Effects of remote stimulation on the modulated activity of cat retinal ganglion cells.

The output of retinal ganglion cells depends on local and global aspects of the visual scene. The local receptive field is well studied and classically consists of a linear excitatory center and a linear antagonistic surround. The global receptive field contains pools of nonlinear subunits that are distributed widely across the retina. The subunit pools mediate in uncertain ways various nonlinear behaviors of ganglion cells, like temporal-frequency doubling, saccadic suppression, and contrast adaptation. To clarify mechanisms of subunit function, we systematically examined the effect of remote grating patterns on the spike activity of cat X- and Y-type ganglion cells in vivo. We present evidence for two distinct subunit types based on spatiotemporal relationships between response nonlinearities elicited by remote drifting and contrast-reversing gratings. One subunit type is excitatory and activated by gratings of approximately 0.1 cycles per degree, while the other is inhibitory and activated by gratings of approximately 1 cycle per degree. The two subunit pools contribute to a global gain control mechanism that differentially modulates ganglion cell response dynamics, particularly for ON-center cells, where excitatory and inhibitory subunit stimulation respectively makes responses to antipreferred and preferred contrast steps more transient. We show that the excitatory subunits also have a profound influence on spatial tuning, turning cells from lowpass into bandpass filters. Based on difference-of-Gaussians model fits to tuning curves, we attribute the increased bandpass selectivity to changes in center-surround strength and relative phase and not center-surround size. A conceptual model of the extraclassical receptive field that could explain many observed phenomena is discussed.

Retinal ganglion cell adaptation to small luminance fluctuations.

To accommodate the wide input range over which the visual system operates within the narrow output range of spiking neurons, the retina adjusts its sensitivity to the mean light level so that retinal ganglion cells can faithfully signal contrast, or relative deviations from the mean luminance. Given the large operating range of the visual system, the majority of work on luminance adaptation has involved logarithmic changes in light level. We report that luminance gain controls are recruited for remarkably small fluctuations in luminance as well. Using spike recordings from the rat optic tract, we show that ganglion cell responses to a brief flash of light are modulated in amplitude by local background fluctuations as little as 15% contrast. The time scale of the gain control is rapid (<125 ms), at least for on cells. The retinal locus of adaptation precedes the ganglion cell spike generator because response gain changes of on cells were uncorrelated with firing rate. The mechanism seems to reside within the inner retinal network and not in the photoreceptors, because the adaptation profiles of on and off cells differed markedly. The response gain changes follow Weber's law, suggesting that network mechanisms of luminance adaptation described in previous work modulates retinal ganglion cell sensitivity, not just when we move between different lighting environments, but also as our eyes scan a visual scene. Finally, we show that response amplitude is uniformly reduced for flashes on a modulated background that has spatial contrast, indicating that another gain control that integrates luminance signals nonlinearly over space operates within the receptive field center of rat ganglion cells.

Selective activation of neuronal targets with sinusoidal electric stimulation.

Electric stimulation of the CNS is being evaluated as a treatment modality for a variety of neurological, psychiatric, and sensory disorders. Despite considerable success in some applications, existing stimulation techniques offer little control over which cell types or neuronal substructures are activated by stimulation. The ability to more precisely control neuronal activation would likely improve the clinical outcomes associated with these applications. Here, we show that specific frequencies of sinusoidal stimulation can be used to preferentially activate certain retinal cell types: photoreceptors are activated at 5 Hz, bipolar cells at 25 Hz, and ganglion cells at 100 Hz. In addition, low-frequency stimulation (≤25 Hz) did not activate passing axons but still elicited robust synaptically mediated responses in ganglion cells; therefore, elicited neural activity is confined to within a focal region around the stimulating electrode. Our results suggest that sinusoidal stimulation provides significantly improved control over elicited neural activity relative to conventional pulsatile stimulation.

High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing.

Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95% transfection efficiency with minimum impact on cell viability and expansion potential. We additionally demonstrated processing of samples comprising up to 500 million T cells at a rate of 20 million cells/min. We anticipate that our device will help to streamline the production of autologous therapies requiring on the order of 10[Formula: see text]-10[Formula: see text] cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.

Miniature electroparticle-cuff for wireless peripheral neuromodulation.

