Steven T. Walston

Electrical Stimulation of the Retina to Produce Artificial Vision

Retinal prostheses aim to restore vision to blind individuals suffering from retinal diseases such as retinitis pigmentosa and age-related macular degeneration. These devices function by electrically stimulating surviving retinal neurons, whose activation is interpreted by the brain as a visual percept. Many prostheses are currently under development. They are categorized as epiretinal, subretinal, and suprachoroidal prostheses on the basis of the placement of the stimulating microelectrode array. Each can activate ganglion cells through direct or indirect stimulation. The response of retinal neurons to these modes of stimulation are discussed in detail and are placed in context of the visual percept they are likely to evoke. This article further reviews challenges faced by retinal prosthesis and discusses potential solutions to address them.

Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina.

Retinitis pigmentosa is a family of inherited retinal diseases identified by the degeneration of photoreceptors, which leads to blindness. In efforts to restore vision lost to retinitis pigmentosa, retinal prostheses have been developed to generate visual percepts by electrically stimulating the surviving retinal bipolar and ganglion cells. The response of retinal ganglion cells to electrical stimulation has been characterized through direct measurement. However, the response of bipolar cells has only been inferred by measuring retinal ganglion cell activity. This investigation reports on a novel tissue preparation technique facilitating bipolar cell patch clamp recordings in wholemount retina. We find that bipolar cells respond to extracellular electrical stimuli with time-locked voltage spike depolarizations, which are likely mediated by voltage-gated calcium channels.

In vivo characterization of genetic expression of virus-transduced calcium indicators in retinal ganglion cells using a low-cost funduscope

Virus-transduced calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. To track the level of in vivo expression of genetically encoded calcium indicators (GECIs) in rodent retina, we developed a noninvasive imaging approach based on a custom-modified low-cost and simple fundus system that enabled us to monitor and characterize in vivo bright-field and fluorescence retinal image. The system clearly resolves individual retinal ganglion cells (RGCs) and axons. RGC fluorescence intensity and number of observable fluorescent cells show a consistent rising trend from week 1 to week 3 after viral injection, indicating a uniform increase of GCaMP6f expression. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array (MEA) and used calcium imaging to identify the optimal time for studying the responsiveness of RGCs to external electrical stimulation. The results show that the fluorescence-endoscopy fundus system is a powerful and widely accessible tool for evaluating in vivo fluorescence reporter expression.Virus-transduced calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. To track the level of in vivo expression of genetically encoded calcium indicators (GECIs) in rodent retina, we developed a noninvasive imaging approach based on a custom-modified low-cost and simple fundus system that enabled us to monitor and characterize in vivo bright-field and fluorescence retinal image. The system clearly resolves individual retinal ganglion cells (RGCs) and axons. RGC fluorescence intensity and number of observable fluorescent cells show a consistent rising trend from week 1 to week 3 after viral injection, indicating a uniform increase of GCaMP6f expression. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array (MEA) and used calcium imaging to identify the optimal time for studying the responsiveness of RGCs to external electrical stimulation. The results show that the fluorescence-endoscopy fundus system is a powerful and widely accessible tool for evaluating in vivo fluorescence reporter expression.

Method to remove photoreceptors from whole mount retina in vitro

Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct in a whole mount retina preparation because surrounding neurons block the path of the patch pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at the cost of severing the lateral connection between neurons. We have developed a technique to remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing access for patch clamp recording. Repeated application to and removal of filter paper from the photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells and, in degenerate retina, hypertrophied Müller cell end feet. Live-dead assays applied to neurons remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner retinal neurons in a whole mount retina preparation for investigation of signal processing. A disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The technique may be useful for investigations of extracellular electrical stimulation, photoreceptor DNA analysis, and nonpharmacological removal of light input.NEW & NOTEWORTHY This study reports a method for removing photoreceptors from rodent whole mount retina while preserving the architecture of the inner retina. The method enables easier access to the inner retina for studies of neural processing, such as by patch clamp recording.

Direct measurement of bipolar cell responses to electrical stimulation in wholemount mouse retina

OBJECTIVE This in vitro investigation examines the response of retinal bipolar cells to extracellular electrical stimulation. APPROACH In vitro investigations characterizing the response of retinal neurons to electrical stimulation have primarily focused on retinal ganglion cells because they are the output neurons of the retina and their superficial position in the retina makes them readily accessible to in vitro recording techniques. Thus, the majority of information regarding the response of inner retinal neurons has been inferred from ganglion cell activity. Here we use patch clamp electrophysiology to directly record electrically-evoked activity in bipolar cells within the inner retina of normal Tg(Gng13-EGFP)GI206Gsat and degenerate rd10 Tg(Gng13-EGFP)GI206Gsat mice using a wholemount preparation. MAIN RESULTS Bipolar cells respond to electrical stimulation with time-locked depolarizing voltage transients. The latency of the response declines with increases in stimulation amplitude. A desensitizing response is observed during repeated stimulation with 25 ms biphasic current pulses delivered at pulse rates greater than 6 pps. A burst of long-latency (200-1000 ms) inhibitory postsynaptic potentials are evoked by the stimulus and the burst exhibits evidence of a lower and upper stimulation threshold. SIGNIFICANCE These results provide insights into the various types of bipolar cell activity elicited by electrical stimulation and may be useful for future retinal prosthesis stimulation protocols. This investigation uses patch clamp electrophysiology to provide direct analysis of ON-type bipolar cell responses to electrical stimulation in a wholemount retina preparation. It explores the effects of variable stimulus amplitudes, pulse widths, and frequencies in both normal and degenerate retina. The analysis adds to a body of work largely based upon indirect measurements of bipolar cell activity, and the methodology demonstrates an alternative retina preparation technique in which to acquire single-cell activity.

