Retinal Circuitry Research Articles

How Diverse Retinal Functions Arise from Feedback at the First Visual Synapse

Many brain regions contain local interneurons of distinct types. How does an interneuron type contribute to the input-output transformations of a given brain region? We addressed this question in the mouse retina by chemogenetically perturbing horizontal cells, an interneuron type providing feedback at the first visual synapse, while monitoring the light-driven spiking activity in thousands of ganglion cells, the retinal output neurons. We uncovered six reversible perturbation-induced effects in the response dynamics and response range of ganglion cells. The effects were enhancing or suppressive, occurred in different response epochs, and depended on the ganglion cell type. A computational model of the retinal circuitry reproduced all perturbation-induced effects and led us to assign specific functions to horizontal cells with respect to different ganglion cell types. Our combined experimental and theoretical work reveals how a single interneuron type can differentially shape the dynamical properties of distinct output channels of a brain region.

Formation of retinal direction-selective circuitry initiated by starburst amacrine cell homotypic contact.

A common strategy by which developing neurons locate their synaptic partners is through projections to circuit-specific neuropil sublayers. Once established, sublayers serve as a substrate for selective synapse formation, but how sublayers arise during neurodevelopment remains unknown. Here, we identify the earliest events that initiate formation of the direction-selective circuit in the inner plexiform layer of mouse retina. We demonstrate that radially migrating newborn starburst amacrine cells establish homotypic contacts on arrival at the inner retina. These contacts, mediated by the cell-surface protein MEGF10, trigger neuropil innervation resulting in generation of two sublayers comprising starburst-cell dendrites. This dendritic scaffold then recruits projections from circuit partners. Abolishing MEGF10-mediated contacts profoundly delays and ultimately disrupts sublayer formation, leading to broader direction tuning and weaker direction-selectivity in retinal ganglion cells. Our findings reveal a mechanism by which differentiating neurons transition from migratory to mature morphology, and highlight this mechanism's importance in forming circuit-specific sublayers.

Convergence and Divergence of CRH Amacrine Cells in Mouse Retinal Circuitry.

Inhibitory interneurons sculpt the outputs of excitatory circuits to expand the dynamic range of information processing. In mammalian retina, >30 types of amacrine cells provide lateral inhibition to vertical, excitatory bipolar cell circuits, but functional roles for only a few amacrine cells are well established. Here, we elucidate the function of corticotropin-releasing hormone (CRH)-expressing amacrine cells labeled in Cre-transgenic mice of either sex. CRH cells costratify with the ON alpha ganglion cell, a neuron highly sensitive to positive contrast. Electrophysiological and optogenetic analyses demonstrate that two CRH types (CRH-1 and CRH-3) make GABAergic synapses with ON alpha cells. CRH-1 cells signal via graded membrane potential changes, whereas CRH-3 cells fire action potentials. Both types show sustained ON-type responses to positive contrast over a range of stimulus conditions. Optogenetic control of transmission at CRH-1 synapses demonstrates that these synapses are tuned to low temporal frequencies, maintaining GABA release during fast hyperpolarizations during brief periods of negative contrast. CRH amacrine cell output is suppressed by prolonged negative contrast, when ON alpha ganglion cells continue to receive inhibitory input from converging OFF-pathway amacrine cells; the converging ON- and OFF-pathway inhibition balances tonic excitatory drive to ON alpha cells. Previously, it was demonstrated that CRH-1 cells inhibit firing by suppressed-by-contrast (SbC) ganglion cells during positive contrast. Therefore, divergent outputs of CRH-1 cells inhibit two ganglion cell types with opposite responses to positive contrast. The opposing responses of ON alpha and SbC ganglion cells are explained by differing excitation/inhibition balance in the two circuits.SIGNIFICANCE STATEMENT A goal of neuroscience research is to explain the function of neural circuits at the level of specific cell types. Here, we studied the function of specific types of inhibitory interneurons, corticotropin-releasing hormone (CRH) amacrine cells, in the mouse retina. Genetic tools were used to identify and manipulate CRH cells, which make GABAergic synapses with a well studied ganglion cell type, the ON alpha cell. CRH cells converge with other types of amacrine cells to tonically inhibit ON alpha cells and balance their high level of excitation. CRH cells diverge to different types of ganglion cell, the unique properties of which depend on their balance of excitation and inhibition.

