Zapping the Retina - Understanding electrical responsiveness and electrical desensitization in mouse retinal ganglion cells

The field of science and technology has come a long way since the famous 70’s science fiction series “The Six Million Dollar Man,” where a disabled pilot was transformed into a bionic superhero after receiving artificial implants. What was indeed once a science fiction has now turned into a science fact with the development of various electronic devices interfacing the human neurons in the brain, retina, and limbs. One such advancement was the development of retinal implants. Over the past two decades, the field of retinal prosthetics has made significant advancement in restoring functional vision in patients blinded by diseases such as Retinitis pigmentosa (RP) and Age-related macular degeneration (AMD). RP and AMD are the two leading cause of degenerative blindness. While there is still no definitive cure for either of these diseases, various treatment strategies are currently being explored. Of the various options, the most successful one has been the retinal implants. Retinal implants are small microelectrode or photodiode arrays, which are implanted in the eye of a patient, to stimulate the degenerating retina electrically. They are broadly classified into three types depending on the placement ̶ epiretinal (close proximity to retinal ganglion cells, RGCs) , subretinal (close proximity to bipolar cells, BP) and suprachoroidal (close proximity to choroid). While the ongoing human trials have shown promising results, there remains a considerable variability among patients concerning the quality of visual percepts which limits the working potential of these implants. One such limitation often reported by the implanted patients is “ fading” of visual percepts. Fading refers to the limited ability to elicit temporally stable visual percepts. While, this is not a primary concern for epiretinal implants , it is often observed in subretinal and suprachoroidal implants which use the remaining retinal network to control the temporal spiking pattern of the ganglion cells. The neural correlate of fading is often referred to as “electrical desensitization”, which is the reduction of ganglion cell responses to repetitive electrical stimulation . While much is known about the temporal component of desensitization ( time constant, τ), the spatial aspects (space constant, λ) has not been well characterized. Further, how both these aspects interact to generate spiking responses, remains poorly understood. These crucial questions formed the critical components of my thesis. To address these questions, we stimulated the retinal network electrically, with voltage and current pulses and recorded the corresponding spiking activity using the microelectrode arrays (MEAs). While addressing the primary question of my thesis, we were able to address few idiosyncrasies which has currently stymied the field of retinal prosthetics. At a conceptual level, we have developed an experimental and analysis framework by which one can identify the single stimulus that will activate the most ganglion cells (Chapter 2, Part 1). This stimulus is optimal for ‘blind’ experiments where the specific response properties of each cell are unknown. Furthermore, we attempted to understand the correspondence between the electrical response patterns and visual response types (Chapter 2, Part2). In Chapter 3, we sought to understand better how the visual responses parameters change during ongoing electrical stimulation. We demonstrated that apart from the adaptation occurring due to visual stimulation and invitro experimental conditions, the electrical stimulation alters the RGC visual responses, suggesting the requirement for stimulation-induced changes to be incorporated in the designing of stimulation paradigms for the implant. Finally addressing the primary question (Chapter 4) of my thesis with which we started, we were able to demonstrate, that the electrical desensitization requires the interaction of both time and distance and is not a global phenomenon of the retina. In the final chapter (Chapter 5) we summarize the results of the thesis, discuss the key outcomes and its relevance to the prosthetic field and other vision restoration strategies and the potential future directions of this research. Therefore, in future, to improve the efficacy of retinal prostheses and patient outcomes, it is crucial to have an in-depth understanding of the responsiveness of the retinal circuitry to electrical stimulation

Photovoltaic restoration of sight with high visual acuity

Patients with retinal degeneration lose sight due to the gradual demise of photoreceptors. Electrical stimulation of surviving retinal neurons provides an alternative route for the delivery of visual information. We demonstrate that subretinal implants with 70-μm-wide photovoltaic pixels provide highly localized stimulation of retinal neurons in rats. The electrical receptive fields recorded in retinal ganglion cells were similar in size to the natural visual receptive fields. Similarly to normal vision, the retinal response to prosthetic stimulation exhibited flicker fusion at high frequencies, adaptation to static images and nonlinear spatial summation. In rats with retinal degeneration, these photovoltaic arrays elicited retinal responses with a spatial resolution of 64 ± 11 μm, corresponding to half of the normal visual acuity in healthy rats. The ease of implantation of these wireless and modular arrays, combined with their high resolution, opens the door to the functional restoration of sight in patients blinded by retinal degeneration

