Membrane depolarization mediates both the inhibition of neural activity and cell-type-differences in response to high-frequency stimulation.

Neuromodulation using high frequency (>1 kHz) electric stimulation (HFS) enables preferential activation or inhibition of individual neural types, offering the possibility of more effective treatments across a broad spectrum of neurological diseases. To improve effectiveness, it is important to better understand the mechanisms governing activation and inhibition with HFS so that selectivity can be optimized. In this study, we measure the membrane potential (Vm) and spiking responses of ON and OFF α-sustained retinal ganglion cells (RGCs) to a wide range of stimulus frequencies (100-2500 Hz) and amplitudes (10-100 µA). Our findings indicate that HFS induces shifts in Vm, with both the strength and polarity of the shifts dependent on the stimulus conditions. Spiking responses in each cell directly correlate with the shifts in Vm, where strong depolarization leads to spiking suppression. Comparisons between the two cell types reveal that ON cells are more depolarized by a given amplitude of HFS than OFF cells-this sensitivity difference enables the selective targeting. Computational modeling indicates that ion-channel dynamics largely account for the shifts in Vm, suggesting that a better understanding of the differences in ion-channel properties across cell types may improve the selectivity and ultimately, enhance HFS-based neurostimulation strategies.

Avoidance of axonal stimulation with sinusoidal epiretinal stimulation.

Objective.Neuromodulation, particularly electrical stimulation, necessitates high spatial resolution to achieve artificial vision with high acuity. In epiretinal implants, this is hindered by the undesired activation of distal axons. Here, we investigate focal and axonal activation of retinal ganglion cells (RGCs) in epiretinal configuration for different sinusoidal stimulation frequencies.Approach.RGC responses to epiretinal sinusoidal stimulation at frequencies between 40 and 100 Hz were tested inex-vivophotoreceptor degenerated (rd10) isolated retinae. Experiments were conducted using a high-density CMOS-based microelectrode array, which allows to localize RGC cell bodies and axons at high spatial resolution.Main results.We report current and charge density thresholds for focal and distal axon activation at stimulation frequencies of 40, 60, 80, and 100 Hz for an electrode size with an effective area of 0.01 mm2. Activation of distal axons is avoided up to a stimulation amplitude of 0.23µA (corresponding to 17.3µC cm-2) at 40 Hz and up to a stimulation amplitude of 0.28µA (14.8µC cm-2) at 60 Hz. The threshold ratio between focal and axonal activation increases from 1.1 for 100 Hz up to 1.6 for 60 Hz, while at 40 Hz stimulation frequency, almost no axonal responses were detected in the tested intensity range. With the use of synaptic blockers, we demonstrate the underlying direct activation mechanism of the ganglion cells. Finally, using high-resolution electrical imaging and label-free electrophysiological axon tracking, we demonstrate the extent of activation in axon bundles.Significance.Our results can be exploited to define a spatially selective stimulation strategy avoiding axonal activation in future retinal implants, thereby solving one of the major limitations of artificial vision. The results may be extended to other fields of neuroprosthetics to achieve selective focal electrical stimulation.

A finite element method framework to model extracellular neural stimulation.

Objective.Increasing complexity in extracellular stimulation experiments and neural implant design also requires realistic computer simulations capable of modeling the neural activity of nerve cells under the influence of an electrical stimulus. Classical model approaches are often based on simplifications, are not able to correctly calculate the electric field generated by complex electrode designs, and do not consider electrical effects of the cell on its surrounding. A more accurate approach is the finite element method (FEM), which provides necessary techniques to solve the Poisson equation for complex geometries under consideration of electrical tissue properties. Especially in situations where neurons experience large and non-symmetric extracellular potential gradients, a FEM solution that implements the cell membrane model can improve the computer simulation results. To investigate the response of neurons in an electric field generated by complex electrode designs, a FEM framework for extracellular stimulation was developed in COMSOL.Approach.Methods to implement morphologically- and biophysically-detailed neurons including active Hodgkin-Huxley (HH) cell membrane dynamics as well as the stimulation setup are described in detail. Covered methods are (a) development of cell and electrode geometries including meshing strategies, (b) assignment of physics for the conducting spaces and the realization of active electrodes, (c) implementation of the HH model, and (d) coupling of the physics to get a fully described model.Main results.Several implementation examples are briefly presented: (a) a full FEM implementation of a HH model cell stimulated with a honeycomb electrode, (b) the electric field of a cochlear electrode placed inside the cochlea, and (c) a proof of concept implementation of a detailed double-cable cell membrane model for myelinated nerve fibers.Significance.The presented concepts and methods provide basic and advanced techniques to realize a full FEM framework for innovative studies of neural excitation in response to extracellular stimulation.