Measuring Retinal Function in the Mouse.

Electroretinography is a crucial assay for studying the function and the functional integrity of the retina. The mouse is an important animal model for studying the retinal neurons and circuitries. In addition, it is often used as animal model for human retinal disorders. Therefore, a good understanding of the procedures in animal handling, of the methods for data analysis and of the requirements for stimulators and for the data acquisition equipment is of importance. Here, the currently most common methods and materials for in vivo electroretinography in the mouse are discussed.

Reduction on OFF-responses of Electroretinogram in Monkeys with Long-term High Intraocular Pressure.

Background:There are ON- and OFF-pathways in the normal vertebrate retina. Short- and long-flash electroretinogram (ERG) are suitable methods to observe the function of ON- and OFF-pathways in vivo, respectively. It is clear that high intraocular pressure (IOP) might cause dysfunction of cone-dominated photopic negative response (PhNR) in monkeys with high IOP in ON-pathway. However, whether cone-dominated OFF-responses are also affected is less known. The aim of this study was to observe photopic OFF-responses of ERG in monkeys with high IOP.Methods:Nine monkeys were involved in the experiment from January 2006 to December 2016. High IOP was induced in the right eye by laser coagulation of the mid-trabecular meshwork in five monkeys. Six years after the laser coagulation, both short- and long-flash of the photopic ERG were recorded. Stimulus light was red flashes superimposed on a blue background. Four normal monkeys were examined under the same ERG protocols as controls. Paired t-test was used to compare the difference of each ERG parameter between the lasered eye and the fellow eye. Analysis of variance (ANOVA) with Tukey adjustment was adopted to calculate the differences among the lasered eye, the fellow eye, and the eyes of normal monkeys.Results:The mean amplitude of a-wave (11.73 ± 2.05) and PhNR (8.67 ± 2.44) in lasered eyes was significantly lower than that of a-wave (21.47 ± 3.15) and PhNR (22.05 ± 3.42) in fellow eyes (P = 0.03 and P = 0.01, respectively) in response to short flash. The mean amplitude of d-wave (1.60 ± 0.59) and i-wave (3.13 ± 0.64) was significantly reduced in the lasered eyes than that of d-wave (4.01 ± 0.56) and i-wave (8.79 ± 1.75) in the fellow eyes (P = 0.02 and P = 0.02, respectively) in response to long flash.Conclusions:Reduced OFF-responses are recorded in monkeys with high IOP when dysfunction of photoreceptor is involved. The reduced OFF-responses to long-flash stimulus show evidence of anomalous retinal circuitry in glaucomatous retinopathy.

It's not all black and white: visual scene parameters influence optokinetic reflex performance in Xenopus laevis tadpoles.

The maintenance of visual acuity during active and passive body motion is ensured by gaze-stabilizing reflexes that aim at minimizing retinal image slip. For the optokinetic reflex (OKR), large-field visual motion of the surround forms the essential stimulus that activates eye movements. Properties of the moving visual world influence cognitive motion perception and the estimation of visual image velocity. Therefore, the performance of brainstem-mediated visuo-motor behaviors might also depend on image scene characteristics. Employing semi-intact preparations of mid-larval stages of Xenopus laevis tadpoles, we studied the influence of contrast polarity, intensity, contour shape and different motion stimulus patterns on the performance of the OKR and multi-unit optic nerve discharge during motion of a large-field visual scene. At high contrast intensities, the OKR amplitude was significantly larger for visual scenes with a positive contrast (bright dots on a dark background) compared with those with a negative contrast. This effect persisted for luminance-matched pairs of stimuli, and was independent of contour shape. The relative biases of OKR performance along with the independence of the responses from contour shape were closely matched by the optic nerve discharge evoked by the same visual stimuli. However, the multi-unit activity of retinal ganglion cells in response to a small single moving vertical edge was strongly influenced by the light intensity in the vertical neighborhood. This suggests that the underlying mechanism of OKR biases related to contrast polarity directly derives from visual motion-processing properties of the retinal circuitry.

