Two mechanisms for direction selectivity in a model of the primate starburst amacrine cell.

In a recent study, visual signals were recorded for the first time in starburst amacrine cells of the macaque retina, and, as for mouse and rabbit, a directional bias observed in calcium signals was recorded from near the dendritic tips. Stimulus motion from the soma toward the tip generated a larger calcium signal than motion from the tip toward the soma. Two mechanisms affecting the spatiotemporal summation of excitatory postsynaptic currents have been proposed to contribute to directional signaling at the dendritic tips of starbursts: (1) a "morphological" mechanism in which electrotonic propagation of excitatory synaptic currents along a dendrite sums bipolar cell inputs at the dendritic tip preferentially for stimulus motion in the centrifugal direction; (2) a "space-time" mechanism that relies on differences in the time-courses of proximal and distal bipolar cell inputs to favor centrifugal stimulus motion. To explore the contributions of these two mechanisms in the primate, we developed a realistic computational model based on connectomic reconstruction of a macaque starburst cell and the distribution of its synaptic inputs from sustained and transient bipolar cell types. Our model suggests that both mechanisms can initiate direction selectivity in starburst dendrites, but their contributions differ depending on the spatiotemporal properties of the stimulus. Specifically, the morphological mechanism dominates when small visual objects are moving at high velocities, and the space-time mechanism contributes most for large visual objects moving at low velocities.

Progress on Designing a Chemical Retinal Prosthesis.

The last major review of progress toward a chemical retinal prosthesis was a decade ago. Many important advancements have been made since then with the aim of producing an implantable device for animal testing. We review that work here discussing the potential advantages a chemical retinal prosthesis may possess, the spatial and temporal resolutions it might provide, the materials from which an implant might be constructed and its likely effectiveness in stimulating the retina in a natural fashion. Consideration is also given to implant biocompatibility, excitotoxicity of dispensed glutamate and known changes to photoreceptor degenerate retinas.

Origins of direction selectivity in the primate retina.

From mouse to primate, there is a striking discontinuity in our current understanding of the neural coding of motion direction. In non-primate mammals, directionally selective cell types and circuits are a signature feature of the retina, situated at the earliest stage of the visual process. In primates, by contrast, direction selectivity is a hallmark of motion processing areas in visual cortex, but has not been found in the retina, despite significant effort. Here we combined functional recordings of light-evoked responses and connectomic reconstruction to identify diverse direction-selective cell types in the macaque monkey retina with distinctive physiological properties and synaptic motifs. This circuitry includes an ON-OFF ganglion cell type, a spiking, ON-OFF polyaxonal amacrine cell and the starburst amacrine cell, all of which show direction selectivity. Moreover, we discovered that macaque starburst cells possess a strong, non-GABAergic, antagonistic surround mediated by input from excitatory bipolar cells that is critical for the generation of radial motion sensitivity in these cells. Our findings open a door to investigation of a precortical circuitry that computes motion direction in the primate visual system.

Hepatic cell mobilization for protection against ischemic myocardial injury.

The heart is capable of activating protective mechanisms in response to ischemic injury to support myocardial survival and performance. These mechanisms have been recognized primarily in the ischemic heart, involving paracrine signaling processes. Here, we report a distant cardioprotective mechanism involving hepatic cell mobilization to the ischemic myocardium in response to experimental myocardial ischemia-reperfusion (MI-R) injury. A parabiotic mouse model was generated by surgical skin-union of two mice and used to induce bilateral MI-R injury with unilateral hepatectomy, establishing concurrent gain- and loss-of-hepatic cell mobilization conditions. Hepatic cells, identified based on the cell-specific expression of enhanced YFP, were found in the ischemic myocardium of parabiotic mice with intact liver (0.2 ± 0.1%, 1.1 ± 0.3%, 2.7 ± 0.6, and 0.7 ± 0.4% at 1, 3, 5, and 10 days, respectively, in reference to the total cell nuclei), but not significantly in the ischemic myocardium of parabiotic mice with hepatectomy (0 ± 0%, 0.1 ± 0.1%, 0.3 ± 0.2%, and 0.08 ± 0.08% at the same time points). The mobilized hepatic cells were able to express and release trefoil factor 3 (TFF3), a protein mitigating MI-R injury as demonstrated in TFF3-/- mice (myocardium infarcts 17.6 ± 2.3%, 20.7 ± 2.6%, and 15.3 ± 3.8% at 1, 5, and 10 days, respectively) in reference to wildtype mice (11.7 ± 1.9%, 13.8 ± 2.3%, and 11.0 ± 1.8% at the same time points). These observations suggest that MI-R injury can induce hepatic cell mobilization to support myocardial survival by releasing TFF3.

