Colocalization of TrkB and brain-derived neurotrophic factor proteins in green-red-sensitive cone outer segments.

PURPOSE: To examine the distribution of neurotrophins (NTs) and their catalytic receptors in adult rat photoreceptors. METHODS: Immunocytochemistry and Western blot analyses were performed using primary antibodies raised against NTs (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], NT-3, and NT-4/5) and NT receptors (TrkA, TrkB, TrkC, and p75NTR). Double-labeling of retinal sections with opsin-specific antibodies was performed to identify each photoreceptor type. Competitive experiments using excess recombinant NT or Trk receptors confirmed the binding specificity of each antibody. RESULTS: TrkB and BDNF immunoreactivity was colocalized in cone outer segments. TrkB and BDNF were detected in all green-red-sensitive cones, but not in blue-UV cones or rods, and other NTs and NT receptors were not detected in any of the photoreceptor types. CONCLUSIONS: The findings suggest a specific role for BDNF through its signaling receptor TrkB in the function and maintenance of green-red cones, the predominant cone type in the rat retina.

Prolonged administration of NT-4/5 fails to rescue most axotomized retinal ganglion cells in adult rats.

The survival of axotomized RGCs was increased by intravitreal NT-4/5 given by repeated injections or osmotic minipumps, but the effects were less complete than predicted. Compared to a single injection of the neurotrophin on day 0, second injections on days 3 or 7 only sustained an additional 10-20% of the RGCs on day 10. Minipumps augmented RGC survival up to 4-fold (50%) at 2 weeks but most RGCs were lost by 1 month. Thus, specific neurotrophins can rescue many RGCs soon after injury but long-term neuronal survival may require a better understanding of changes in neurotrophin receptors and interactions with other molecules.

Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected Müller cells temporarily rescues injured retinal ganglion cells.

In this study, we demonstrate that: (i) injection of an adenovirus (Ad) vector containing the brain-derived neurotrophic factor (BDNF) gene (Ad.BDNF) into the vitreous chamber of adult rats results in selective transgene expression by Müller cells; (ii) in vitro, Müller cells infected with Ad.BDNF secrete BDNF that enhances neuronal survival; (iii) in vivo, Ad-mediated expression of functional BDNF by Müller cells, temporarily extends the survival of axotomized retinal ganglion cells (RGCs); 16 days after axotomy, injured retinas treated with Ad.BDNF showed a 4.5-fold increase in surviving RGCs compared with control retinas; (iv) the transient expression of the BDNF transgene, which lasted approximately 10 days, can be prolonged with immunosuppression for at least 30 days, and such Ad-mediated BDNF remains biologically active, (v) persistent expression of BDNF by infected Müller cells does not further enhance the survival of injured RGCs, indicating that the effect of this neurotrophin on RGC survival is limited by changes induced by the lesion within 10-16 days after optic nerve transection rather than the availability of BDNF. Thus, Ad-transduced Müller cells are a novel pathway for sustained delivery of BDNF to acutely-injured RGCs. Because these cells span the entire thickness of the retina, Ad-mediated gene delivery to Müller cells may also be useful to influence photoreceptors and other retinal neurons.

Regenerated retinal ganglion cell axons form normal numbers of boutons but fail to expand their arbors in the superior colliculus.

Regenerated retinal ganglion cell (RGC) axons can re-form functional synapses with target neurons in the superior colliculus (SC). Because preterminal axon branching determines the size, shape and density of innervation fields, we investigated the branching patterns and bouton formation of individual RGC axons that had regrown along peripheral nerve (PN) grafts to the SC. Within the superficial layers of the SC, the regenerated axons formed terminal arbors with average numbers of terminal boutons that were similar to the controls. However, axonal branches were shorter than normal so that the mean area of the regenerated arbors was nearly one-tenth that of control arbors and the resulting fields of innervation contained greater than normal numbers of synapses concentrated in small areas of the target. Our results have delineated a critical defect in the reconstitution of retino-collicular circuitry in adult mammals: the failure of terminal RGC branches to expand appropriately. Because recent studies have documented that brain-derived neurotrophic factor (BDNF) can specifically lengthen RGC axonal branches not only during development in the SC but also within the adult retina after axotomy, the present quantitative studies should facilitate experimental attempts to correct this deficit of the regenerative response.

Brain-derived neurotrophic factor modulates GAP-43 but not T alpha1 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 T alpha1 alpha-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 T alpha1. Axotomy alone resulted in a general decrease in T alpha1 alpha-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 microg) to axotomized RGCs on the day of injury did not affect T alpha1 alpha-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 T alpha1 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 T alpha1 tubulin, correlate with branching of injured neurons as opposed to long distance regrowth.