Imaging and quantifying ganglion cells and other transparent neurons in the living human retina.

Ganglion cells (GCs) are fundamental to retinal neural circuitry, processing photoreceptor signals for transmission to the brain via their axons. However, much remains unknown about their role in vision and their vulnerability to disease leading to blindness. A major bottleneck has been our inability to observe GCs and their degeneration in the living human eye. Despite two decades of development of optical technologies to image cells in the living human retina, GCs remain elusive due to their high optical translucency. Failure of conventional imaging-using predominately singly scattered light-to reveal GCs has led to a focus on multiply-scattered, fluorescence, two-photon, and phase imaging techniques to enhance GC contrast. Here, we show that singly scattered light actually carries substantial information that reveals GC somas, axons, and other retinal neurons and permits their quantitative analysis. We perform morphometry on GC layer somas, including projection of GCs onto photoreceptors and identification of the primary GC subtypes, even beneath nerve fibers. We obtained singly scattered images by: (i) marrying adaptive optics to optical coherence tomography to avoid optical blurring of the eye; (ii) performing 3D subcellular image registration to avoid motion blur; and (iii) using organelle motility inside somas as an intrinsic contrast agent. Moreover, through-focus imaging offers the potential to spatially map individual GCs to underlying amacrine, bipolar, horizontal, photoreceptor, and retinal pigment epithelium cells, thus exposing the anatomical substrate for neural processing of visual information. This imaging modality is also a tool for improving clinical diagnosis and assessing treatment of retinal disease.

Comparison of Monophasic and Biphasic Electrical Stimulation by Using Temporal Analysis for Different Inter-electrode Spacings in the Hexagonal Arrays

Recent multidisciplinary studies in the field of visual prosthesis rely on electrically stimulating retinal tissue by placing electrode array into different part of it, bypassing the nerve cells of the visual pathway which have lost their functionality due to various degenerative diseases. The visual prosthesis systems could be more efficient by mimicking physiologically natural electrical signals. In this study, the responses of the retina to biphasic and monophasic electrical stimulations were compared temporally using in-vitro rabbit retina. The retinal tissue was electrically stimulated with charge-balanced biphasic and monophasic pulses. When temporal diversity was comparatively analyzed, spike activity was observed to intensify in the first 20 ms after the moment of stimulation for biphasic stimulation. On the other hand, the spike activity was observed to form in a way that it can be classified into two categories, the primary one forming in the first 20 ms and the secondary delayed one forming between 80 and 100 ms for monophasic stimulation. Moreover, the effect of retina stimulation amplitude was also analyzed. The spike distribution was observed to cumulate mostly in the post-stimulation time interval of the first 20 ms as the stimulation amplitude for biphasic stimulation increased. In monophasic stimulation, secondary delayed spike activity was observed over 80 \({\upmu }\hbox {A}\). The obtained experimental findings suggested that lower amplitude charge-balanced short biphasic waveforms could better elicit the precise and proper stimulation patterns necessary to mimic the neural activity of retinal circuitry that resembles the natural ones when it was compared with monophasic stimulation.

Asymmetry between ON and OFF α ganglion cells of mouse retina: integration of signal and noise from synaptic inputs.