Characterization of Retinal Ganglion Cell Responses to Electrical Stimulation Using White Noise

Retinitis pigmentosa and age-related macular degeneration are two leading causes of degenerative blindness. While there is still not a definitive course of treatment for either of these diseases, there is currently the world over, many different treatment strategies being explored. Of these various strategies, one of the most successful has been retinal implants. Retinal implants are microelectrode or photodiode arrays, that are implanted in the eye of a patient, to electrically stimulate the degenerating retina. Clinical trials have shown that many patients implanted with such a device, are able to regain a certain degree of functional vision. However, while the results of these ongoing clinical trials have been promising, there are still many technical challenges that need to be overcome. One of the biggest challenges facing present implants is the inability to preferentially stimulate different retinal pathways. This is because retinal implants use large-amplitude current or voltage pulses. This in turn leads to the indiscriminate activation of multiple classes of retinal ganglion cells (RGCs), and therefore, an overall reduction in the restored visual acuity. To tackle this issue, we decided to explore a novel stimulus paradigm, in which we present to the retina, a stream of smaller-amplitude subthreshold voltage pulses. By then correlating the retinal spikes to the stimuli preceding them, we calculate temporal input filters for various classes of RGCs, using a technique called spike-triggered averaging (STA). In doing this, we found that ON and OFF RGCs have electrical filters, which are very distinct from each other. This finding creates the possibility for the selective activation of the retina through the use of STA-based waveforms. Finally, using statistical models, we verify how well these temporal filters can predict RGC responses to novel electrical stimuli. In a broad sense, our work represents the successful application of systems engineering tools to retinal prosthetics, in an attempt to answer one of the field’s most difficult questions, namely selective stimulation of the retina

Classifying Retinal Ganglion Cells for Bionic Vision

Current retinal implants implement pulsate stimuli to activate the neural circuits of the retina. This type of stimulation can activate antagonist retinal pathways which lead to the improper perception of the visual scene. Developing a precise stimulation strategy with the ability to preferentially target retinal neural circuits is one of the alternative methods to improve the accuracy of restored vision. Previous studies tried to decipher the electrical properties of different retina ganglion cell (RGC) types by applying electrical Gaussian noise and estimating the electrical input filter of the cells. Sekhar et al reported that ON and OFF cells have different electrical input filters. In this study, we aimed to pursue the same goal by using a similar approach to assess the electrical profiles for a wider range of ganglion cell types. We implemented an array of visual stimuli along with an electrical noise stimulus to fully characterize the light and electrical response properties of both healthy and degenerated retina ganglion cells

Glia Excitation in the CNS Modulates Intact Behaviors and Sensory-CNS-Motor Circuitry

Glial cells play a role in many important processes, though the mechanisms through which they affect neighboring cells are not fully known. Insights may be gained by selectively activating glial cell populations in intact organisms utilizing the activatable channel proteins channel rhodopsin (ChR2XXL) and TRPA1. Here, the impacts of the glial-specific expression of these channels were examined in both larval and adult Drosophila. The Glia \u3e ChR2XXL adults and larvae became immobile when exposed to blue light and TRPA1-expressed Drosophila upon heat exposure. The chloride pump expression in glia \u3e eNpHR animals showed no observable differences in adults or larvae. In the in situ neural circuit activity of larvae in the Glia \u3e ChR2XXL, the evoked activity first became more intense with concurrent light exposure, and then the activity was silenced and slowly picked back up after light was turned off. This decrease in motor nerve activity was also noted in the intact behaviors for Glia \u3e ChR2XXL and Glia \u3e TRPA1 larvae. As a proof of concept, this study demonstrated that activation of the glia can produce excessive neural activity and it appears with increased excitation of the glia and depressed motor neuron activity