Neuromodulation using electroosmosis.

Objective.Our laboratory has proposed chemical stimulation of retinal neurons using exogenous glutamate as a biomimetic strategy for treating vision loss caused by photoreceptor (PR) degenerative diseases. Although our previousin-vitrostudies using pneumatic actuation indicate that chemical retinal stimulation is achievable, an actuation technology that is amenable to microfabrication, as needed for anin-vivoimplantable device, has yet to be realized. In this study, we sought to evaluate electroosmotic flow (EOF) as a mechanism for delivering small quantities of glutamate to the retina. EOF has great potential for miniaturization.Approach.An EOF device to dispense small quantities of glutamate was constructed and its ability to drive retinal output tested in anin-vitropreparation of PR degenerate rat retina.Main results.We built and tested an EOF microfluidic system, with 3D printed and off-the-shelf components, capable of injecting small volumes of glutamate in a pulsatile fashion when a low voltage control signal was applied. With this device, we produced excitatory and inhibitory spike rate responses in PR degenerate rat retinae. Glutamate evoked spike rate responses were also observed to be voltage-dependent and localized to the site of injection.Significance.The EOF device performed similarly to a previously tested conventional pneumatic microinjector as a means of chemically stimulating the retina while eliminating the moving plunger of the pneumatic microinjector that would be difficult to miniaturize and parallelize. Although not implantable, the prototype device presented here as a proof of concept indicates that a retinal prosthetic based on EOF-driven chemical stimulation is a viable and worthwhile goal. EOF should have similar advantages for controlled dispensing of charged neurochemicals at any neural interface.

Correlation between retinal ganglion cell loss and nerve crush force-impulse established with instrumented tweezers in mice.

Objectives: Rodent models of optic nerve crush (ONC) have often been used to study degeneration and regeneration of retinal ganglion cells (RGCs) and their axons as well as the underlying molecular mechanisms. However, ONC results from different laboratories exhibit a range of RGC injury with varying degree of axonal damage. We developed instrumented tweezers to measure optic nerve (ON) crush forces in real time and studied the correlation between RGC axon loss and force-impulse, the product of force and duration, applied through the instrumented tweezers in mice.Methods: A pair of standard self-closing #N7 tweezers were instrumented with miniature foil strain gauges at optimal locations on both tweezers' arms. The instrumented tweezers were capable of recording the tip closure forces in the form of voltages, which were calibrated through load cells to corresponding tip closure forces over the operating range. Using the instrumented tweezers, the ONs of multiple mice were crushed with varied forces and durations and the axons in the immunostained sections of the crushed ONs were counted.Results: We found that the surviving axon density correlated with crush force, with longer duration and stronger crush forces producing consistently more axon damage.Discussion: The instrumented tweezers enable a simple technique for measurement of ONC forces in real-time for the first time. Using the instrumented tweezers, experimenters can quantify crush forces during ONC to produce consistent and predictable post-crush cell death. This should permit future studies a way to produce nerve damage more consistently than is available now.

Investigation of Injection Depth for Subretinal Delivery of Exogenous Glutamate to Restore Vision via Biomimetic Chemical Neuromodulation.

Chemical neuromodulation of the retina using native neurotransmitters to biomimetically activate target retinal neurons through chemical synapses is a promising biomimetic alternative to electrical stimulation for restoring vision in blindness caused by photoreceptor degenerative diseases. Recent research has shown that subretinal chemical stimulation could be advantageous for treating photoreceptor degenerative diseases but many of the parameters for achieving efficacious chemical neuromodulation are yet to be explored. In this paper, we investigated how the depth at which neurotransmitter is injected subretinally affects the success rate, spike rate characteristics (i.e., amplitude, response latency, and time width), and spatial resolution of chemical stimulation in wild-type Long Evans and photoreceptor degenerated S334ter-3 transgenic rat retinas in vitro. We compared the responses to injections of glutamate at the subretinal surface and two subsurface depths near the outer and inner plexiform layers and found that while injections at all depths elicited robust retinal ganglion cell responses, they differed significantly in terms of the spike rate characteristics and spatial resolutions across injection depths. Shallow subsurface injections near the outer plexiform layer evoked the highest spike rate amplitudes and had the highest spatial resolution and success rates, while deep subsurface injections near the inner plexiform layer elicited the shortest latencies and narrowest time widths. Our results suggest that surface injections are suboptimal for subretinal chemical neuromodulation, while shallow subsurface and deep subsurface injections may optimize high spatial and high temporal resolution, respectively. These findings have great significance for the design and development of a potential neurotransmitter-based subretinal prosthesis.