Bipolar and amacrine cells presynaptic to the ON sustained α cell of mouse retina provide currents with a higher signal-to-noise power ratio (SNR) than those presynaptic to the OFF sustained α cell. Yet the ON cell loses proportionately more SNR from synaptic inputs to spike output than the OFF cell does. The higher SNR of ON bipolar cells at the beginning of the ON pathway compensates for losses incurred by the ON ganglion cell, and improves the processing of positive contrasts. ON and OFF pathways in the retina include functional pairs of neurons that, at first glance, appear to have symmetrically similar responses to brightening and darkening, respectively. Upon careful examination, however, functional pairs exhibit asymmetries in receptive field size and response kinetics. Until now, descriptions of how light-adapted retinal circuitry maintains a preponderance of signal over the noise have not distinguished between ON and OFF pathways. Here I present evidence of marked asymmetries between members of a functional pair of sustained α ganglion cells in the mouse retina. The ON cell exhibited a proportionately greater loss of signal-to-noise power ratio (SNR) from its presynaptic arrays to its postsynaptic currents. Thus the ON cell combines signal and noise from its presynaptic arrays of bipolar and amacrine cells less efficiently than the OFF cell does. Yet the inefficiency of the ON cell is compensated by its presynaptic arrays providing a higher SNR than the arrays presynaptic to the OFF cell, apparently to improve visual processing of positive contrasts. Dynamic clamp experiments were performed that introduced synaptic conductances into ON and OFF cells. When the amacrine-modulated conductance was removed, the ON cell's spike train exhibited an increase in SNR. The OFF cell, however, showed the opposite effect of removing amacrine input, which was a decrease in SNR. Thus ON and OFF cells have different modes of synaptic integration with direct effects on the SNR of the spike output.

Intraocular Injection of ES Cell-Derived Neural Progenitors Improve Visual Function in Retinal Ganglion Cell-Depleted Mouse Models.

Retinal ganglion cell (RGC) transplantation is a promising strategy to restore visual function resulting from irreversible RGC degeneration occurring in glaucoma or inherited optic neuropathies. We previously demonstrated FGF2 induced differentiation of mouse embryonic stem cells (ESC) to RGC lineage, capable of retinal ganglion cell layer (GCL) integration upon transplantation. Here, we evaluated possible improvement of visual function by transplantation of ES cell derived neural progenitors in RGC depleted glaucoma mice models. ESC derived neural progenitors (ES-NP) were transplanted into N-Methyl-D-Aspartate (NMDA) injected, RGC-ablated mouse models and a pre-clinical glaucoma mouse model (DBA/2J) having sustained higher intra ocular pressure (IOP). Visual acuity and functional integration was evaluated by behavioral experiments and immunohistochemistry, respectively. GFP-expressing ES-NPs transplanted in NMDA-injected RGC-depleted mice differentiated into RGC lineage and possibly integrating into GCL. An improvement in visual acuity was observed after 2 months of transplantation, when compared to the pre-transplantation values. Expression of c-Fos in the transplanted cells, upon light induction, further suggests functional integration into the host retinal circuitry. However, the transplanted cells did not send axonal projections into optic nerve. Transplantation experiments in DBA/2J mouse showed no significant improvement in visual functions, possibly due to both host and transplanted retinal cell death which could be due to an inherent high IOP. We showed that, ES NPs transplanted into the retina of RGC-ablated mouse models could survive, differentiate to RGC lineage, and possibly integrate into GCL to improve visual function. However, for the survival of transplanted cells in glaucoma, strategies to control the IOP are warranted.

Elevated IOP alters the space–time profiles in the center and surround of both ON and OFF RGCs in mouse

Glaucoma is a leading cause of blindness worldwide, and is characterized by progressive retinal ganglion cell (RGC) death. An experimental model of glaucoma has been established by elevating the intraocular pressure (IOP) via microbead occlusion of ocular fluid outflow in mice. Studies in this model have found visual dysfunction that varied with adaptational state, occurred before anatomical changes, and affected OFF RGCs more than ON RGCs. These results indicate subtle alterations in the underlying retinal circuitry that could help identify disease before irreversible RGC changes. Therefore, we looked at how RGC function was altered with elevated IOP under both photopic and scotopic conditions. We first found that responses to light offset are diminished with IOP elevation along with a concomitant decrease in receptive field center size for OFF RGCs. In addition, the antagonistic surround strength and size was reduced in ON RGCs. Furthermore, elevation of IOP significantly accelerated the photopic temporal tuning of RGC center responses in both ON and OFF RGCs. We found that some of the IOP-induced functional changes to OFF RGCs relied on ON cross-over pathways, indicating dysfunction in inner retinal circuitry. Overall, these results suggest that IOP alters multiple functions in the retina depending on the adaptational state. They provide a basis for designing multiple functional tests for early detection of glaucoma and for circuit-specific therapeutic targets in treatment of this blinding disease.

