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During mammalian visual system development, retinal ganglion cells (RGCs) undergo extensive apoptotic death. In mouse retina, approximately 50% of RGCs present at birth (postnatal day 0; P0) die by P5, at a time when axons innervate central targets such as the superior colliculus (SC). We examined whether RGCs that make short-range axonal targeting errors within the contralateral SC are more likely to be eliminated during the peak period of RGC death (P1-P5), compared with RGCs initially making more accurate retinotopic connections. A small volume (2.3 nL) of the retrograde nucleophilic dye Hoechst 33342 was injected into the superficial left SC of anesthetized neonatal C57Bl/6J mice at P1 (n = 5) or P4 (n = 8), and the contralateral retina wholemounted 12 hr later. Retrogradely labelled healthy and dying (pyknotic) RGCs were identified by morphological criteria and counted. The percentage of pyknotic RGCs was analyzed in relation to distance from the area of highest density RGC labelling, presumed to represent the most topographically accurate population. As expected, pyknotic RGC density at P1 was significantly greater than P4 (p < 0.05). At P4, the density of healthy RGCs 500-750 µm away from the central region was significantly less, although this was not reflected in altered pyknotic rates. However, at P1 there was a trend (p = 0.08) for an increased proportion of pyknotic RGCs, specifically in temporal parts of the retina outside the densely labelled center. Overall, the lack of consistent association between short-range targeting errors and cell death suggests that most postnatal RGC loss is not directly related to topographic accuracy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 51-60, 2018., (© 2017 Wiley Periodicals, Inc.)

Lampreys are one of two extant representatives of the earliest group of vertebrates, the agnathans or jawless fishes. The single species of the southern hemisphere lamprey family Geotriidae, Geotria australis, possesses the potential for pentachromatic color discrimination opposed to the mono- or dichromacy found in other lampreys. However, little is known of the retinal ganglion cell types that contribute to visual processing in G. australis. A quantitative morphological approach was used to distinguish and describe retinal ganglion cell types in G. australis. The morphology of retinal ganglion cells was revealed by retrograde biocytin labeling from the optic disc. Cells were digitally reconstructed, and somatic area and position and dendritic field size, density, tortuosity, and stratification were subjected to quantitative morphometric analyses. Cluster analysis, in conjunction with similarity profile analysis (SIMPROF), statistically identified five discrete monostratified retinal ganglion cell types, one of which may comprise two subtypes. Two bistratified types were identified separately, including a biplexiform and a bistratified subtype. The use of cluster analysis with SIMPROF provided a robust statistical technique for objectively identifying cell types whose characteristics were similar and significantly different from those of other types and thus provides an objective resolution of the problems posed by "lumpers vs. splitters" when designating cell types. The diversity of retinal ganglion cells suggests that visual information in the lamprey G. australis is processed in parallel streams, as in gnathostomes. These findings, together with the results of previous studies, indicate that the visual system of the lamprey G. australis represents the upper limit of visual complexity in extant agnathans., (© 2013 Wiley Periodicals, Inc.)

Purpose: In adult rats, intravitreal injections of the Rho-GTPase inhibitor C3 transferase (BA-210), or a cocktail of recombinant ciliary neurotrophic factor (CNTF) and a cyclic AMP analogue (CPTcAMP), increase retinal ganglion cell (RGC) survival and axonal regeneration. Here we examined whether these treatments also affect the dendritic architecture of regrowing RGCs., Methods: In Fischer F344 rats, one optic nerve was cut and an autologous peripheral nerve graft was sutured onto it. Rats then received intravitreal injections (4 μl) of saline, BA-210 or CNTF + CPTcAMP four and eleven days after the PN graft surgery. After 5 weeks, regenerating RGCs were retrogradely labelled with fluorogold (FG) and in living wholemounts the dendritic trees of FG labelled RGCs were visualised by intracellular injection of 2% Lucifer Yellow., Results: Injection of BA-210 or CNTF + CPTcAMP resulted in significantly more regenerating RGCs with abnormal dendritic morphologies, including abnormally long looping processes. Compared to saline-injected regenerating controls, RGCs in BA-210 injected eyes had significantly smaller dendritic field areas and sparser dendrites, while in CNTF + CPTcAMP injected eyes there was increased branching of more distal dendrites., Conclusions: While both intraocular treatments enhance RGC axonal regrowth, they also induce significant changes in RGC dendritic morphology. It remains to be determined if such changes alter the function of the regenerating neuronal population.