Mechanical Stimulation of the Retina: Therapeutic Feasibility and Cellular Mechanism.

Retinal prostheses that seek to restore vision by artificially stimulating retinal neurons with electrical current are an emerging treatment for photoreceptor degenerative diseases but face difficulties achieving naturalistic vision with high spatial resolution. Here, we report the unexpected discovery of a technique for mechanically stimulating retinal neurons with the potential to bypass the limitations of electrical stimulation. We found that pulsatile injections of standard Ames medium solution into explanted retinas of wild type rats under certain injection conditions (pulse-width > 50ms at 0.69 kPa pressure) elicit spatially localized retinal responses similar to light-evoked responses. The same injections made into photoreceptor degenerated retinas of transgenic S334ter-3 rats also elicit robust neural responses. We investigated the cellular mechanism causing these responses, by repeating the injections after treating the retinas with a pharmacological blocker of the transient receptor potential vanilloid (TRPV) channel group, a common mechanoreceptor found on retinal neurons, and observed a significant reduction in retinal ganglion cell spike rate response amplitudes. Together, these data reveal that therapeutic mechanical stimulation of the retina, occurring in part through TRPV channel activation, is feasible and this little explored neurostimulation paradigm could be useful in stimulating photoreceptor degenerated retinas for vision restoration.

Microfluidics-Based Subretinal Chemical Neuromodulation of Photoreceptor Degenerated Retinas.

Purpose: Retinal prostheses can restore rudimentary vision in cases of photoreceptor degeneration through electrical stimulation, but face difficulties achieving high spatial resolution because electrical current is an inherently unnatural stimulus. We investigated the therapeutic feasibility of using patterned delivery of the glutamate neurotransmitter, a primary agent of natural synaptic communication of the retina, as a biomimetic chemical alternative to electrical current for neuromodulation of photoreceptor degenerate retina. Methods: We injected small quantities of the neurotransmitter glutamate into the subretina of 20 explanted photoreceptor degenerated S334ter-3 rat retinas using glass micropipettes and a prototype multiport microfluidic device to accomplish single- and multisite stimulation in vitro. The effects of chemical stimulation were characterized by recording neural responses from retinal ganglion cells (RGCs) using a multielectrode array. Results: Subretinally injected exogenous glutamate activates RGCs, despite the substantial anatomic and physiologic changes caused by retinal remodeling, eliciting robust neural responses. The presence of excitatory and inhibitory RGC responses provides evidence that exogenous glutamate differentially activated neurons presynaptic to RGCs, likely inner retinal neurons belonging to the OFF and ON pathways. We also demonstrate that glutamate injections can evoke focal RGC responses with spatial resolutions comparable to or better than current generation electrical prostheses and, when applied at multiple sites simultaneously with the multiport microfluidic device, can produce spatially patterned neural responses. Conclusions: These significant results establish that chemical stimulation of degenerated retinas with neurotransmitters is an effective neuromodulation strategy with the potential of restoring high-resolution visual perception in patients rendered blind through photoreceptor degeneration.

Nonselective Wiring Accounts for Red-Green Opponency in Midget Ganglion Cells of the Primate Retina.