A distinct pattern of trophic factor expression in myelin-deficient nerves of Trembler mice: implications for trophic support by Schwann cells.

Distal to a peripheral nerve transection, myelin degradation and Schwann cell (SC) proliferation are accompanied by a marked upregulation of brain-derived neurotrophic factor (BDNF) and a decrease of ciliary neurotrophic factor (CNTF) in non-neuronal cells. To investigate the role of SC differentiation in trophic factor regulation, we studied BDNF and CNTF expression in sciatic nerves from Trembler-J (Tr-J) mice. In these animals, a mutation in the pmp-22 gene causes a failure of myelination and continuous SC proliferation, but axonal continuity is preserved. In spite of the severe abnormalities in Tr-J nerves, BDNF levels remained as low as in the intact controls. Thus, the primary SC disorder in Tr-J produces a different pattern of BDNF expression from that caused by axonal breakdown due to nerve transection. Furthermore, the upregulation of BDNF mRNA triggered by transection was 70-fold in control nerves, but only 30-fold in Tr-J sciatic nerves. Because these results raised the possibility that axonal loss may influence neurotrophin expression only in SCs that have differentiated toward a myelinating phenotype, we measured BDNF mRNA after axotomy in the cervical sympathetic trunk (CST), a predominantly unmyelinated autonomic nerve. In contrast to the sciatic nerves, the BDNF mRNA level barely increased in the injured CST, supporting the idea that not all SCs are equal sources of trophic molecules. In Tr-J sciatic nerves, CNTF mRNA levels were fourfold lower than normal, implying that the downregulation of this cytokine is a sensitive indicator of a spectrum of SC perturbations that affect myelinating cells.

Brain-derived neurotrophic factor and neurotrophin-4/5 stimulate growth of axonal branches from regenerating retinal ganglion cells.

To investigate the influences of growth factors on axonal regeneration in the mammalian CNS, we used intracellular tracers to quantitate the effects of brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-4/5, or NT-3 on individual retinal ganglion cell (RGC) axons in the retinas of adult rats after optic nerve transection. A single injection of BDNF or the prolonged administration of NT-4/5 by mini-pump increased axon branch median lengths by eightfold but had no effect on the number of branches formed by the RGC axons. NT-3 did not significantly influence axonal regrowth. These specific in vivo effects of BDNF and NT-4/5 on axonal regeneration from injured RGCs may be used to promote growth and expand the abnormally small terminal arbors observed when RGCs regrow into their CNS targets.

Effects of neurotrophins on the survival and regrowth of injured retinal neurons.

The focus of this short review is the role of certain neurotrophins and their receptors on the survival and regrowth of retinal ganglion cells (RGCs) whose axons are damaged in the optic nerve. Initial experiments in our laboratory documented patterns of RGC death after axotomy. Subsequent studies were designed to investigate the distribution of high-affinity neurotrophin receptors in neurons and glial cells of the retina and optic nerve. This information was used both in vitro and in vivo to study the effects of specific trophic molecules on the survival and regrowth of injured RGCs. During the course of experiments involving neurotrophin administration, an endogenous source of trophic support--independent of the exogenous administration of growth factors--was found within the eye. Several experiments were subsequently undertaken to define further this survival effect and determine its nature and source within the eye. Finally, anatomical techniques that help visualize fine axonal processes within the retina have provided insights into the specific effects of neurotrophins on the growth and branching of injured CNS axons.

Trophic factors.

The various neurotrophic factors influence a wide range of cell functions in the developing, mature and injured nervous system. Recent studies have provided valuable insights on the receptors that mediate these effects and on the intracellular events that follow the binding of the ligand. Although growth factors were known to be expressed by non-neuronal cells in the targets and pathways of neuronal projections, it is now clear that the neurons themselves can also be a source of these molecules. A better understanding of the mechanisms of action of trophic factors on the survival and differentiation of neurons, coupled with advances in methods for the delivery of these molecules to the nervous system have provided an impetus for exploring their use as aids to the protection and regeneration of the injured nervous system.

Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth in vitro.

Retinal ganglion cell (RGC) survival and neurite outgrowth were investigated in retinal explants from adult rats. Neurotrophin-4/5 (NT-4/5) caused dose-dependent increases in neurite outgrowth with one-half maximal effects at approximately 0.5 ng/ml and maximal effects at 5 ng/ml. In explants treated for 7 days, the actions of NT-4/5 were similar to those of brain-derived neurotrophic factor (BDNF); with either neurotrophin, nearly twice as many RGCs survived and there was a two- to threefold increase in the number of neurites formed by RGCs. Combinations of saturating concentrations of NT-4/5 and BDNF did not enhance these in vitro effects, implying that both neurotrophins share a common signaling pathway. In contrast, nerve growth factor (NGF), neurotrophin-3 (NT-3), or ciliary neurotrophic factor (CNTF) appeared to exert minimal influences on RGC survival or neurite outgrowth.

Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats.

Using quantitative anatomical techniques, we show that after intraorbital optic nerve transection in adult rats, virtually all retinal ganglion cells (RGCs) survive for 5 d and then die abruptly in large numbers, reducing the RGC population to approximately 50% of normal by day 7 and to less than 10% on day 14. During this period of rapid cell loss, some RGCs show cytochemical alterations indicative of apoptosis ("programmed cell death"), a change not previously categorized after axotomy in adult mammals. With intracranial lesions 8-9 mm from the eye, the onset of cell death is delayed until day 8 and is greater with cut than crush. The demonstration that axotomy results in apoptosis, the long interval between axonal injury and RGC death, and the different time of onset of the massive RGC loss with optic nerve lesions near or far from the eye suggest that axonal interruption triggers a cascade of molecular events whose outcome may be critically dependent on the availability of neuronal trophic support from endogenous or exogenous sources. The role of such molecules in RGC survival and the reversible nature of these injury-induced changes is underscored by the temporary rescue of most RGCs by a single intravitreal injection of brain-derived neurotrophic factor during the first 5 d after intraorbital optic nerve injury (Mansour-Robaey et al., 1994). The delayed pattern of RGC loss observed in the present experiments likely explains such a critical period for effective neurotrophin administration.

Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells.

Optic nerve transection in adult rats results in the death of approximately 50% of the axotomized retinal ganglion cells (RGCs) by 1 week and nearly 90% by 2 weeks after injury. The capacity of brain-derived neurotrophic factor (BDNF) to prevent this early, severe loss of RGCs was investigated in vivo by intravitreal injections of BDNF [5 micrograms in 5 microliters of bovine serum albumin/phosphate-buffered saline (BSA/PBS)] or vehicle (5 microliters of BSA/PBS). Using quantitative anatomical techniques, we show that (i) all RGCs survived 1 week after a single injection of BDNF at the time of axotomy. (ii) RGC densities decreased in the BDNF-treated retinas by 2 weeks but remained significantly greater than in the untreated controls. (iii) An enhanced RGC survival was obtained with single injections of BDNF from 6 days before to 5 days after axotomy. (iv) Repeated injections resulted in greater numbers of surviving RGCs, an effect that declined to undetectable levels by 6 weeks. (v) There were indications for an endogenous local source of trophic support whose expression was triggered by ocular injury, particularly to the anterior part of the eye. (vi) With multiple BDNF injections, there was profuse axonal sprouting around the optic disc. This remarkable intraretinal growth was not, however, reflected in increased RGC innervation of the peripheral nerve grafts, which are known to facilitate regeneration when used as optic nerve substitutes.

Long-term growth and remodeling of regenerated retino-collicular connections in adult hamsters.

The capacity of regenerating axons for long-term growth and synaptic plasticity was investigated in the visual system of adult hamsters. Four to six and 8-10 months after the eye and the superior colliculus (SC) were linked by a peripheral nerve (PN) graft, the retinal ganglion cell (RGC) axons that had regrown into the SC were examined ultrastructurally. Together with the data from hamsters with similar PN grafts for 2 months (Carter et al., 1989), this study spans most of the life of these animals. The overall findings indicate that (1) the RGC axons extended twice as far into the SC and the number of RGC terminals increased 30-fold between 2 and 4-6 months. These parameters did not change thereafter. The highest density of regenerated RGC terminals observed in the SC was 11.5% of controls. (2) The new RGC terminals acquired most of their normal ultrastructural characteristics by 2 months. (3) The mean size of the terminals was larger than in controls but decreased gradually, and there was a small increase in the size of the regenerated synapses. (4) At all times, the RGC terminals remained confined to the layers of the SC that normally receive retinal inputs, and their synapses were formed in normal proportions with the dendritic shafts and spines of SC neurons. Thus, there is a protracted long-term growth and remodeling of the RGC axons that have regenerated into the SC of these adult mammals.(ABSTRACT TRUNCATED AT 250 WORDS)

Marked increase in beta-tubulin mRNA expression during regeneration of axotomized retinal ganglion cells in adult mammals.