Block Phenomena During Electric Micro-Stimulation of Pyramidal Cells and Retinal Ganglion Cells.

Electric micro-stimulation of the nervous system is a means to restore various body functions. The stimulus amplitude necessary to generate action potentials, the lower threshold (LT), is well characterized for many neuronal populations. However, electric overstimulation above an upper threshold (UT) prevents action potential generation and therefore hinders optimal neuro-rehabilitation. Previous studies demonstrated the impact of the UT in micro-stimulation of retinal ganglion cells (RGCs). The observed phenomenon is mostly explained by (i) reversed sodium ion flow in the soma membrane, and (ii) anodal surround block that hinders spike conduction in strongly hyperpolarized regions of the axon at high stimulus intensities. However, up to now, no detailed study of the nature of these phenomena has been presented, particularly for different cell types. Here, we present computational analyses of LT and UT for layer 5 pyramidal cells (PCs) as well as alpha RGCs. Model neurons were stimulated in close vicinity to the cell body and LTs and UTs as well as the ratio UT/LT were compared. Aside from a simple point source electrode and monophasic stimuli also realistic electrode and pulse configurations were examined. The analysis showed: (i) in RGCs, the soma contributed to action potential initiation and block for small electrode distances, whereas in PCs the soma played no role in LTs or UTs. (ii) In both cell types, action potential always initiated within the axon initial segment at LT. (iii) In contrast to a complete block of spike conductance at UT that occurred in RGCs, an incomplete block of spiking appeared in PC axon collaterals. (iv) PC axon collateral arrangement influenced UTs but had small impact on LTs. (v) Population responses of RGCs change from circular regions of activation to ring-shaped patterns for increasing stimulus amplitude. A better understanding of the stimulation window that can reliably activate target neurons will benefit the future development of neuroprostheses.

Morphological Factors that Underlie Neural Sensitivity to Stimulation in the Retina.

Retinal prostheses are a promising therapeutic intervention for patients afflicted by outer retinal degenerative diseases like retinitis pigmentosa and age-related macular degeneration. While significant advances in the development of retinal implants have been made, the quality of vision elicited by these devices remains largely sub-optimal. The variability in the responses produced by retinal devices is most likely due to the differences between the natural cell type-specific signaling that occur in the healthy retina vs. the non-specific activation of multiple cell types arising from artificial stimulation. In order to replicate these natural signaling patterns, stimulation strategies must be capable of preferentially activating specific RGC types. To design more selective stimulation strategies, a better understanding of the morphological factors that underlie the sensitivity to prosthetic stimulation must be developed. This review will focus on the role that different anatomical components play in driving the direct activation of RGCs by extracellular stimulation. Briefly, it will (1) characterize the variability in morphological properties of α-RGCs, (2) detail the influence of morphology on the direct activation of RGCs by electric stimulation, and (3) describe some of the potential biophysical mechanisms that could explain differences in activation thresholds and electrically evoked responses between RGC types.

Differential Responses to High-Frequency Electrical Stimulation in Brisk-Transient and Delta Retinal Ganglion Cells.

Retinal microprostheses strive to evoke a sense of vision in individuals blinded by outer retinal degenerative diseases, by electrically stimulating the surviving retina. It is widely suspected that a stimulation strategy that can selectively activate different retinal ganglion cell types will improve the quality of evoked phosphenes. Previous efforts towards this goal demonstrated the potential for selective ON and OFF brisk-transient cell activation using high-rate (2000 pulses per second, PPS) stimulation. Here, we build upon this earlier work by testing an additional rate of stimulation and additional cell populations. We find considerable variability in responses both within and across individual cell types, but show that the sensitivity of a ganglion cell to repetitive stimulation is highly correlated to its single-pulse threshold. Consistent with this, we found thresholds for both stimuli to be correlated to soma size, and thus likely mediated by the properties of the axon initial segment. The ultimate efficacy of high-rate stimulation will likely depend on several factors, chief among which are (a) the residual ganglion types, and (b) the stimulation frequency.