In primate retina, "red-green" color coding is initiated when signals originating in long (L) and middle (M) wavelength-sensitive cone photoreceptors interact antagonistically. The center-surround receptive field of "midget" ganglion cells provides the neural substrate for L versus M cone-opponent interaction, but the underlying circuitry remains unsettled, centering around the longstanding question of whether specialized cone wiring is present. To address this question, we measured the strength, sign, and spatial tuning of L- and M-cone input to midget receptive fields in the peripheral retina of macaque primates of either sex. Consistent with previous work, cone opponency arose when one of the cone types showed a stronger connection to the receptive field center than to the surround. We implemented a difference-of-Gaussians spatial receptive field model, incorporating known biology of the midget circuit, to test whether physiological responses we observed in real cells could be captured entirely by anatomical nonselectivity. When this model sampled nonselectively from a realistic cone mosaic, it accurately reproduced key features of a cone-opponent receptive field structure, and predicted both the variability and strength of cone opponency across the retina. The model introduced here is consistent with abundant anatomical evidence for nonselective wiring, explains both local and global properties of the midget population, and supports a role in their multiplexing of spatial and color information. It provides a neural basis for human chromatic sensitivity across the visual field, as well as the maintenance of normal color vision despite significant variability in the relative number of L and M cones across individuals.SIGNIFICANCE STATEMENT Red-green color vision is a hallmark of the human and nonhuman primate that starts in the retina with the presence of long (L)- and middle (M)-wavelength sensitive cone photoreceptor types. Understanding the underlying retinal mechanism for color opponency has focused on the broad question of whether this characteristic can emerge from nonselective wiring, or whether complex cone-type-specific wiring must be invoked. We provide experimental and modeling support for the hypothesis that nonselective connectivity is sufficient to produce the range of red-green color opponency observed in midget ganglion cells across the retina. Our nonselective model reproduces the diversity of physiological responses of midget cells while also accounting for systematic changes in color sensitivity across the visual field.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro.

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Different functional susceptibilities of mouse retinal ganglion cell subtypes to optic nerve crush injury.

In optic neuropathies, the progressive deterioration of retinal ganglion cell (RGC) function leads to irreversible vision loss. Increasing experimental evidence suggests differing susceptibility for RGC functional subtypes. Here with multi-electrode array recordings, RGC functional loss was characterized at multiple time points in a mouse model of optic nerve crush. Firing rate, latency of response and receptive field size were analyzed for ON, OFF and ON-OFF RGCs separately. It was observed that responses and receptive fields of OFF cells were impaired earlier than ON cells after the injury. For the ON-OFF cells, the OFF component of response was also more susceptible to optic nerve injury than the ON component. Moreover, more ON transient cells survived than ON sustained cells post the crush, implying a diversified vulnerability for ON cells. Together, these data support the contention that RGCs' functional degeneration in optic nerve injury is subtype dependent, a fact that needs to be considered when developing treatments of glaucomatous retinal ganglion cell degeneration and other optic neuropathies.

Overexpression of Brain-Derived Neurotrophic Factor Protects Large Retinal Ganglion Cells After Optic Nerve Crush in Mice.

Brain-derived neurotrophic factor (BDNF), a neurotrophin essential for neuron survival and function, plays an important role in neuroprotection during neurodegenerative diseases. In this study, we examined whether a modest increase of retinal BDNF promotes retinal ganglion cell (RGC) survival after acute injury of the optic nerve in mice. We adopted an inducible Cre-recombinase transgenic system to up-regulate BDNF in the mouse retina and then examined RGC survival after optic nerve crush by in vivo imaging. We focused on one subtype of RGC with large soma expressing yellow fluorescent protein transgene that accounts for ∼11% of the total SMI-32-positive RGCs. The median survival time of this subgroup of SMI-32 cells was 1 week after nerve injury in control mice but 2 weeks when BDNF was up-regulated. Interestingly, we found that the survival time for RGCs taken as a whole was 2 weeks, suggesting that these large-soma RGCs are especially vulnerable to optic nerve crush injury. We also studied changes in axon number using confocal imaging, confirming first the progressive loss reported previously for wild-type mice and demonstrating that BDNF up-regulation extended axon survival. Together, our results demonstrate that the time course of RGC loss induced by optic nerve injury is type specific and that overexpression of BDNF prolongs the survival of one subgroup of SMI-32-positive RGCs.

Prototype chemical synapse chip for spatially patterned neurotransmitter stimulation of the retina ex vivo.

