Bcl-2 is not required in retinal ganglion cells surviving optic nerve axotomy.

Axotomy of the optic nerve in rodents induces the majority of retinal ganglion cells (RGCs) to undergo apoptosis: Only 10-15% survive 14 days past lesion. The molecular mechanism allowing this survival is not known. To test whether expression of the anti-apoptotic proto-oncogene bcl-2 gene is required in those RGCs, we examined the effect of optic nerve axotomy in bcl-2-/- mice. 7 days and 14 days post-lesion, the same number of surviving RGCs was detected in mutant and wild type retinas. Thus, the bcl-2 gene is not necessary for the survival of the subpopulation of retinal ganglion cells resisting axotomy-induced apoptosis in adult mice, nor does its normal expression delay retinal ganglion cell degeneration.     

Electrical stimulation enhances the survival of axotomized retinal ganglion cells in vivo

In view of recent reports on the survival promotion of damaged spiral ganglion cells and motoneurons by electrical stimulation, we hypothesized that an electrical stimulation of the cut optic nerve (ON) may promote the survival of axotomized retinal ganglion cells (RGCs) in vivo. To test this hypothesis, we examined 1 week after ON transection the RGC densities in the retinas with or without electrical stimulation. The densities of surviving RGCs in the retinas with the electrical stimulation increased as compared with those without the electrical stimulation. We concluded that electrical stimulation of the ON enhances the survival of axotomized RGCs in vivo, probably due to electrical activation of their soma.     

Is nerve growth factor required for the survival of retinal ganglion cells during development?

Staining for nerve growth factor receptor was observed in the ferret's retinal ganglion cell layer, optic nerve and tract, and in the lateral geniculate nucleus and superficial layers of the superior colliculus in the prenatal period, but had disappeared by birth. Thus the incidence of this transient staining does not correspond with the ganglion cell death that is known to occur in the ferret retina during the first postnatal week.     

Inner hair cells are not required for survival of spiral ganglion neurons in the adult cochlea

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.     

The effect of PKC activation on the survival of rat retinal ganglion cells in culture

Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C (PKC), an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept "in vitro" for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate (PMA), a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ng/ml produced the maximal effect (a two-fold increase on ganglion cells survival compared with 48 h control). This effect was totally abolished by 1.25 microM chelerythrine chloride (an inhibitor of PKC) and 30 microM genistein (an inhibitor of tyrosine kinase enzymes). Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 microM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, or 25 microM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells "in vitro" may be mediated by a mechanism that involves PKC activation.     

Interleukin-6 increases the survival of retinal ganglion cells in vitro.

Interleukin-6 is a pleiotropic cytokine that mediates cellular communication both in physiological and pathological states. In this work, we demonstrate that 50 ng/mL IL-6 increases the survival of retinal ganglion cells (RGCs) after 48 h in culture. This effect was blocked by an intracellular Ca(+2) chelator, by inhibition of ryanodinic receptors and by an inhibitor of L-type Ca(+2) channels. IL-6 effect is mediated by PKC, tyrosine kinase, PI3-kinase and MEK activity. The blockade of polypeptide release also abolished the effect of IL-6. These results suggest a role for this cytokine during the development of the central nervous system (CNS).     

Retinal ganglion cell death is delayed by activation of retinal intrinsic cell survival program

Neuronal cells undergo apoptosis when deprived of neurotrophic factors due to injury, trauma, or neurodegenerative disease. This study examined cell death in the retina after chronic elevation of intraocular pressure (IOP) in an experimental rat model of human glaucomatous disease. Three episcleral veins on the ocular surface of rats were cauterized. Activation of several cell death programs represented by Fas ligand, FADD (Fas Associated Death Domain/Mort1) and the caspase cascade (caspase-8 and -3) and survival programs represented by phosphorylated protein kinase B (PKB/Akt), Bcl-2 associated death domain (BAD), and cAMP responsive element binding protein (CREB) were examined using immunohistochemistry and Western blotting. Following injury, two major events occurred simultaneously in the retina: activation of programmed cell death pathways and activation of survival mechanisms to maintain the cellular homeostasis of the retina. At the later stage of injury, markers of an activated cell death program appeared to be concentrated in the retinal ganglion cells. In conclusion, we suggest that endogenous cell survival factors triggered at the early stage of injury play a critical role in control of the death or survival of retinal ganglion cells and that the manipulation of this decision phase is one of the therapeutic targets for glaucoma.     