Inhibitory Interneurons in the Retina: Types, Circuitry, and Function.

Visual signals in the vertebrate retina are shaped by feedback and feedforward inhibition in two synaptic layers. In one, horizontal cells establish fundamental center-surround receptive-field properties via morphologically and physiologically complex synapses with photoreceptors and bipolar cells. In the other, a panoply of amacrine cells imbue ganglion cell responses with spatiotemporally complex information about the visual world. Here, I review current ideas about horizontal cell signaling, considering the evidence for and against the leading, competing theories. I also discuss recent work that has begun to make sense of the remarkable morphological and physiological diversity of amacrine cells. These latter efforts have been aided tremendously by increasingly complete connectivity maps of inner retinal circuitry and new genetic tools that enable study of individual, sparsely expressed amacrine cell types.

Dendro-dendritic cholinergic excitation controls dendritic spike initiation in retinal ganglion cells

The retina processes visual images to compute features such as the direction of image motion. Starburst amacrine cells (SACs), axonless feed-forward interneurons, are essential components of the retinal direction-selective circuitry. Recent work has highlighted that SAC-mediated dendro-dendritic inhibition controls the action potential output of direction-selective ganglion cells (DSGCs) by vetoing dendritic spike initiation. However, SACs co-release GABA and the excitatory neurotransmitter acetylcholine at dendritic sites. Here we use direct dendritic recordings to show that preferred direction light stimuli evoke SAC-mediated acetylcholine release, which powerfully controls the stimulus sensitivity, receptive field size and action potential output of ON-DSGCs by acting as an excitatory drive for the initiation of dendritic spikes. Consistent with this, paired recordings reveal that the activation of single ON-SACs drove dendritic spike generation, because of predominate cholinergic excitation received on the preferred side of ON-DSGCs. Thus, dendro-dendritic release of neurotransmitters from SACs bi-directionally gate dendritic spike initiation to control the directionally selective action potential output of retinal ganglion cells.

Photopharmacological control of bipolar cells restores visual function in blind mice.

Photopharmacological control of neuronal activity using synthetic photochromic ligands, or photoswitches, is a promising approach for restoring visual function in patients suffering from degenerative retinal diseases. Azobenzene photoswitches, such as AAQ and DENAQ, have been shown to restore the responses of retinal ganglion cells to light in mouse models of retinal degeneration but do not recapitulate native retinal signal processing. Here, we describe diethylamino-azo-diethylamino (DAD), a third-generation photoswitch that is capable of restoring retinal ganglion cell light responses to blue or white light. In acute brain slices of murine layer 2/3 cortical neurons, we determined that the photoswitch quickly relaxes to its inactive form in the dark. DAD is not permanently charged, and the uncharged form enables the photoswitch to rapidly and effectively cross biological barriers and thereby access and photosensitize retinal neurons. Intravitreal injection of DAD restored retinal light responses and light-driven behavior to blind mice. Unlike DENAQ, DAD acts upstream of retinal ganglion cells, primarily conferring light sensitivity to bipolar cells. Moreover, DAD was capable of generating ON and OFF visual responses in the blind retina by utilizing intrinsic retinal circuitry, which may be advantageous for restoring visual function.

Color percepts elicited by stimulation of individual targeted cones

To study the circuits underlying color vision near the fovea, we developed a system with adaptive optics and high-speed eye tracking that enables tracking, targeting and stimulation of cones in living eyes. In two subjects with classified mosaics, cones stimulated with 543 nm light against a white background yield expected and unexpected percepts. Stimulated M cones yield either green or achromatic percepts, and the individual responses are pure - largely falling into color or achromatic reporting classes. L cones respond similarly, except the fraction of color-reporting cones yield red percepts. Similar results are found against a blue background, but the M cones convey distinctly blue percepts rather than green. What is unexpected is that the arrangement of the color and achromatic reporting cones are distributed in a way that is inconsistent with simple chromatically-opponent, center-surround midget ganglion cells. To understand how lateral interactions influence these percepts, we're measuring cone sensitivity thresholds against different adapting background conditions. Collectively, these studies shed light on how foveal cone signals are transformed by retinal circuitry.