Biomimetic stimulation of the retina with neurotransmitters, the natural agents of communication at chemical synapses, could be more effective than electrical stimulation for treating blindness from photoreceptor degenerative diseases. Recent studies have demonstrated the feasibility of neurotransmitter stimulation by injecting glutamate, a primary retinal neurotransmitter, into the retina at isolated single sites. Here, we demonstrate spatially patterned multisite stimulation of the retina with glutamate, offering the first experimental evidence for applicability of this strategy for translating visual patterns into afferent neural signals. To accomplish pattern stimulation, we fabricated a special microfluidic device comprising an array of independently addressable microports connected to tiny on-chip glutamate reservoirs via microchannels. The device prefilled with glutamate was interfaced with explanted rat retinas placed over a multielectrode array (MEA) with the retinal ganglion cells (RGC) contacting the electrodes and photoreceptor surface contacting the microports. By independently and simultaneously activating a subset of the microports with modulated pressure pulses, small boluses of glutamate were convectively injected at multiple sites in alphabet patterns over the photoreceptor surface. We found that the glutamate-driven RGC responses recorded through the MEA system were robust and spatially laid out in patterns strongly resembling the injection patterns. The stimulations were also highly localized with spatial resolutions comparable to or better than electrical retinal prostheses. Our findings suggest that surface stimulation of the retina with neurotransmitters in pixelated patterns of visual images is feasible and an artificial chemical synapse chip based on this approach could potentially circumvent the limitations of electrical retinal prostheses.

Differential stimulation of the retina with subretinally injected exogenous neurotransmitter: A biomimetic alternative to electrical stimulation.

Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no subretinal electrical or chemical stimulation study has stimulated the OFF and ON pathways differentially through inner retinal activation. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses.

Long-Term Protection of Retinal Ganglion Cells and Visual Function by Brain-Derived Neurotrophic Factor in Mice With Ocular Hypertension.

PURPOSE: Glaucoma, frequently associated with elevated intraocular pressure (IOP), is characterized by progressive retinal ganglion cell (RGC) death and vision loss. Brain-derived neurotrophic factor (BDNF) has been studied as a candidate for neuroprotection in rodent models of experimental glaucoma, yet it remains to be determined whether BDNF exerts long-term protection for subtype RGCs and vision against chronic IOP elevation. METHODS: We induced modest and sustained IOP elevation by laser illumination and microbead injection in mice. Using a tamoxifen-induced Cre recombinase system, BDNF was upregulated in the mouse retina when sustained IOP elevation was induced. We then examined whether overexpression of BDNF protected RGCs and vision during the period of ocular hypertension. Given that BDNF modulates axon growth and dendritic formation in a subtype-dependent manner, we tested whether BDNF protects RGC dendritic structure against the hypertensive insult also in a subtype-dependent manner. RESULTS: Sustained IOP elevation was induced and lasted up to 6 months. Overexpression of BDNF delayed progressive RGC and axon loss in hypertensive eyes. Brain-derived neurotrophic factor overexpression also helped to preserve acuity against the chronic hypertensive insult. We classified RGCs into ON and ON-OFF subtypes based on their dendritic lamination pattern in the inner plexiform layer and found that BDNF prevented ON-RGC dendritic degeneration in mice with sustained ocular hypertension. CONCLUSIONS: Our data demonstrated that BDNF can protect the dendritic fields of ON RGCs and reduce RGC and vision loss in mice with sustained ocular hypertension.

Thalamic Visual Prosthesis.

Glaucoma is a neurological disorder leading to blindness initially through the loss of retinal ganglion cells, followed by loss of neurons higher in the visual system. Some work has been undertaken to develop prostheses for glaucoma patients targeting tissues along the visual pathway, including the lateral geniculate nucleus (LGN) of the thalamus, but especially the visual cortex. This review makes the case for a visual prosthesis that targets the LGN. The compact nature and orderly structure of this nucleus make it a potentially better target to restore vision than the visual cortex. Existing research for the development of a thalamic visual prosthesis will be discussed along with the gaps that need to be addressed before such a technology could be applied clinically, as well as the challenge posed by the loss of LGN neurons as glaucoma progresses.

Retinal Ganglion Cell Loss is Delayed Following Optic Nerve Crush in NLRP3 Knockout Mice.

The NLRP3 inflammasome, a sensor for a variety of pathogen- and host-derived threats, consists of the adaptor ASC (Apoptosis-associated Speck-like protein containing a Caspase Activation and Recruitment Domain (CARD)), pro-caspase-1, and NLRP3 (NOD-Like Receptor family Pyrin domain containing 3). NLRP3-induced neuroinflammation is implicated in the pathogenesis and progression of eye diseases, but it remains unclear whether activation of NLRP3 inflammasome contributes to retinal ganglion cell (RGC) death. Here we examined NLRP3-induced neuroinflammation and RGC survival following partial optic nerve crush (pONC) injury. We showed that NLRP3 was up-regulated in retinal microglial cells following pONC, propagating from the injury site to the optic nerve head and finally the entire retina within one day. Activation of NLRP3-ASC inflammasome led to the up-regulation of caspase-1 and a proinflammatory cytokine, interleukin-1ß (IL-1ß). In NLRP3 knockout mice, up-regulation of ASC, caspase-1, and IL-1ß were all reduced, and, importantly, RGC and axon loss was substantially delayed following pONC injury. The average survival time of RGCs in NLRP3 knockout mice was about one week longer than for control animals. Taken together, our study demonstrated that ablating the NLRP3 gene significantly reduced neuroinflammation and delayed RGC loss after optic nerve crush injury.

