intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an inital abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A. persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons. © 1993 John Wiley & Sons, Inc.     

Neurotrophic factor regulation of developing avian oculomotor neurons: differential effects of BDNF and GDNF.

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target.     

Embryonic lens repels retinal ganglion cell axons.

During development of the vertebrate visual system, retinal ganglion cell (RGC) axons follow a precise path toward their midbrain targets. Although much is known about the cues that direct RGC axons once they have left the optic disc, less is known about the guidance of axons at earlier stages, when RGCs first send out their axons to navigate within the developing retina. Using collagen gel coculture experiments, we find that the embryonic lens produces a powerful diffusible repulsive activity for RGC axons. We also find that this activity is localized to the lens epithelium and not the lens fiber layer, while the pigmented epithelium and vitreous humour are devoid of activity. The further observation that the lens also chemorepels primary sensory axons, but does not repel olfactory bulb axons, shows that this activity is specific for subsets of axons. Our experiments have excluded two candidate repellents for RGC axons (collapsin-1/sema III and chondroitin sulfate proteoglycans). These results implicate the lens in the earliest stages of RGC axon guidance. One function of the lens repellent may be to prevent aberrant targeting toward the lens, and it may also be involved in the directional guidance of RGC axons toward the optic disc.