Connectomics of synaptic microcircuits: lessons from the outer retina.

Photoreceptors form a sophisticated synaptic complex with bipolar and horizontal cells, transmitting the signals generated by the phototransduction cascade to downstream retinal circuitry. The cone photoreceptor synapse shows several characteristic anatomical connectivity motifs that shape signal transfer: typically, ON-cone bipolar cells receive photoreceptor input through invaginating synapses; OFF-cone bipolar cells form basal synapses with photoreceptors. Both ON- and OFF-cone bipolar cells are believed to sample from all cone photoreceptors within their dendritic span. Electron microscopy and immunolabelling studies have established the robustness of these motifs, but have been limited by trade-offs in sample size and spatial resolution, respectively, constraining precise quantitative investigation to a few individual cells. 3D-serial electron microscopy overcomes these limitations and has permitted complete sets of neurons to be reconstructed over a comparatively large section of retinal tissue. Although the published mouse dataset lacks labels for synaptic structures, the characteristic anatomical motifs at the photoreceptor synapse can be exploited to identify putative synaptic contacts, which has enabled the development of a quantitative description of outer retinal connectivity. This revealed unexpected exceptions to classical motifs, including substantial interaction between rod and cone pathways at the photoreceptor synapse, sparse photoreceptor sampling and atypical contacts. Here, we summarize what was learned from this study in a more general context: we consider both the implications and limitations of the study and identify promising avenues for future research.

Detailed electroretinographic findings in rd8 mice.

Previous work has suggested that the retinal degeneration mutant rd8 mouse lacks an electroretinographic (ERG) phenotype until about 9months of age. We evaluated the ERG phenotype of these mice by measuring both conventional ERG responses and scotopic threshold responses. Groups of 4-month-old wild-type (WT) and mutant (rd8) mice were anesthetized and tested for mass retinal responses (ERGs) to several types of visual stimuli. Scotopic threshold responses were accumulated with brief scotopic flashes at a series of very dim intensities. Dark-adapted (scotopic) and light-adapted (photopic) responses to brief flashes at a series of higher intensities were recorded, along with long flashes and random modulations of light levels under photopic conditions. Negative scotopic threshold responses (nSTRs) had lower amplitudes in rd8 mice compared to WTs. Positive scotopic threshold responses were similar in the two groups. With the more intense stimuli, a- and c-wave amplitudes were smaller in rd8 mice. Both scotopic and photopic b-wave amplitudes tended to be larger in rd8 mice, though generally not significantly. The striking decrease in nSTR amplitudes was surprising, given that the main retinal effects of the rd8 mutation occur in the outer retina, at the external limiting membrane. The primary source of nSTRs in mice is thought to be at the amacrine cell level in the inner retina. Investigation of how this mutation leads to inner retinal dysfunction might reveal unexpected aspects of retinal cell biology and circuitry.

Bright flash response recovery of mammalian rods in vivo is rate limited by RGS9.

The temporal resolution of scotopic vision is thought to be constrained by the signaling kinetics of retinal rods, which use a highly amplified G-protein cascade to transduce absorbed photons into changes in membrane potential. Much is known about the biochemical mechanisms that determine the kinetics of rod responses ex vivo, but the rate-limiting mechanisms in vivo are unknown. Using paired flash electroretinograms with improved signal-to-noise, we have recorded the amplitude and kinetics of rod responses to a wide range of flash strengths from living mice. Bright rod responses in vivo recovered nearly twice as fast as all previous recordings, although the kinetic consequences of genetic perturbations previously studied ex vivo were qualitatively similar. In vivo, the dominant time constant of recovery from bright flashes was dramatically reduced by overexpression of the RGS9 complex, revealing G-protein deactivation to be rate limiting for recovery. However, unlike previous ex vivo recordings, dim flash responses in vivo were relatively unaffected by RGS9 overexpression, suggesting that other mechanisms, such as calcium feedback dynamics that are strongly regulated by the restricted subretinal microenvironment, act to determine rod dim flash kinetics. To assess the consequences for scotopic vision, we used a nocturnal wheel-running assay to measure the ability of wild-type and RGS9-overexpressing mice to detect dim flickering stimuli and found no improvement when rod recovery was speeded by RGS9 overexpression. These results are important for understanding retinal circuitry, in particular as modeled in the large literature that addresses the relationship between the kinetics and sensitivity of retinal responses and visual perception.