Tailoring of the axon initial segment shapes the conversion of synaptic inputs into spiking output in OFF-α T retinal ganglion cells.

Recently, mouse OFF-α transient (OFF-α T) retinal ganglion cells (RGCs) were shown to display a gradient of light responses as a function of position along the dorsal-ventral axis; response differences were correlated to differences in the level of excitatory presynaptic input. Here, we show that postsynaptic differences between cells also make a strong contribution to response differences. Cells in the dorsal retina had longer axon initial segments (AISs)-the greater number of Nav1.6 channels in longer AISs directly mediates higher rates of spiking and helps avoid depolarization block that terminates spiking in ventral cells with shorter AISs. The pre- and postsynaptic specializations that shape the output of OFF-α T RGCs interact in different ways: In dorsal cells, strong inputs and the long AISs are both necessary to generate their strong, sustained spiking outputs, while in ventral cells, weak inputs or the short AISs are both sufficient to limit the spiking signal.

The relationship between morphological properties and thresholds to extracellular electric stimulation in α RGCs.

OBJECTIVE: Retinal prostheses strive to restore vison to patients that are blind from retinal degeneration by electrically stimulating surviving retinal ganglion cells (RGCs). The quality of elicited percepts remains limited however and it is desirable to develop improved stimulation strategies. Here, we examine how the anatomical and biophysical properties of RGCs influence activation thresholds, including the effects of variations found naturally. APPROACH: Detailed reconstructions were made of a large number of mouse α RGCs and were used to create an array of model cells; the models were used to study the effects of individual anatomical features on activation threshold to electric stimulation. Stimulation was delivered epiretinally from a point-source or disk electrode and consisted of monophasic or biphasic rectangular pulses. MAIN RESULTS: Modeling results show that the region of minimum threshold always is within the axon initial segment (AIS). The properties of this region as well as the absolute value of the minimum threshold are dependent on the length of the AIS as well as on the relative composition of sodium channels within the AIS. Other morphological features, including cell size, dendritic field size and the distance between the AIS and the soma had only a minimal influence on thresholds. Introducing even a small number of low-threshold Nav1.6 channels into the AIS was sufficient to lower minimum thresholds substantially although further increases in Nav1.6 had diminishing effects. The distance between the AIS and the electrode affects threshold levels while alignment of the electrode with the axon or dendritic parts of the RGC can result in lower thresholds, even if the distance to the cell remains the same. SIGNIFICANCE: Intrinsic morphological features can influence activation thresholds with the AIS having the strongest influence. However, the combined influence remains limited and may not be large enough to allow for selective activation between different RGC types.

On optimal coupling of the 'electronic photoreceptors' into the degenerate retina.

Objective: To restore sight in atrophic age-related macular degeneration, the lost photoreceptors can be replaced with electronic implants, which replicate their two major functions: (1) converting light into an electric signal, and (2) transferring visual information to the secondary neurons in the retinal neural network­the bipolar cells (BC). We study the selectivity of BC activation by subretinal implants and dynamics of their response to pulsatile waveforms in order to optimize the electrical stimulation scheme such that retinal signal processing with 'electronic photoreceptors' remains as close to natural as possible. Approach: A multicompartmental model of a BC was implemented to simulate responses of the voltage-gated calcium channels and subsequent synaptic vesicle release under continuous and pulsatile stimuli. We compared the predicted response under various frequencies, pulse durations, and alternating gratings to the corresponding experimental measurements. In addition, electric field was computed for various electrode configurations in a 3-d finite element model to assess the stimulation selectivity via spatial confinement of the field. Main results: The modeled BC-mediated retinal responses were, in general, in good agreement with previously published experimental results. Kinetics of the calcium pumps and of the neurotransmitter release in ribbon synapses, which underpin the BC's temporal filtering and rectifying functions, allow mimicking the natural BC response with high frequency pulsatile stimulation, thereby preserving features of the retinal signal processing, such as flicker fusion, adaptation to static stimuli and non-linear summation of subunits in receptive field. Selectivity of the BC stimulation while avoiding direct activation of the downstream neurons (amacrine and ganglion cells­RGCs) is improved with local return electrodes. Significance: If the retinal neural network is preserved to a large extent in age-related macular degeneration, selective stimulation of BCs with proper spatial and temporal modulation of the extracellular electric field may retain many features of the natural retinal signal processing and hence allow highly functional restoration of sight.