Changes in gene expression were investigated in axotomized CNS neurons under conditions that inhibit or permit regrowth of their damaged axons. Levels of mRNA encoding beta-tubulin, the 150 kDa neurofilament subunit (NF-M), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined by quantitative in situ hybridization of adult rat retinal ganglion cells (RGCs) after axotomy in the optic nerve or during regeneration in a peripheral nerve (PN) graft. Soon after optic nerve section beta-tubulin, NF-M, and GAPDH mRNA levels decreased and remained low during the 1 month studied. In these retinas beta-tubulin mRNA fell to approximately 50% of normal controls. However, in the PN-grafted retinas, where approximately 20% of the surviving axotomized RGCs regenerate their axons, there were "hot spots" of beta-tubulin mRNAs where neuronal levels were nearly 300% higher than in controls. By retrograde neuronal labeling these hot spots were shown to correspond to the injured RGCs that regrew their axons into the PN graft; beta-tubulin mRNA levels in nonregenerating RGCs of the same retinas averaged 63% of controls. We suggest that interactions of RBC axons and components of the grafts' non-neuronal environment play a key role in the over fourfold differences in beta-tubulin mRNA levels observed between injured and regenerating RGCs.

Different forms of the neurotrophin receptor trkB mRNA predominate in rat retina and optic nerve.

The expression of TrkB mRNAs was investigated in rat retina and optic nerve. A 11.5 kb transcript that encodes full-length TRKB was found to predominate in Northern blots of retinal RNA. By in situ hybridization, this trkB expression was concentrated in the ganglion cell and inner nuclear layers. Furthermore, an antibody to the full-length TRKB immunostained retinal ganglion cells and their axons. In contrast, Northern blots of optic nerve RNA showed a prominent 9.5 kb band that encoded a form of the TRKB receptor lacking the tyrosine kinase domain. This species was also detected in both the sciatic nerve and cultured astrocytes and C6 glioma cells. These results suggest that neurons express the full-length TRKB containing the tyrosine kinase domain, while non-neuronal cells express the truncated form of the receptor. These two classes of TRKB may mediate different neurotrophic actions in the retina and optic nerve.

Temporal changes in beta-tubulin and neurofilament mRNA levels after transection of adult rat retinal ganglion cell axons in the optic nerve.

Axons of adult mammalian retinal ganglion cells (RGCs) do not regenerate spontaneously after injury in the optic nerve and show a persistent decrease in the rate of transport of tubulin and neurofilament proteins. To investigate further the expression of cytoskeletal proteins in these axotomized CNS neurons, mRNA levels of beta-tubulin and the 150 kDa neurofilament subunit (NF-M) were measured after interrupting the optic nerve 9 mm from the eye. Northern blots of RNA extracted from whole retinas after optic nerve transection showed that the total level of both of these mRNAs fell after injury. To determine if this decrease was a result of the death of axotomized RGCs or reflected changes in individual neurons, RNA probes were hybridized to radial cryostat sections of normal and axotomized retinas from 1 d to 6 months after injury. Grain counts revealed two trends of tubulin expression in RGCs. An early increase in tubulin mRNAs in the axotomized RGCs was followed by a later decrease. Such an increase in tubulin mRNA levels has been correlated with regenerative growth in other neurons. By 1 week after injury, the beta-tubulin mRNA levels decreased to 70% of the control value. Moreover, the time of this fall coincided with the onset of a marked slowing of cytoskeletal transport that follows injury in the optic nerve. In contrast, NF-M mRNA levels dropped immediately after axotomy, and remained at 80% of the control level. It is suggested that the transient increase in tubulin mRNAs may reflect an early regenerative response whose persistence depends on further growth cone interactions with the substrate.

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 marker diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or 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 initial 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.

Synapse formation and preferential distribution in the granule cell layer by regenerating retinal ganglion cell axons guided to the cerebellum of adult hamsters.

To investigate constraints and preferences for synaptogenesis in the injured mammalian CNS, regenerating retinal ganglion cell (RGC) axons of adult hamsters were guided through a peripheral nerve (PN) graft to a target they do not usually innervate: the cerebellum (Cb). When identified by the presence of HRP anterogradely transported from the retina 2-9 months later, such RGC axons were found to have extended into the cerebellar cortex for up to 650 microns. Most of this growth was in the granule cell layer (GCL) and only a few axons entered the molecular layer. The preference for the GCL could not be explained by the position of the PN graft in the Cb, a selective denervation of the GCL, local damage to other neurons, or the distribution of reactive gliosis in the vicinity of the graft. Furthermore, by EM, more than 95% of the labeled retinocerebellar terminals and synapses were in the GCL. Retinocerebellar terminals were larger and contained more synapses than the regenerated RGC terminals previously studied in the superior colliculus. These results indicate that regenerating axons of CNS neurons can form persistent synapses with novel targets. The preferential synaptogenesis in the GCL suggests that such unusual connections are not formed randomly in the CNS of these adult mammals.