Quantitative electroencephalograms and neuro-optometry: a case study that explores changes in electrophysiology while wearing therapeutic eyeglasses.

The brain is equipped with a complex system for processing sensory information, including retinal circuitry comprising part of the central nervous system. Retinal stimulation can influence brain function via customized eyeglasses at both subcortical and cortical levels. We investigated cortical effects from wearing therapeutic eyeglasses, hypothesizing that they can create measureable changes in electroencephalogram (EEG) tracings. A Z-BellSM test was performed on a participant to select optimal lenses. An EEG measurement was recorded before and after the participant wore the eyeglasses. Equivalent quantitative electroencephalography (QEEG) analyses (statistical analysis on raw EEG recordings) were performed and compared with baseline findings. With glasses on, the participant's readings were found to be closer to the normed database. The original objective of our investigation was met, and additional findings were revealed. The Z-bellSM test identified lenses to influence neurotypical brain activity, supporting the paradigm that eyeglasses can be utilized as a therapeutic intervention. Also, EEG analysis demonstrated that encephalographic techniques can be used to identify channels through which neuro-optomertric treatments work. This case study's preliminary exploration illustrates the potential role of QEEG analysis and EEG-derived brain imaging in neuro-optometric research endeavors to affect brain function.

Outer Plexiform Layer Structures Are Not Altered Following AAV-Mediated Gene Transfer in Healthy Rat Retina.

Ocular gene therapy approaches have been developed for a variety of different diseases. In particular, clinical gene therapy trials for RPE65 mutations, X-linked retinoschisis, and choroideremia have been conducted at different centers in recent years, showing that adeno-associated virus (AAV)-mediated gene therapy is safe, but limitations exist as to the therapeutic benefit and long-term duration of the treatment. The technique of vector delivery to retinal cells relies on subretinal injection of the vector solution, causing a transient retinal detachment. Although retinal detachments are known to cause remodeling of retinal neuronal structures as well as significant cell loss, the possible effects of this short-term therapeutic retinal detachment on retinal structure and circuitry have not yet been studied in detail. In this study, retinal morphology and apoptotic status were examined in healthy rat retinas following AAV-mediated gene transfer via subretinal injection with AAV2/5.CMV.d2GFP or sham injection with fluorescein. Outer plexiform layer (OPL) morphology was assessed by immunohistochemical labeling, laser scanning confocal microscopy, and electron microscopy. The number of synaptic contacts in the OPL was quantified after labeling with structural markers. To assess the apoptotic status, inflammatory and pro-apoptotic markers were tested and TUNEL assay for the detection of apoptotic nuclei was performed. Pre- and postsynaptic structures in the OPL, such as synaptic ribbons or horizontal and bipolar cell processes, did not differ in size or shape in injected versus non-injected areas and control retinas. Absolute numbers of synaptic ribbons were not altered. No signs of relevant gliosis were detected. TUNEL labeling of retinal cells did not vary between injected and non-injected areas, and apoptosis-inducing factor was not delocalized to the nucleus in transduced areas. The neuronal circuits in the OPL of healthy rat retinas undergoing AAV-mediated gene transfer were not altered by the temporary retinal detachment caused by subretinal injection, the presence of viral particles, or the expression of green fluorescent protein as a transgene. This observation likely requires further investigations in the dog model for RPE65 deficiency in order to determine the impact of RPE65 transgene expression on diseased retinas in animals and men.