The adult central nervous system (CNS) has only a limited capacity to regenerate axons after injury. This is due to a number of factors including the presence of extrinsic inhibitory factors that limit plasticity, lack of effective trophic support, and intrinsic changes in neuronal responsiveness. In this review, we describe the expression and role of neurotrophins in retinal ganglion cells (RGCs) during development and adulthood, and the receptors and miscellaneous signaling systems that influence axonal regeneration after injury. The impact of exogenous neurotrophic factors on adult RGCs injured at different sites in the visual pathway is described for several modes of delivery, including recombinant factors, viral vectors, cell transplantation, as well as combinatorial treatments involving other pharmacotherapeutic agents. Indirect, off-target effects of neurotrophic factors on RGC axonal regeneration are also considered. There remain unresolved issues relating to optimal delivery of neurotrophic factors, and we emphasize the need to develop safe, reliable methods for the regulation of exogenous supply of these factors to the injured CNS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5-8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.

Low intensity repetitive Transcranial Magnetic Stimulation (LI-rTMS), a non-invasive form of brain stimulation, has been shown to induce structural and functional brain plasticity, including short distance axonal sprouting. However, the potential for LI-rTMS to promote axonal regeneration following neurotrauma has not been investigated. This study examined the effect of LI-rTMS on retinal ganglion cell (RGC) survival, axon regeneration and levels of BDNF in an optic nerve crush neurotrauma model. Adult C57Bl/6J mice received a unilateral intraorbital optic nerve crush. Mice received 10 minutes of sham (handling control without stimulation) (n=6) or LI-rTMS (n = 8) daily stimulation for 14 days to the operated eye. Immunohistochemistry was used to assess RGC survival (β-3 Tubulin) and axon regeneration across the injury (GAP43). Additionally, BDNF expression was quantified in a separate cohort by ELISA in the retina and optic nerve of injured (optic nerve crush) (sham n = 5, LI-rTMS n = 5) and non-injured mice (sham n = 5, LI-rTMS n = 5) that received daily stimulation as above for 7 days. Following 14 days of LI-rTMS there was no significant difference in mean RGC survival between sham and treated animals (p>0.05). Also, neither sham nor LI-rTMS animals showed GAP43 positive labelling in the optic nerve, indicating that regeneration did not occur. At 1 week, there was no significant difference in BDNF levels in the retina or optic nerves between sham and LI-rTMS in injured or non-injured mice (p>0.05). Although LI-rTMS has been shown to induce structural and molecular plasticity in the visual system and cerebellum, our results suggest LI-rTMS does not induce neuroprotection or regeneration following a complete optic nerve crush. These results help define the therapeutic capacity and limitations of LI-rTMS in the treatment of neurotrauma. [ABSTRACT FROM AUTHOR]