Scaling of the AIS and Somatodendritic Compartments in α S RGCs.

The anatomical properties of the axon initial segment (AIS) are tailored in certain types of CNS neurons to help optimize different aspects of neuronal function. Here, we questioned whether the AISs of retinal ganglion cells (RGC) were similarly customized, and if so, whether they supported specific RGC functions. To explore this, we measured the AIS properties in alpha sustained RGCs (α S RGCs) of mouse; α S RGCs sizes vary systematically along the nasal temporal axis of the retina, making these cells an attractive population with which to study potential correlations between AIS properties and cell size. Measurements of AIS length as well as distance from the soma revealed that both were scaled to cell size, i.e., cells with large dendritic fields had long AISs that were relatively far from the soma. Within the AIS, the percentage of Na v 1.6 voltage-gated sodium channels remained highly consistent, regardless of cell size or other AIS properties. Although ON RGCs were slightly larger than OFF cells at any given location of the retina, the level of scaling and relative distribution of voltage-gated sodium channels were highly similar. Computational modeling revealed that AIS scaling influenced spiking thresholds, spike rate as well as the kinetics of individual action potentials, Interestingly, the effect of individual features of the AIS varied for different neuronal functions, e.g., AIS length had a larger effect on the efficacy by which the AIS initiated spike triggered the somatic spike than it did on repetitive spiking. The polarity of the effect varied for different properties, i.e., increases to soma size increased spike threshold while increases to AIS length decreased threshold. Thus, variations in the relative level of scaling for individual components could fine tune threshold or other neuronal functions. Light responses were highly consistent across the full range of cell sizes suggesting that scaling may post-synaptically shape response stability, e.g., in addition to several well-known pre-synaptic contributors.

Response of Mouse Visual Cortical Neurons to Electric Stimulation of the Retina.

Retinal prostheses strive to restore vision to the blind by electrically stimulating the neurons that survive the disease process. Clinical effectiveness has been limited however, and much ongoing effort is devoted toward the development of improved stimulation strategies, especially ones that better replicate physiological patterns of neural signaling. Here, to better understand the potential effectiveness of different stimulation strategies, we explore the responses of neurons in the primary visual cortex to electric stimulation of the retina. A 16-channel implantable microprobe was used to record single unit activities in vivo from each layer of the mouse visual cortex. Layers were identified by electrode depth as well as spontaneous rate. Cell types were classified as excitatory or inhibitory based on their spike waveform and as ON, OFF, or ON-OFF based on the polarity of their light response. After classification, electric stimulation was delivered via a wire electrode placed on the surface of cornea (extraocularly) and responses were recorded from the cortex contralateral to the stimulated eye. Responses to electric stimulation were highly similar across cell types and layers. Responses (spike counts) increased as a function of the amplitude of stimulation, and although there was some variance across cells, the sensitivity to amplitude was largely similar across all cell types. Suppression of responses was observed for pulse rates ≥3 pulses per second (PPS) but did not originate in the retina as RGC responses remained stable to rates up to 5 PPS. Low-frequency sinusoids delivered to the retina replicated the out-of-phase responses that occur naturally in ON vs. OFF RGCs. Intriguingly, out-of-phase signaling persisted in V1 neurons, suggesting key aspects of neural signaling are preserved during transmission along visual pathways. Our results describe an approach to evaluate responses of cortical neurons to electric stimulation of the retina. By examining the responses of single cells, we were able to show that some retinal stimulation strategies can indeed better match the neural signaling patterns used by the healthy visual system. Because cortical signaling is better correlated to psychophysical percepts, the ability to evaluate which strategies produce physiological-like cortical responses may help to facilitate better clinical outcomes.

Comparison of electrically elicited responses in rabbit and mouse retinal ganglion cells.