Progressive degeneration of retinal and superior collicular functions in mice with sustained ocular hypertension.

PURPOSE: We investigated the progressive degeneration of retinal and superior collicular functions in a mouse model of sustained ocular hypertension. METHODS: Focal laser illumination and injection of polystyrene microbeads were used to induce chronic ocular hypertension. Retinal ganglion cell (RGC) loss was characterized by in vivo optical coherence tomography (OCT) and immunohistochemistry. Retinal dysfunction was also monitored by the full-field ERG. Retinal ganglion cell light responses were recorded using a 256-channel multielectrode array (MEA), and RGC subtypes were characterized by noncentered spike-triggered covariance (STC-NC) analysis. Single-unit extracellular recordings from superficial layers of the superior colliculus (SC) were performed to examine the receptive field (RF) properties of SC neurons. RESULTS: The elevation of intraocular pressure (IOP) lasted 4 months in mice treated with a combination of laser photocoagulation and microbead injection. Progressive RGC loss and functional degeneration were confirmed in ocular hypertensive (OHT) mice. These mice had fewer visually responsive RGCs than controls. Using the STC-NC analysis, we classified RGCs into ON, OFF, and ON-OFF functional subtypes. We showed that ON and OFF RGCs were more susceptible to the IOP elevation than ON-OFF RGCs. Furthermore, SC neurons of OHT mice had weakened responses to visual stimulation and exhibited mismatched ON and OFF subfields and irregular RF structure. CONCLUSIONS: We demonstrated that the functional degeneration of RGCs is subtype-dependent and that the ON and OFF pathways from the retina to the SC were disrupted. Our study provides a foundation to investigate the mechanisms underlying the progressive vision loss in experimental glaucoma.

Chemical stimulation of rat retinal neurons: feasibility of an epiretinal neurotransmitter-based prosthesis.

OBJECTIVE: No cure currently exists for photoreceptor degenerative diseases, which cause partial or total blindness in millions of people worldwide. Electrical retinal prostheses have been developed by several groups with the goal of restoring vision lost to these diseases, but electrical stimulation has limitations. It excites both somas and axons, activating retinal pathways nonphysiologically, and limits spatial resolution because of current spread. Chemical stimulation of retinal ganglion cells (RGCs) using the neurotransmitter glutamate has been suggested as an alternative to electrical stimulation with some significant advantages. However, sufficient scientific data to support developing a chemical-based retinal prosthesis is lacking. The goal of this study was to investigate the feasibility of a neurotransmitter-based retinal prosthesis and determine therapeutic stimulation parameters. APPROACH: We injected controlled amounts of glutamate into rat retinas from the epiretinal side ex vivo via micropipettes using a pressure injection system and recorded RGC responses with a multielectrode array. Responsive units were identified using a spike rate threshold of 3 Hz. MAIN RESULTS: We recorded both somal and axonal units and demonstrated successful glutamatergic stimulation across different RGC subtypes. Analyses show that exogenous glutamate acts on RGC synapses similar to endogenous glutamate and, unlike electrical prostheses, stimulates only RGC somata. The spatial spread of glutamate stimulation was ≈ 290 µm from the injection site, comparable to current electrical prostheses. Further, the glutamate injections produced spatially differential responses in OFF, ON, and ON-OFF RGC subtypes, suggesting that differential stimulation of the OFF and ON systems may be possible. A temporal resolution of 3.2 Hz was obtained, which is a rate suitable for spatial vision. SIGNIFICANCE: We provide strong support for the feasibility of an epiretinal neurotransmitter-based retinal prosthesis. Our findings suggest that chemical stimulation of RGCs is a viable alternative to electrical stimulation and could offer distinct advantages such as the selective stimulation of RGC somata.