Foxn4 is required for retinal ganglion cell distal axon patterning

The regulation of retinal ganglion cell (RGC) axon growth and patterning in vivo is thought to be largely dependent on interactions with visual pathway and target cells. Here we address the hypothesis that amacrine cells, RGCs' presynaptic partners, regulate RGC axon growth or targeting. We asked whether amacrine cells play a role in RGC axon growth in vivo using Foxn4(-/-) mice, which have fewer amacrine cells, but a normal complement of RGCs. We found that Foxn4(-/-) mice have a similar reduction in most subtypes of amacrine cells examined. Remarkably, spontaneous retinal waves were not affected by the reduction of amacrine cells in the Foxn4(-/-) mice. There was, however, a developmental delay in the distribution of RGC projections to the superior colliculus. Furthermore, RGC axons failed to penetrate into the retinorecipient layers in the Foxn4(-/-) mice. Foxn4 is not expressed by RGCs and was not detectable in the superior colliculus itself. These findings suggest that amacrine cells are critical for proper RGC axon growth in vivo, and support the hypothesis that the amacrine cell-RGC interaction may contribute to the regulation of distal projections and axon patterning.     

Central retinal area is not the site where ganglion cells are generated first

The development of retinal ganglion cells (RGC) was studied in the chick from stage 18 to adulthood. Our main objectives were to identify the retinal site where the first RGCs differentiate, to locate this site relative to the optically defined central retinal area, and to map the spatial arrangement of the RGC field at different stages in development. The eyes of the experimental animals were fixed and serially sectioned. The borders of RGC fields were determined from the presence of either ganglion cell perikarya or ganglion cell axons. In seven cases between stages 21 and 26, the borders of the RGC fields were confirmed electron microscopically. The serial sections together with the RGC fields were then reconstructed in three dimensions. The reconstructed retinae were projected onto a plane by using the radially equidistant polar azimuthal projection. First, RGCs appear dorsal to the apex of the optic fissure. Ganglion cell development then initially spreads out symmetrically with respect to the optic fissure. However, from stage 29 on, the nasal half of the retina expands much more than the temporal half. This asymmetrical growth entails that the optic fissure is eventually located in the temporal half of the retina in the mature animal. The RGC fields of the embryonic stages were superimposed on the retina of a visually active animal according to their real size and position. It turned out that the central retinal area was at least 2 mm away from the site where the first RGCs were generated. It is not before stage 28 that the prospective central retinal area is included into the expanding ganglion cell field. The fact that RGCs at the central retinal area are generated 2.5 days later than first RGCs near the apex of the optic fissure has important implications for the formation of the retinotectal projection. © 1993 Wiley-Liss, Inc.     

Separation of calcium currents in retinal ganglion cells from postnatal rat

A culture system of the postnatal rat retina was established to investigate Ca²⁺ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present study was to identify channel specific components in whole-cell Ca²⁺ currents of RGNs and to clarify the potential use of the lanthanide Gd³⁺ as a selective Ca²⁺ channel blocker. About one third of freshly dissociated RGNs generated both low voltage activated Ca²⁺ currents (I_Ca(LVA)) and high voltage activated Ca²⁺ currents (I_Ca(HVA)). The remaining 2/3 of RGNs in short term culture and most RGNs in long-term culture displayed only I_Ca(HVA). The latter comprised at least three different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of I_Ca(HVA) was irreversible blocked by the N channel antagonist ω-CgTx (5 μM). The L channel antagonist nifedipine (10 μM) eliminated about 25% of I_Ca(HVA). Thus, about 1/3 of the HVA Ca²⁺ or Ba²⁺ current remained unaffected by either ω-CgTx or nifedipine. ω-AgaTx (200 nM) completely failed to block HVA Ca²⁺ or Ba²⁺ currents in RGNs. Gd³⁺ exerted contrasting actions on LVA and HVA Ca²⁺ currents. While I_Ca(LVA) consistently increased in the presence of Gd³⁺ (0.32–3.2 μM), I_Ca(HVA) always decreased, especially when using higher concentrations of Gd³⁺ (10–32 μM). The blocking action of Gd³⁺ was not restricted to the ω-CgTx-sensitive HVA current component, but also concerned ω-CgTx- and nifedipine-resistant components. The decay of Ca²⁺ currents was accelerated in the presence of Gd³⁺. Even in RGNs lacking I_Ca(LVA), application of 3.2 μM Gd³⁺ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from −90 to 0 mV; 10 mM [Ca²⁺]₀; 26°C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed by Gd³⁺. Gd³⁺ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd³⁺ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca²⁺ currents, that resemble in some aspects T, N and L channel currents. A significant component of the HVA Ca²⁺ current was resistant to the available HVA channel blockers suggesting the presence of a pharmacologically distinct type of HVA Ca²⁺ channel type in RGNs. Our experiments also show that Gd³⁺ is not suitable for isolation of HVA subcomponents in RGNs, but it can be used to distinguish between LVA and HVA Ca²⁺ currents, as these currents reacted to Gd³⁺ in an opposite way. The purely depressive effect of this lanthanide on spontaneous synaptic activity is consistent with the assumption that in retinal neurons LVA Ca²⁺ channels are not involved in the regulation of glutamate release.     