TrkB is an important receptor for brain-derived neurotrophic factor and NT4, members of the neurotrophin family. TrkB signaling is crucial in many activity-dependent and activity-independent processes of neural development. Here, we investigate the role of trkB signaling in the development of two distinct, organizational features of retinal projections—the segregation of crossed and uncrossed retinal inputs along the “lines of projection” that represent a single point in the visual field and the “retinotopic” mapping of retinofugal axons within their cerebral targets. Using anterograde tracing, we obtained quantitative measures of the distribution of retinal projections in the dorsal nucleus of the lateral geniculate body (LGd) and superior colliculus (SC) of wild-type mice and mice homozygous for constitutive null mutation (knockout) of the full-length trkB receptor (trkB). In trkB mice, uncrossed retinal projections cluster normally but there is a topographic expansion in the distribution of these clusters across the SC. By contrast, the absence of trkB signaling has no significant effect on the segregation of crossed and uncrossed retinal projections along the lines of projection in LGd or SC. We conclude that the normal topographic organization of uncrossed retinal projections depends upon trkB signaling, whereas the segregation of crossed and uncrossed retinal projections is trkB-independent. We also found that in trkB mice, neuronal number was reduced in the LGd and SC and in the caudate-putamen. Previous studies by ourselves and others have shown that the number of retinal ganglion cells (RGCs) is unchanged in trkB mice. Together, these results demonstrate that there is no matching of the numbers of RGCs with neuronal numbers in the LGd or SC. [ABSTRACT FROM AUTHOR]

Topographically ordered projections are established by molecular guidance cues and refined by neuronal activity. Retinal input to a primary visual center, the superior colliculus (SC), is bilateral with a dense contralateral projection and a sparse ipsilateral one. Both projections are topographically organized, but in opposing anterior-posterior orientations. This arrangement provides functionally coherent input to each colliculus from the binocular visual field, supporting visual function. When guidance cues involved in contralateral topography (ephrin-As) are absent, crossed retinal ganglion cell (RGC) axons form inappropriate terminations within the SC. However, the organization of the ipsilateral projection relative to the abnormal contralateral input remains unknown, as does the functional capacity of both projections. We show here that in ephrin-A-/- mice, the SC contains an expanded, diffuse ipsilateral projection. Electrophysiological recording demonstrated that topography of visually evoked responses recorded from the contralateral superior colliculus of ephrin-A-/- mice displayed similar functional disorder in all genotypes, contrasting with their different degrees of anatomical disorder. In contrast, ipsilateral responses were retinotopic in ephrin-A2-/- but disorganized in ephrin-A2/A5-/- mice. The lack of integration of binocular input resulted in specific visual deficits, which could be reversed by occlusion of one eye. The discrepancy between anatomical and functional topography in both the ipsilateral and contralateral projections implies suppression of inappropriately located terminals. Moreover, the misalignment of ipsilateral and contralateral visual information in ephrin-A2/A5-/-mice suggests a role for ephrin-As in integrating convergent visual inputs. [ABSTRACT FROM AUTHOR]

Abstract: The cytokine hormone erythropoietin (EPO) is neuroprotective in models of brain injury and disease, and protects retinal ganglion cells (RGC) from cell death after axotomy. Here, we assessed EPO''s neuroprotective properties in vivo by examining RGC survival and axon regeneration at 4 weeks following intraorbital optic nerve transection in adult rat. EPO was administered as a single intravitreal injection at the time of transection (5, 10, 25, 50 units, PBS control). Intravitreal EPO (5, 10 units) significantly increased RGC somata and axon survival between the eye and transection site. Twenty five units did not improve survival of RGC somata but did increase axon survival between the eye and transection site. In addition, a small proportion of axons penetrated the transection site and regenerated up to 1 mm into the distal nerve. In a second series, intravitreal EPO (25 units) doubled the number of RGC axons regenerating along a length of peripheral nerve grafted onto the retrobulbar optic nerve. Our in vivo evidence of both neuroregeneration and neuroprotection, taken together with the natural occurrence of EPO within the body and its ability to cross the blood–brain barrier, suggests that it offers promise as a therapeutic agent for central nerve repair. [Copyright &y& Elsevier]