Establishing optogenetic tools in the auditory system of the Mongolian Gerbil

The Mongolian Gerbil (Meriones unguiculatus) serves as a popular and widely used model organism for the human auditory system. Its hearing range largely overlaps with that of human’s and even extends below 1 kHz, frequencies very important for human hearing. Like humans, gerbils can localize sounds based on their interaural time difference (ITD) or interaural level difference (ILD) and also show perceptual suppression of the spatial source of reverberations (precedence effect). The auditory circuitries underlying the computation of ITDs and ILDs are very well described in the gerbil, although the exact mechanisms for the extraction of ITDs are still under debate. The contribution of the medial nucleus of the trapezoid body (MNTB) in tuning neurons sensitive to ITDs is still unclear. Similarly, the precedence effect is well known and thought to greatly facilitate listening in reverberant environments, yet the neural substrate of the precedence effect is still elusive. A circuitry that might subserve the precedence effect is hypothesized to be formed by the dorsal nucleus of the lateral lemniscus (DNLL) and the inferior colliculus (IC). However, a precise and reversible manipulation of the DNLL-IC circuitry or the ITD circuitry has not been possible due to the lack of technical means. With the advent of optogenetics, tools are becoming available that would allow to specifically activate and silence nuclei within both circuitries. Yet, transgenic lines or genetic tools are neither disposable nor established for the Mongolian Gerbil. Hence, in order to express optogenetic tools in the gerbil auditory brainstem and midbrain, a reliable and neuron specific gene delivery system needs to be established as a major prerequisite. Only when this important first step is taken, the actual optogenetical tools can be applied and tested. In this study, the first hurdle of gene delivery into the Mongolian Gerbil was successfully cleared by using recombinant adeno-associated viruses (rAAV) as vectors. Via the stereotactic injection of rAAVs into the DNLL, IC and MNTB, not only reliable and efficient transduction of neurons was achieved but also neuronal specific expression of transgenes was attained. As a second accomplishment, the channelrhodopsin mutant CatCH as well as the halorhodopsin NpHR3.0 were characterized in acute brain slices by performing whole cell patch-clamp recordings of transduced neurons. As a final step and proof of principle experiment, sound evoked neural responses in the DNLL and IC were successfully manipulated with light in vivo, as could be demonstrated by single cell extracellular recordings from anaesthetized animals. In sum, this study successfully adapted and established gene delivery and optogenetic tools in the auditory system of the Mongolian Gerbil. This represents a fully functional and highly versatile toolbox that not only paves the way to further elucidate the ITD as well as the DNLL-IC circuitry but is also applicable to other questions

PROFILING THE ACTION OF ACETYLCHOLINE IN THE DROSOPHILA MELANOGASTER LARVAL MODEL: HEART, BEHAVIOR, AND THE DEVELOPMENT AND MAINTENANCE OF SENSORIMOTOR CIRCUITS

