Mammalian central nervous system (CNS) neurons lose their ability to regenerate their axons after injury during development. For example, optic nerve injury studies in hamsters have shown that optic nerve axons injured around the time of birth retain the ability to regenerate to their target, but this ability is lost during development (So et al., 1981). The development of an inhibitory CNS environment has been implicated in the inability of the adult CNS to regenerate, however there is also support for this loss being a result of changes in developmental programs intrinsic to the neurons themselves (Goldberg et al., 2002a; Goldberg, 2004). While some molecules have been identified as being involved in intrinsic mechanisms controlling axon growth, there is still much to be discovered. Using genes shown to be regulated in retinal ganglion cells (RGCs) during development (Wang et al., 2007), I performed an overexpression screen in embryonic primary neurons measuring changes in neurite growth. Of these genes, the most significant effect in neurite growth was seen with overexpression of Krüppel-like factor 4 (KLF4), resulting in a greater than 50% decrease in growth. KLF4 is a member of the KLF family of transcription factors which all possess a DNA binding domain containing 3 zinc finger motifs. Outside of the nervous system, KLF4 has been implicated in cancer (Black et al., 2001; Rowland and Peeper, 2006), mitotic growth arrest (Shields et al., 1996) and most recently in the induction of pluripotency (Yamanaka, 2008; Zhao and Daley, 2008). In the CNS, KLF4 has recently been implicated in increasing the sensitivity of cortical neurons to NMDA insult (Zhu et al, 2009), though no effect of KLF4 on neurite growth or regeneration has yet been described. I found that KLF4 overexpression in RGCs results in decreased neurite growth and neurite initiation. KLF4 overexpression also leads to decreases in polarity acquisition in hippocampal neurons, though even when they acquire polarity, they still display decreased neurite growth. Additionally, KLF4 knockout targeted to RGCs leads to an increased neurite growth ability and increased neurite initiation in vitro. In vivo, KLF4 knockout increases RGC axon regeneration after optic nerve injury. Interestingly, KLF4 is one of 17 members of the KLF family, known for their ability to act redundantly and competitively amongst family members for their binding sites. Therefore, we looked to see if other KLFs could affect neurite growth ability. 15 of 17 KLF family members are expressed in RGCs, and their overexpression results in differential effects on neurite growth in both cortical neurons and RGCs. Additionally, many of the family members are developmentally regulated in a manner that typically correlates with their ability to affect neurite growth. For example, KLF6 and -7, whose expression decreases during development, when overexpressed, increase neurite growth, whereas KLF9, whose expression increases developmentally, when overexpressed, decreases neurite growth. Surprisingly, there are multiple KLFs expressed in RGCs that are neurite growth-suppressors, and further study has revealed that the combination of KLF growth enhancers with KLF growth suppressors results in a suppressive or neutral phenotype (Moore et al., 2009), suggesting that to further enhance regeneration after injury in vivo, we will need to additionally remove the growth suppression from other KLF family members. Taken together, these data suggest that KLFs may play an important role in the intrinsic loss of axon growth and regeneration seen during development. Further characterization of downstream targets of KLF4 and other KLF family members may reveal specific neuronal gene targets that could mediate the phenotypic effects of these transcription factors. It is my hope that by determining the developmental programs that underlie the loss of intrinsic axon growth ability of CNS neurons, we may ultimately determine how to revert adult CNS neurons to their embryonic axon growth ability.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

Purpose: Retinal ganglion cell (RGC) loss is a measure of the progression of many visual disorders. It is important to identify RGCs with good specificity, so RGC numbers can be reliably analyzed. The purpose of this study was to analyze the effectiveness of two current RGC markers: Brn3a immunohistochemistry and the expression of Thy1-CFP in the Thy1-CFP transgenic mouse. Methods: Rhodamine-?-isothiocyanate (RITC) retrograde labeling, immunohistochemistry, wholemount retinal imaging, western blot, cross sectional analysis and cell densities in uninjured control animals and 3, 5, 7 and 14 days post-optic nerve crush (ONC) or transection (ONT) were tabulated. Results: Brn3a positive (Brn3a+) cell density was significantly less than RITC positive (RITC+) cell density in control mice. After ON injury, Brn3a+ cell density did not decrease at the same rate as RITC+ cell density. The density of RGCs that express Brn3a was significantly less than the individual Brn3a+ and RITC+ cell density at all experimental time points. Thy1-CFP positive (Thy1-CFP+) cell density was significantly less than RITC+ in control mice and significantly more than RITC+ cell density 14 days after ON injury. Thy1-CFP co-localized with ChAT positive (ChAT+) cells 7 days after ONT. Conclusion: Brn3a and Thy1-CFP are not reliable markers of RGCs. Retrograde labeling remains one of the most reliable methods of labeling RGCs in mice.

No description available.