Morphological properties of mouse retinal ganglion cells during postnatal development

Quantitative methods were used to assess dendritic stratification and other structural features of developing mouse retinal ganglion cells from birth to after eye opening. Cells were labeled by transgenic expression of yellow fluorescent protein, DiOlistics or diffusion of DiI, and subsequently imaged in three dimensions on a confocal microscope followed by morphometric analysis of 13 different structural properties. At postnatal day 1 (P1), the dendrites of all cells ramified across the vertical extent of the inner plexiform layer (IPL). By P3/4, dendrites were largely confined to different strata of the IPL. The stratification of dendrites initially reflected a retraction of widely ramifying dendritic processes, but for the most part this was due to the subsequent vertical expansion of the IPL. By P8, distinct cell classes could be recognized, although these had not yet attained adult-like properties. The structural features differentiating cell classes were found to follow three different developmental trends. The mean values of one set of morphological parameters were essentially unchanged throughout postnatal development; another set of measures showed a rapid rise with age to adult values; and a third set of measures first increased with age and later decreased, with the regressive events initiated around the time of eye opening. These findings suggest that the morphological development of retinal ganglion cells is regulated by diverse factors operating during different but overlapping time periods. Our results also suggest that dendritic stratification may be more highly specified in the developing mammalian retina than has been previously realized.     

Regulation of caspase activation in axotomized retinal ganglion cells

Transection of the optic nerve initiates massive death of retinal ganglion cells (RGCs). Interestingly, despite the severity of the injury, RGC loss was not observed until several days after axotomy. The mechanisms responsible for this initial lack of RGC death remained unknown. In the current study, immunohistochemical analysis revealed that caspases-3 and -9 activation in the RGCs were not detected until day 3 post-axotomy, coinciding with the onset of axotomy-induced RGC loss. Interestingly, elevated Akt phosphorylation was observed in axotomized retinas during the absence of caspase activation. Inhibiting the increase in Akt phosphorylation by intravitreal injection of wortmannin and LY294002, inhibitors of PI3K, resulted in premature nuclear fragmentation, caspases-3 and -9 activation in the ganglion cell layer. Our findings thus indicate that the PI3K/Akt pathway may serve as an endogenous regulator of caspase activation in axotomized RGCs, thereby, contributing to the late onset of RGC death following axotomy.     

Polarization and orientation of retinal ganglion cells in vivo

In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.     

In vivo imaging of murine retinal ganglion cells

Current methods for in vivo retinal ganglion cells (RGCs) imaging involve either retrograde or intravitreal injection of chemical or biological tracers, which are invasive and may require repeated injection for serial long-term assessment. We have developed a confocal scanning laser ophthalmoscope technique (blue-light CSLO or bCSLO) to image retinal ganglion cells (RGCs) in mice expressing cyan fluorescent protein under the control of a Thy-1 promoter. Fluorescent spots corresponding to CFP-expressing retinal ganglion cells were discernable with the bCSLO. 96.1+/-2.6% of CFP expressing cells also were retrograde labeled with DiI indicating the bCSLO imaged fluorescent spots are RGCs. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator to reflect the integrity of RGCs, and provides a non-invasive method for longitudinal study of the mechanism of RGC degeneration and the effect of neuroprotective agents.     

