Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives.

Protocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate-buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have found that these fixatives induce the formation of bead-like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage-gated Na(+) channel toxin, extracellular Ca(2+) ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde-based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry.

AAV-mediated, optogenetic ablation of Müller Glia leads to structural and functional changes in the mouse retina.

Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.

Short hairpin RNA-mediated knockdown of VEGFA in Müller cells reduces intravitreal neovascularization in a rat model of retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Müller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30eembedded shRNA against VEGFA that targeted Müller cells. Reduction of VEGFA by lentivector VEGFA-shRNAetargeting Müller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Müller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Müller cells safely reduces IVNV in a relevant ROP model.

Colocalization of hyperpolarization-activated, cyclic nucleotide-gated channel subunits in rat retinal ganglion cells.

The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (I(h)) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring I(h) in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage sensitivity of I(h) less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and I(h) activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native I(h) properties with the previously reported presence of HCN4 in these cells, and indicate that I(h) is biophysically similar in morphologically diverse retinal ganglion cells and differs from I(h) in rods, cones, and bipolar cells.

Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness.

Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.

Intravitreal injection of AAV2 transduces macaque inner retina.

PURPOSE: Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans. METHODS: In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters. RESULTS: AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-ß-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters--human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)--AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis. CONCLUSIONS: The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.

Towards optogenetic sensory replacement.

Over the last several years we have developed a rapidly-expanding suite of genetically-encoded reagents (e.g., ChR2, Halo, Arch, Mac, and others) that, when expressed in specific neuron types in the nervous system, enable their activities to be powerfully and precisely activated and silenced in response to light. If the genes that encode for these reagents can be delivered to cells in the body using gene therapy methods, and if the resultant protein payloads operate safely and effectively over therapeutically important periods of time, these molecules could subserve a set of precise prosthetics that use light as the trigger of information entry into the nervous system, e.g. for sensory replacement. Here we discuss the use of ChR2 to make the photoreceptor-deprived retina, as found in diseases such as retinitis pigmentosa, sensitive to light, enabling restoration of functional vision in a mouse model of blindness. We also discuss arrays of light sources that could be useful for delivering patterned sensory information into the nervous system.

In vivo imaging of microscopic structures in the rat retina.

PURPOSE: The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. The authors have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. METHODS: Enhanced green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague-Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. RESULTS: Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics. Adaptive optics correction reduced the total root mean square wavefront error, on average, from 0.30 microm to 0.05 microm (measured at 904 nm, 1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was approximately 1.84 microm, approximately 82% greater than the FWHM of the diffraction-limited LSF. CONCLUSIONS: With perfect aberration compensation, the in vivo resolution in the rat eye could be approximately 2x greater than that in the human eye because of its large numerical aperture (approximately 0.43). Although the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using adaptive optics to investigate the rodent eye in vivo with high resolution.

Rapid and stable knockdown of an endogenous gene in retinal pigment epithelium.

The selective silencing of target genes in specific cell types by RNA interference (RNAi) represents a powerful approach both to gene therapy of dominantly active mutant alleles, and to the investigation of normal gene function in animal models in vivo. We established a simple and versatile in vitro method for screening the efficacy of DNA-based short hairpin RNAs (shRNAs), and identified a highly effective shRNA targeting basic fibroblast growth factor (bFGF), a gene thought to play important roles in endogenous neuroprotective responses in the rat retina. We used two viral vectors, based on lentivirus and adeno-associated virus (AAV), to deliver shRNAs and silence bFGF in retinal pigment epithelial cells in vivo. The AAV experiments made use of a "stabilized double-stranded" version of these vectors with rapid onset of gene expression. In the rat retinal pigment epithelium, shRNAs delivered by either vector reduced bFGF immunoreactivity to undetectable levels in transduced cells, whereas a nonfunctional control construct incorporating a two-base pair mutation had no measurable effect on bFGF expression. Silencing commenced within a few days after injection of virus and remained stable throughout the period of observation, as long as 60 days. Viral delivery of RNAi constructs offers a powerful and versatile approach for both gene therapy and the analysis of fundamental questions in retinal biology.