No / Purpose: Does a physiologically plausible model of the retinal ganglion cell (RGC) receptive field (RF) predict the spatial tuning properties of the Hermann Grid Illusion (HGI)? Methods: The spatial tuning of a single intersection HGI was measured psychophysically in normal observers using a nulling technique at different vertical grid line luminances. We used a model based upon a standard RGC RF, balanced to produce zero response under uniform illumination, to predict the response of the model cell to the equivalent range of stimulus conditions when placed in either the 'street' or the 'intersection' of a single element of a Hermann grid. We determined the equivalent of the nulling luminance required to balance these responses and minimise the HGI. Results: The model and the psychophysical data demonstrated broad spatial tuning with similarly shaped tuning profiles and similar strengths of illusion. The line width at the peak of the model tuning function was around twice the model RGC RF centre size. Modelling the psychophysical functions gave RF centre sizes smaller than expected from human anatomical evidence but similar to that suggested by primate physiological evidence. In the model and psychophysically the strength of the illusion varied with the luminance of the vertical grid line when HGI strength was expressed as a Michelson nulling contrast, but this effect was smaller when HGI strength was expressed as a nulling luminance. Conclusions: The shape, width, height and position of the spatial tuning function of the HGI can be well modelled by a RGC RF based model. The broad tuning of these functions does not appear to require a broad range of different cell sizes either in the retina or later in the visual pathway.

yes / Purpose.: To establish whether a clinically exploitable relationship exists between surrogate measures of retinal ganglion cell number and functional sampling density and visual contrast sensitivity in healthy young eyes. Methods.: Psychometric functions for contrast detection were measured at 9° eccentricity in superior and inferior visual field from 20 healthy adults (age 23–43, median 26 years). Functions were compared with corresponding localized regions of retinal nerve fiber layer (RNFL) thickness measured by optical coherence tomography, a surrogate of retinal ganglion cell number, and to grating resolution acuity, a psychophysical surrogate of retinal ganglion cell sampling density. Correlations between psychometric function parameters and retinal ganglion cell surrogates were measured by Spearman's rank correlation. Results.: All measures exhibited a 2- to 4-fold variation in our sample. Despite this, correlations between measures were weak. Correlations between psychometric function parameters (threshold, spread) and RNFL thickness ranged in magnitude from 0.05 to 0.19 (P = 0.43–0.85). Grating resolution was sampling limited for 16 of 20 participants in superior visual field, and for 12 of 20 participants in inferior visual field. Correlations between psychometric function parameters and grating resolution acuities ranged in magnitude from 0.05 to 0.36 (P = 0.12–0.85) when all data were considered, and from 0.06 to 0.36 (P = 0.26–0.87) when only sampling-limited data were considered. Conclusions.: Despite considerable variation in both psychometric functions for contrast detection and surrogate measures of retinal ganglion cell number and sampling density among healthy eyes, relationships between these measures are weak. These relationships are unlikely to be exploitable for improving clinical tests in healthy populations.

No description available.

In the developing visual system, the axon of a pre-synaptic cell must be guided to a post-synaptic partner. Retinal ganglion cells (RGCs) in the eye are an excellent model to study this process. Multiple classes exist that respond to specific types of light input, and these project to different destinations in the brain that process distinct types of information. The RGC axons that navigate to the lateral geniculate nucleus (LGN) do so in a class-specific manner. Axons from RGCs that mediate non-image forming functions innervate the ventral LGN (vLGN) and the intergeniculate leaflet (IGL). Axons from RGCs that process image-forming information bypass these regions to innervate the dorsal LGN (dLGN). The extracellular protein reelin was identified as a potential factor in RGC axonal targeting of the vLGN and IGL, and the reeler mutant mouse used to study the effects of its functional absence. Anterograde labeling of RGCs and their axons with Cholera toxin B (CTB) revealed reduced patterns of retinal innervation to the vLGN and IGL in mutant mice. Moreover, the absence of functional reelin resulted in axons incorrectly growing into inappropriate regions of the thalamus. We identified these misrouted axons as those of the intrinsically photosensitive RGCs (ipRGCS), a class of RGCs known to project to the affected subnuclei. In contrast to defects in ipRGC targeting, no deficits were seen in retinogeniculate or corticothalamic projections in classes of axons that normally target the dLGN. Immunohistochemistry did not reveal any effects of the absence of the functional reelin on the LGN cytoarchitecture, which is unlike many other brain regions altered in the reeler. In summary, results suggest that intact reelin is required for class-specific retinogeniculate targeting to the vLGN and IGL. The defects are likely to be in targeting and not in neuronal positioning.

Experimental and clinical data have documented the adverse effects of perinatal ethanol intoxication on peripheral organs and the central nervous system. There is little known, however, about potential damaging effects of perinatal ethanol on the developing visual system. The purpose of this study was to examine the effects of neonatal ethanol intoxication on RGC morphology, estimate the total number of neurons in RGC layer and dorsolateral geniculate nucleus (dLGN), and on the eye-specific fiber segregation in the dLGN), in YFP and C57BL/6 mice pups. Ethanol (3 g/kg/day) was administered by intragastric intubation throughout postnatal days (PD) 3-20 or 3-10. Intubation control (IC) and untreated control (C) groups were included. Blood alcohol concentration (BAC) was measured in separate groups of pups on PD3, PD10, and PD20 at 4 different time points, 1, 1.5, 2 and 3 h after the second intubation. Numbers neurons in the RGCs and dLGN were quantified on PD10, PD20 using unbiased stereological procedures. The RGC images were taken using a confocal microscope and images were traced using Neurolucida software. On PD9, intraocular injections of cholera toxin-