Abstract: Retinotectal topography is established during development and relies on the sequential recruitment of glutamate receptors within postsynaptic tectal cells. NMDA receptors underpin plastic changes at early stages when retinal ganglion cell (RGC) terminal arbors are widespread and topography is coarse; AMPA/kainate receptors mediate fast secure neurotransmission characteristic of mature circuits once topography is refined. Here, we have examined the relative contributions of these receptors to visually evoked activity in normal adult goldfish, in which retinotectal topography is constantly adjusted to compensate for the continual neurogenesis and the addition of new RGC arbors. Furthermore, we examined animals at two stages of optic nerve regeneration. In the first, RGC arbors are widespread and receptive fields large resulting in coarse topography; in the second, RGC arbors are pruned to reduce receptive fields leading to refined topography. Antagonists were applied to the tectum during multiunit recording of postsynaptic responses. Normal goldfish have low levels of NMDA receptor-mediated activity and high levels of AMPA/kainate. When coarse topography has been restored, NMDA receptor-mediated activity is increased and that of AMPA/kainate decreased. Once topography has been refined, the balance of NMDA and AMPA/kainate receptor-mediated activity returns to normal. The data suggest that glutamatergic neurotransmission in normal adult goldfish is dual with NMDA receptors fine-tuning topography and AMPA receptors allowing stable synaptic function. Furthermore, the normal operation of both receptors allows a response to injury in which the balance can be transiently reversed to restore topography and vision. [Copyright &y& Elsevier]

Peripheral nerve (PN) grafts can be used to bridge tissue defects in the CNS. Using a PN-to-optic nerve (ON) graft model, we combined gene therapy with pharmacotherapy to promote the long-distance regeneration of injured adult retinal ganglion cells (RGCs). Autologous sciatic nerve was sutured onto the transected ON and the distal end immediately inserted into contralateral superior colliculus (SC). Control rats received intraocular injections of saline or adeno-associated virus (AAV) encoding GFP. In experimental groups, three bi- cis tronic AAV vectors encoding ciliary neurotrophic factor (CNTF) were injected into different regions of the grafted eye. Each vector encoded a different fluorescent reporter to assess retinotopic order in the regenerate projection. To encourage sprouting/synaptogenesis, after 6 weeks some AAV–CNTF injected rats received an intravitreal injection of recombinant brain-derived neurotrophic factor (rBDNF) or AAV–BDNF. Four months after surgery, cholera toxin B was used to visualize regenerate RGC axons. RGC viability and axonal regrowth into SC were significantly greater in AAV–CNTF groups. In some cases, near the insertion site, regenerate axonal density resembled retinal terminal densities seen in normal SC. Complex arbors were seen in superficial but not deep SC layers and many terminals were immunopositive for presynaptic proteins vGlut2 and SV2. There was improvement in visual function via the grafted eye with significantly greater pupillary constriction in both AAV–CNTF + BDNF groups. In both control and AAV–CNTF + rBDNF groups the extent of light avoidance correlated with the maximal distance of axonal penetration into superficial SC. Despite the robust regrowth of RGC axons back into the SC, axons originating from different parts of the retina were intermixed at the PN graft/host SC interface, indicating that there remained a lack of order in this extensive regenerate projection. [ABSTRACT FROM AUTHOR]

Abstract: In the developing visual system, growing retinal ganglion cell (RGC) axons are exposed to multiple guidance and growth factors. Furthermore, the relative levels of these factors are differentially regulated as topography is roughly established and then refined. We have shown that during the establishment of rough topography (P3), growth cones of pure and explanted RGCs treated with combinations of BDNF and ephrin-A5-Fc responded differently than RGCs treated with BDNF or ephrin-A5-Fc alone (p =0.0083). The response to the combined treatment mimicked that of RGCs cultured with ephrin-A5-Fc alone once topography refines. The guidance cue receptors EphA and TrkB were shown to co-localise in RGCs in vitro. Furthermore, EphA and TrkB receptors interacted directly in in vitro binding assays. Our results suggest that the conversion of growth cone responses from collapse to stabilisation as topography refines, occurs as a result of interactions between EphA and TrkB receptors. [Copyright &y& Elsevier]