Understanding the role of various chemical messengers in altering behaviors and physiological processes is a common goal for scientists across multiple disciplines. The main focus of this dissertation is on characterizing the action of an important neurotransmitter, acetylcholine (ACh), modulating larval Drosophila melanogaster neural circuits and heart. In this dissertation, I provide important insights into the mechanisms by which ACh influences the formation and performance of select neural circuits, while also revealing significant details regarding its role in additional physiological functions, including cardiac pace making. In Chapter 1, I provide a general overview of ACh action in mammals and flies with a particular focus on the physiological and behavioral effects of cholinergic signaling in the context of modulation of neural circuits and developmental impacts. Chapters 2 and 3 are dedicated to the role of ACh in modulating larval Drosophila heart rate (HR). Previous analysis has been performed identifying neuromodulator influence on larval heart rate, and I add to the current understanding of chemical modulation of cardiac function utilizing a pharmacological approach to assess ACh regulation of HR. I provide evidence that ACh modulates larval HR primarily through muscarinic receptors. I follow this by employing an optogenetic approach to assess ACh and additional neuroendocrine modulation of HR in an intact system in Chapter 3, further illuminating ACh regulation of larval HR. Chapter 4 is dedicated to describing the role of ACh in modulation of neural circuits underlying larval locomotion, feeding behavior, and sensorimotor circuit activity. I discuss the pharmacological approach taken to address this topic. Here, behavioral as well as electrophysiological approaches reveal a contribution from both ACh receptor subtypes in regulation of these behaviors. I leverage this information and describe the influence of a specific receptor subtype, the muscarinic acetylcholine receptor (mAChR) on the function of these circuits by using combined pharmacological and genetic approaches to strengthen the pharmacological assessment, discussed in Chapter 8. An additional goal of this work is to refine the optogenetic technique in the larval Drosophila model. Chapter 5 discusses useful experimental paradigms that allow for investigation of repetitively activating light-sensitive opsins on neuronal physiology in the larval model. Chapter 6 discusses an intriguing, previously undefined identification of Glutamic acid decarboxylase1 expression in larval body wall muscle, which was identified using optogenetic approaches in concert with electrophysiology. Furthermore, I combine these approaches to discuss the development of an experimental paradigm to address the developmental impacts of altering sensory (cholinergic) input on the formation and maintenance of a specific mechanosensory circuit (Chapter 8). Chapter 7 discusses the implication of deep tissue injury on proprioceptive sensory function in two model proprioceptive organs in crab and crayfish

Excitation and Excitability of Unipolar Brush Cells

__Abstract__ The cerebellum is a distinct brain structure that ensures the spatial accuracy and temporal coordination of movements. It is located superimposed on the brainstem and has an appearance and organization unlike that of the cerebral cortex: its surface has a highly regular foliation pattern, and its neural circuitry is organized in repeated structured modules. Neural activity enters the cerebellum via two excitatory pathways, the mossy ber system and the climbing ber system. Climbing bers originate from the inferior olivary nucleus in the brainstem, and assert a powerful in uence on cerebellar output and long-term adaptation processes. Mossy bers originate from a large number of sources, and carry contextual information on sensory inputs, aspects of motor planning and commands, and proprioceptive feedback. In the cerebellum this information is evaluated and integrated, to produce neural output that in uences ongoing movement directly. Mossy ber signals are processed in the cerebellum in three stages. In the granular layer, the input stage of the cerebellum, mossy ber signals undergo a recoding step where they are combined and expanded by granule cells. Next, in the molecular layer, granule cell signals are integrated with climbing ber signals in Purkinje cells. Together, the granular la