Cytochrome oxidase activity in rat retinal ganglion cells during postnatal development

In this study, the metabolic activity of rat retinal ganglion cells during postnatal development has been examined in vivo using cytochrome oxidase histochemistry. The intensity of staining was measured by optical densitometry. The activity of cytochrome oxidase in retinal ganglion cells progressively increased from postnatal day 0 (P0) and reached a peak during the second week of postnatal development (P10-P14) and declined thereafter. Our data show that the increased levels of cytochrome oxidase seen in developing retinal ganglion cells occur at the same time, when neuronal maturity and synaptogenesis reach their peaks.     

Long-term effect of inhibition of ced 3-like caspases on the survival of axotomized retinal ganglion cells in vivo.

There is growing evidence that caspase inhibition exerts neuroprotective effects in various models of neuronal injury in vivo. However, whether caspase inhibition provides long-term neuroprotection is not known yet. In the present study, we therefore investigated the effects of prolonged caspase inhibition on the survival of adult rat retinal ganglion cells (RGCs) following optic nerve (ON) transection. Four weeks following ON transection the number of surviving RGCs in untreated animals declined to 11% of controls. Treatment for the initial 2 weeks with z-DEVD-cmk, an irreversible inhibitor of ced 3-like caspases, increased the number of surviving RGCs 4 weeks postlesion to 24%. Z-DEVD-cmk treatment over the entire experimental period of 4 weeks had no additional effect. Thus, we still found a neuroprotective effect of caspase inhibition on axotomized RGCs after extended survival time. However, in comparison to our recent observations 2 weeks after optic nerve transection, in which z-DEVD-cmk rescued 46% of RGCs (P. Kermer, N. Kl?cker, M. Labes, and M. B?hr, 1998, J. Neurosci. 18(12), 4656-4662) the positive effect clearly decreased. In conclusion, our results indicate that the therapeutical approach presented here results in a significant delay of secondary death rather than providing a permanent and complete rescue of axotomized RGCs.     

Downregulation of STAT3 activation is required for presumptive rod photoreceptor cells to differentiate in the postnatal retina

Ciliary neurotrophic factor (CNTF) has been known to inhibit the differentiation of presumptive rod photoreceptor cells; however, the underlying mechanisms have remained to be elucidated. We demonstrated that STAT3 activation, but not SHP2 activation, is responsible for the CNTF/gp130 signaling that inhibits expression of Rhodopsin and its upstream activator, crx, in the retinal explants derived from P0 mice (P0 retinal explants), utilizing STAT3-deficient retina and electroporation of dominant-negative form of STAT3 (STAT3F). We also demonstrated that STAT3 activation in presumptive rod photoreceptor cells at E18.5 is rapidly downregulated at P0, when Rhodopsin expression starts during retinal development. Persistent STAT3 activation in the P0 retinal explants prevented Rhodopsin expression and rapid upregulation of crx expression. STAT3-deficient retinas did not exhibit precocious rod photoreceptor cell differentiation as a whole, although they occasionally exhibited precocious upregulation of crx mRNA. Thus, we conclude that downregulation of STAT3 activation is required, but insufficient, for rod photoreceptor cell differentiation in the postnatal retina.     

Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo

After transection of the optic nerve (ON) in adult rats, retinal ganglion cells (RGC) progressively degenerate until, after two months, a residual population of only about 5% of these cells survives. In this study, we investigated the effect of regeneration-associated factors from sciatic nerve (ScN), BDNF, and CNTF on the survival of adult rat RGC after intraorbital ON transection. Neurotrophic factors were injected into the vitreous body. Rats were allowed to survive 3, 5, or 7 weeks, and the remaining viable RGC were then labelled by retrograde staining with the carbocyanine dye, 4Di-10Asp, which was applied onto the proximal nerve stump in vivo. The animals were sacrificed 3 days later and RGC counted in retinal whole mounts. Due to progressive degeneration following nerve transection the number of surviving RGC decreased to about 10% of the initially labelled population after 3 weeks, to about 8% after 5 weeks, and to about 5% after 7 weeks. Survival of axotomized cells could be prolonged using either of the neurotrophic factors: after 3 weeks a 2–3-fold increase in the number of viable RGC could be obtained compared to uninjected controls and to those which received injection of buffer. The prolonged survival effect vanished after 5 and 7 weeks, and no additive effect could be seen when combining brain-derived neurotrophic factor (BDNF) and ciliary neuronotrophic factor (CNTF) treatment. Morphometric analysis of labelled cells revealed that all neurotrophic factors supported predominantly large RGC with somal areas > 250 μm². In retinae from rats that survived the ON transection for several months, a characteristic population of axotomy-resistant RGC remained alive. Their few, very large, and often curled dendrites showed signs of placticity in the depleted inner nuclear layer of the adult rat retina. We conclude that the intraocular injection of CNTF, BDNF, and ScN-derived medium, which retard the process of lesion-induced RGC degeneration, may be successfully used as a subsidiary strategy in transplantation protocols. This would result in larger populations of RGC which can be recruited to regenerate their axons and provide a basis for functional recovery.     