Retinal implants are currently the only commercially available devices that can restore vision in patients suffering from a wide range of outer retinal degenerations. In order to improve the clinical outcome, i.e. the quality of elicited vision, a large number of in-vitro experiments probing the impact of electric stimulation on activation of the retina have been conducted. In these studies, however, retinas from many different species have been used which impedes comparisons between studies. Therefore, we measured the responses from four major ganglion cell types to light and electric stimulation in rabbit and mouse retina and compared their responses. We found strong similarities between the two species in transient cells whereas responses in sustained cell types typically did not match as well.

Visual and electric spiking responses of seven types of rabbit retinal ganglion cells.

Electric stimulation of the retina via retinal implants is currently the only commercially available method to restore vision in patients suffering from a wide range of outer retinal degenerations. To improve the quality of retinal implants, it is desirable to better understand how different retinal cell classes and types respond to electric stimuli so that more effective stimulation strategies can be developed. Here, we measured the response of seven major types of retinal ganglion cells to electric stimulation. A simple series of light stimuli were used to classify cells into known types. Electric stimulation produced unique responses in almost all ganglion cell types and the electric responses typically matched elements of the corresponding light responses.

Electric stimulus duration alters network-mediated responses depending on retinal ganglion cell type.

OBJECTIVE: To improve the quality of artificial vision that arises from retinal prostheses, it is important to bring electrically-elicited neural activity more in line with the physiological signaling patterns that arise normally in the healthy retina. Our previous study reported that indirect activation produces a closer match to physiological responses in ON retinal ganglion cells (RGCs) than in OFF cells (Im and Fried 2015 J. Physiol. 593 3677-96). This suggests that a preferential activation of ON RGCs would shape the overall retinal response closer to natural signaling. Recently, we found that changes to the rate at which stimulation was delivered could bias responses towards a stronger ON component (Im and Fried 2016a J. Neural Eng. 13 025002), raising the possibility that changes to other stimulus parameters can similarly bias towards stronger ON responses. Here, we explore the effects of changing stimulus duration on the responses in ON and OFF types of brisk transient (BT) and brisk sustained (BS) RGCs. APPROACH: We used cell-attached patch clamp to record RGC spiking in the isolated rabbit retina. Targeted RGCs were first classified as ON or OFF type by their light responses, and further sub-classified as BT or BS types by their responses to both light and electric stimuli. Spiking in targeted RGCs was recorded in response to electric pulses with durations varying from 5 to100 ms. Stimulus amplitude was adjusted at each duration to hold total charge constant for all experiments. MAIN RESULTS: We found that varying stimulus durations modulated responses differentially for ON versus OFF cells: in ON cells, spike counts decreased significantly with increasing stimulus duration while in OFF cells the changes were more modest. The maximum ratio of ON versus OFF responses occurred at a duration of ~10 ms. The difference in response strength for BT versus BS cells was much larger in ON cells than in OFF cells. SIGNIFICANCE: The stimulation rates preferred by subjects during clinical trials are similar to the rates that maximize the ON/OFF response ratio in in vitro testing (Im and Fried 2016a J. Neural Eng. 13 025002). Here, we determine the stimulus duration that produces the strongest bias towards ON responses and speculate that it will further enhance clinical effectiveness.

The impact of calcium current reversal on neurotransmitter release in the electrically stimulated retina.

OBJECTIVE: In spite of intense theoretical and experimental investigations on electrical nerve stimulation, the influence of reversed ion currents on network activity during extracellular stimulation has not been investigated so far. APPROACH: Here, the impact of calcium current reversal on neurotransmitter release during subretinal stimulation was analyzed with a computational multi-compartment model of a retinal bipolar cell (BC) that was coupled with a four-pool model for the exocytosis from its ribbon synapses. Emphasis was laid on calcium channel dynamics and how these channels influence synaptic release. MAIN RESULTS: Stronger stimulation with anodic pulses caused transmembrane voltages above the Nernst potential of calcium in the terminals and, by this means, forced calcium ions to flow in the reversed direction from inside to the outside of the cell. Consequently, intracellular calcium concentration decreased resulting in a reduced vesicle release or preventing release at all. This mechanism is expected to lead to a pronounced ring-shaped pattern of exocytosis within a group of neighbored BCs when the stronger stimulated cells close to the electrode fail in releasing vesicles. SIGNIFICANCE: Stronger subretinal stimulation causes failure of synaptic exocytosis due to reversal of calcium flow into the extracellular space in cells close to the electrode.