Optimal electrical activation of retinal ganglion cells

Retinal prostheses are emerging as a viable therapy option for those blinded by degenerative eye conditions that destroy the photoreceptors of the retina but spare the retinal ganglion cells (RGCs). My research sought to address the issue of how a retinal prosthesis might best activate these cells by way of electrical stimulation. Whole-cell patch clamp recordings were made in explanted retinal wholemount preparations from normally-sighted rats. Stimulating electrodes were fabricated from nitrogen-doped ultra-nanocrystalline diamond (N-UNCD) and placed on the epiretinal surface, adjacent to the cell soma. Electrical stimuli were delivered against a distant monopolar return electrode. Using rectangular, biphasic constant current waveforms as employed by modern retinal prostheses, I examined which waveform parameters had the greatest effect on RGC activation thresholds. In a second set of experiments intracellular current injection was employed to assess the effectiveness of sinusoidal current waveforms in selectively activating different RGC subsets. These recordings were also used to validate a biophysical model of RGC activation. Where possible, recorded cells were identified and classified based on 3D confocal reconstruction of their morphology. Electrodes fabricated from N-UNCD were able to electrically activate RGCs while remaining well within the electrochemical limits of the material. They were found to exhibit high electrochemical stability and were resistant to morphological and electrochemical changes over one week of continuous pulsing at charge injection limits. Retinal ganglion cells invariably favoured cathodic-first biphasic current pulses of short first-phase duration, with a small interphase interval. The majority of cells (63\%) were most sensitive to a highly asymmetric waveform: a short-cathodic phase followed by a longer duration, lower amplitude anodic phase. Using the optimal interphase interval led to median charge savings of 14\% compared to the charge required in the absence of any inter-phase interval. Optimising phase duration resulted in median charge savings of 22\%. All RGCs became desensitised to repetitive electrical stimulation. The efficacy of a given stimulus delivered repeatedly decreased after the first stimulus, stabilising at a lower efficacy by the thirtieth pulse. This asymptotic efficacy decreased with increasing stimulus frequency. Cells with smaller somas and dendritic fields were better able to sustain repetitive activation at high frequency. Intracellular sinusoidal stimulation was used to demonstrate that certain RGC subsets, defined on the basis of morphological type, stratification, and size, were more responsive to high frequency stimulation. Simulated RGC responses were validated by experimental data, which confirmed that ON cell responses were heavily suppressed by stimulus frequencies of 20 Hz and higher. OFF cells, on the other hand, were able to sustain repetitive activation over all tested frequencies. Additional simulations suggest this difference may be plausibly attributed to the presence of low-voltage-activated calcium channels in OFF but not ON RGCs. The results of my work demonstrate that (a) N-UNCD is a suitable material for retinal prosthesis applications; (b) a careful choice of electrical waveform parameters can significantly improve prosthesis efficacy; and (c) it may be possible to bias neural activation for certain RGC populations by varying the frequency of stimulation

Characterising Functionally-Distinct Retinal Ganglion Cell Responses to High Frequency Electrical Stimulation

Retinal implants aim to provide artificial vision to those profoundly blind by stimulating the residual network to elicit visual percepts. While human clinical trials have demonstrated encouraging results including the presence of visual percepts as well as partial visual restoration, the vision quality provided remains limited. One potential cause of this poor performance has been attributed to the indiscriminate activation of functionally-different retinal ganglion cell (RGC) types. To combat this problem, a promising strategy has been to design stimulation strategies that are capable of selectively, or preferentially, activating different cell types. One such approach to realise this goal has been through the use of high frequency stimulation (HFS) which was shown to be effective in preferentially activating two major retinal ganglion cell types– ON and OFF. While encouraging, the utility of the technique to target a broader range of cell types, and under different stimulation conditions and environments was still unclear. The studies presented in this thesis were designed to improve the understanding of HFS based preferential activation. Using in vitro whole-cell patch clamp of RGCs in mice (C57BL/6J and rd1), an investigation into whether HFS could be used to preferentially activate four major RGC types namely, ON-sustained (ONS), ON-transient (ONT), OFF-sustained (OFFS), and OFF-transient (OFFT), was undertaken. Results suggested that three of the four targeted cell types could be preferentially activated against the remaining population. A subsequent study documented the responses and the preferential activation capabilities of the aforementioned cell types when the high frequencies were modulated with short stimulation bursts, varying sequence orders and in a continuous waveform. It was shown that the ON (sustained and transient) RGCs typically exhibited more consistent responses and preferential activation regions irrespective of the frequency order, or when presented as a continuous waveform. A final study examined the responses of rd1 ON and OFF RGCs to HFS both with and without the presynaptic degenerate network. The network did appear to have an effect on the HFS evoked responses, and particularly increased the variability of the responses which in turn affected the preferential activation of the cell types. Additionally, a comparison into the specific intrinsic properties between the rd1 and healthy RGCs found that these properties may differ between the cell groups. Overall, this thesis investigated the usefulness of HFS to preferentially activate different cell types and across various stimulation conditions and environments and found that HFS remains a viable stimulation technique to reduce indiscriminate activation of functionally-distinct cell types