Expression of Hsp27 in retinal ganglion cells of the rat during postnatal development

The small heat shock protein Hsp27 has been shown to protect neurons from apoptosis. We have recently shown the expression of Hsp27 in a subset of injured adult retinal ganglion cells (RGCs), a response that is muted by the administration of brain-derived neurotrophic factor. This work has suggested a role for Hsp27 in the long-term survival of RGCs following injury. The purpose of this study was to investigate the expression of Hsp27 during postnatal retinal development, based on Hsp27's role as a neuronal survival factor and on its up-regulation in the adult injured retina. Expression of Hsp27 in the developing retina was examined at various times postnatally (between P0 and P24) by using immunohistochemical techniques. We report that Hsp27 expression peaks in the ganglion cell layer between P6 and P12 and is not detected at earlier (P0-P3) or later (P15-P24) times. Double labeling of the Hsp27-positive cells with Fluorogold applied to the superior colliculus confirmed that Hsp27-positive cells in the ganglion cell layer are RGCs. We have shown developmentally regulated expression of Hsp27 in RGCs of the postnatal rat. The retinal expression of Hsp27 correlates temporally with innervation of the tectum by late-born RGCs and with onset of spontaneous retinotectal activity. We propose that the expression of Hsp27 may play an important role in retinal development during a critical period of RGC functional connectivity with the superior colliculus.     

Brain-derived neurotrophic factor/neurotrophin-4 receptor TrkB is localized on ganglion cells and dopaminergic amacrine cells in the vertebrate retina

The tyrosine kinase TrkB is a receptor for the neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). Retinal ganglion cells are responsive to BDNF, and TrkB has been localized in ganglion cells as well as in a subpopulation of amacrine cells in the retina of the chicken and the rat. In the present paper, we analyzed the distribution of TrkB immunoreactivity in the retina of marmoset monkeys, ferrets, rabbits, rats, mice, chickens, pigeons, barn owls, Pseudemys turtles, Xenopus frogs, goldfishes, and carps. TrkB antibodies gave a positive reaction in all of these vertebrates. TrkB immunoreactivity was detected in the majority of retinal ganglion cells. Some amacrine cells also contained TrkB immunoreactivity; they were located mainly at the vitreal border of the inner nuclear layer, and their relative abundance varied in the different species. Until now, no information has been available concerning the neurochemical identity of the amacrine neurons containing TrkB. In some species (marmoset monkeys, rats, pigeons), we observed that the morphology and location of TrkB-immunoreactive amacrine cells was reminiscent of that of the well-described dopaminergic cells. To determine whether dopaminergic amacrine cells contained TrkB immunoreactivity, we therefore performed double-labelling immunohistochemistry by using tyrosine hydroxylase (TH) antibodies in combination with TrkB antibodies in marmoset monkeys, rats, pigeons, Pseudemys turtles, and goldfishes. The most novel finding of the present paper is that, in all of these species, the majority of dopaminergic neurons were found to contain TrkB immunoreactivity. Dopaminergic neurons, on the other hand, represented only a fraction of the TrkB⁺ amacrine cells. Our data suggest that BDNF and/or NT-4 might modulate expression of TH in the retina and may therefore influence the retinal dopaminergic system. Whatever the action of TrkB ligands on the retinal dopaminergic system, it was conserved during vertebrate evolution. J. Comp. Neurol. 386:149–160, 1997. © 1997 Wiley-Liss, Inc.