Separation of photoreceptor cell compartments in mouse retina for protein analysis

BackgroundLight exposure triggers movement of certain signaling proteins within the cellular compartments of the highly polarized rod photoreceptor cell. This redistribution of proteins between the inner and outer segment compartments affects the performance and physiology of the rod cell. In addition, newly synthesized phototransduction proteins traverse from the site of their synthesis in the inner segment, through the thin connecting cilium, to reach their destination in the outer segment. Processes that impede normal trafficking of these abundant proteins lead to cell death. The study of movement and unique localization of biomolecules within the different compartments of the rod cell would be greatly facilitated by techniques that reliably separate these compartments. Ideally, these methods can be applied to the mouse retina due to the widespread usage of transgenic mouse models in the investigation of basic visual processes and disease mechanisms that affect vision. Although the retina is organized in distinct layers, the small and highly curved mouse retina makes physical separation of retinal layers a challenge. We introduce two peeling methods that efficiently and reliably isolate the rod outer segment and other cell compartments for Western blots to examine protein movement across these compartments.MethodsThe first separation method employs Whatman® filter paper to successively remove the rod outer segments from isolated, live mouse retinas. The second method utilizes ScotchTM tape to peel the rod outer segment layer and the rod inner segment layer from lyophilized mouse retinas. Both procedures can be completed within one hour.ResultsWe utilize these two protocols on dark-adapted and light-exposed retinas of C57BL/6 mice and subject the isolated tissue layers to Western blots to demonstrate their effectiveness in detecting light-induced translocation of transducin (GNAT1) and rod arrestin (ARR1). Furthermore, we provide evidence that RGS9 does not undergo light-induced translocation.ConclusionsThese results demonstrate the effectiveness of the two different peeling protocols for the separation of the layered compartments of the mouse retina and their utility for investigations of protein compositions within these compartments.

GCaMP expression in retinal ganglion cells characterized using a low-cost fundus imaging system

OBJECTIVE Virus-transduced, intracellular-calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. Due to the existence of an optimal time window for the expression of calcium indicators, a suitable tool for tracking GECI expression in vivo following transduction is highly desirable. APPROACH We developed a noninvasive imaging approach based on a custom-modified, low-cost fundus viewing system that allowed us to monitor and characterize in vivo bright-field and fluorescence images of the mouse retina. AAV2-CAG-GCaMP6f was injected into a mouse eye. The fundus imaging system was used to measure fluorescence at several time points post injection. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array and used calcium imaging to evaluate the responsiveness of retinal ganglion cells (RGCs) to external electrical stimulation. MAIN RESULTS The noninvasive fundus imaging system clearly resolves individual (RGCs and axons. RGC fluorescence intensity and the number of observable fluorescent cells show a similar rising trend from week 1 to week 3 after viral injection, indicating a consistent increase of GCaMP6f expression. Analysis of the in vivo fluorescence intensity trend and in vitro neurophysiological responsiveness shows that the slope of intensity versus days post injection can be used to estimate the optimal time for calcium imaging of RGCs in response to external electrical stimulation. SIGNIFICANCE The proposed fundus imaging system enables high-resolution digital fundus imaging in the mouse eye, based on off-the-shelf components. The long-term tracking experiment with in vitro calcium imaging validation demonstrates the system can serve as a powerful tool monitoring the level of genetically-encoded calcium indicator expression, further determining the optimal time window for following experiment.

In vivo investigation of the evolution of skin barrier repair after mechanical injury

The stratum corneum (SC) serves a primary function of skin barrier and its maintenance is vital for the existence of terrestrial life. Few studies have been performed for evaluation of human SC repair in vivo, non-invasively. In the present study tape stripping was performed on the arms and legs of seven volunteers until all the SC was removed. The injured site and a control adjacent site were measured over a period of 10 days after the injury to assess functionality and repair. Transepidermal water loss (TEWL), tryptophan fluorescence and reflectance confocal microscopy were used to determine permeability of the skin barrier, cell turnover and epidermis morphology, respectively. The results show an exponential rate of recovery for the skin permeability (TEWL) which contrasted with a linear increase in the thickness of the SC as determined by confocal microscopy. Cell turnover increased rapidly immediately after the injury to 2.5 times the levels of the control site, attaining a maximum of 3.5-4 times greater levels after three days and slowly returned to baseline levels after the ten days. Correlation of the cell turnover to the thickness of the viable epidermis was observed and further studies are under way to interpret these results.