OBJECTIVE: Recent developments in peripheral nerve electrodes allow the efficient and selective neuromodulation of somatic and autonomic nerves, which has proven beneficial in specific bioelectronic medical applications. However, current most clinical devices are wired and powered by implantable batteries which suffer from several limitations. We recently developed a sub-millimeter inductively powered neural stimulator (electroparticle; EP), and in this study, we report the integration of the EP onto commercial cuff electrodes (EP-C) allowing the wireless activation of peripheral nerves. APPROACH: The current output of this device was defined at different magnetic field strenghts, and with respect to external antenna distance and activation angles. In acute in vivo testing, stimulation of the rat sciatic nerve (ScN) with the EP-C was able to evoke motor responses quantified by 3D tracking of the hind limb movement. Motor recruitment curves were obtained in response to variations in magnetic field strength (0-92.91 A m-1), stimulation frequencies (2-7 Hz), and pulse widths (50-200 µs). MAIN RESULTS: The results show constant output voltage throughout 50 400 stimulating cycles on a benchtop setting, and successful ScN motor activation with a 4 cm distance between external antenna and receiver. We achieved optimal motor recruitment indicated by maximizing range of hindlimb movement (6.01 ± 2.92 mm) with a magnetic field of 40.02 ± 2.85 A m-1 and 150 µs pulse width. Stimulating pulse width or frequency did not significantly influence motor recruitment. SIGNIFICANCE: We confirmed that continuous stimulation for 14 min using monophasic pulses did not deleteriously affect the evoked motor responses when compared to wired charge-balanced biphasic electrical stimulation. We observed, however, a 36%-44% decrease in the evoked limb movement in both groups over time due to muscle fatigue. This study shows that the EP-C device can be used effectively for peripheral nerve neuromodulation.

Behavioral validation of a wireless low-power neurostimulation technology in a conditioned place preference task.

OBJECTIVE: Neurostimulation technologies are important for studying neural circuits and the connections that underlie neurological and psychiatric disorders. However, current methods come with limitations such as the restraint on movement imposed by the wires delivering stimulation. The objective of this study was to assess whether the e-Particle (EP), a novel wireless neurostimulator, could sufficiently stimulate the brain to modify behavior without these limitations. APPROACH: Rats were implanted with the EP and a commercially available stimulating electrode. Animals received rewarding brain stimulation, and performance in a conditioned place preference (CPP) task was measured. To ensure stimulation-induced neuronal activation, immediate early gene c-fos expression was also measured. MAIN RESULTS: The EP was validated in a commonly used CPP task by demonstrating that (1) wireless stimulation via the EP induced preference behavior that was comparable to that induced by standard wired electrodes and (2) neuronal activation was observed in projection targets of the stimulation site. SIGNIFICANCE: The EP may help achieve a better understanding of existing brain stimulation methods while overcoming their limitations. Validation of the EP in a behavioral model suggests that the benefits of this technology may extend to other areas of animal research and potentially to human clinical applications.

The maintained discharge of rat retinal ganglion cells.

Action potentials were recorded from rat retinal ganglion cell fibers in the presence of a uniform field, and the maintained discharge pattern was characterized. Spike trains recorded under ketaminexylazine. The majority of cells had multimodal interval distributions, with the first peak in the range of 25.00.97). Both ON and OFF cells show serial correlations between adjacent interspike intervals, while ON cells also showed second-order correlations. Cells with multimodal interval distribution showed a strong peak at high frequencies in the power spectra in the range of 28.9-41.4 Hz. Oscillations were present under both anesthetic conditions and persisted in the dark at a slightly lower frequency, implying that the oscillations are generated independent of any light stimulus but can be modulated by light level. The oscillation frequency varied slightly between cells of the same type and in the same eye, suggesting that multiple oscillatory generating mechanisms exist within the retina. Cells with high-frequency oscillations were described well by an integrate-and-fire model with the input consisting of Gaussian noise plus a sinusoid where the phase was jittered randomly to account for the bandwidth present in the oscillations.

Altering alpha-frequency brain oscillations with rapid analog feedback-driven neurostimulation.

Oscillations of the brain's local field potential (LFP) may coordinate neural ensembles and brain networks. It has been difficult to causally test this model or to translate its implications into treatments, because there are few reliable ways to alter LFP oscillations. We developed a closed-loop analog circuit to enhance brain oscillations by feeding them back into cortex through phase-locked transcranial electrical stimulation. We tested the system in a rhesus macaque with chronically implanted electrode arrays, targeting 8-15 Hz (alpha) oscillations. Ten seconds of stimulation increased alpha oscillatory power for up to 1 second after stimulation offset. In contrast, open-loop stimulation decreased alpha power. There was no effect in the neighboring 15-30 Hz (beta) LFP rhythm or on a neighboring array that did not participate in closed-loop feedback. Analog closed-loop neurostimulation might thus be a useful strategy for altering brain oscillations, both for basic research and the treatment of neuro-psychiatric disease.

A Sub-millimeter, Inductively Powered Neural Stimulator.

Wireless neural stimulators are being developed to address problems associated with traditional lead-based implants. However, designing wireless stimulators on the sub-millimeter scale (<1 mm3) is challenging. As device size shrinks, it becomes difficult to deliver sufficient wireless power to operate the device. Here, we present a sub-millimeter, inductively powered neural stimulator consisting only of a coil to receive power, a capacitor to tune the resonant frequency of the receiver, and a diode to rectify the radio-frequency signal to produce neural excitation. By replacing any complex receiver circuitry with a simple rectifier, we have reduced the required voltage levels that are needed to operate the device from 0.5 to 1 V (e.g., for CMOS) to ~0.25-0.5 V. This reduced voltage allows the use of smaller receive antennas for power, resulting in a device volume of 0.3-0.5 mm3. The device was encapsulated in epoxy, and successfully passed accelerated lifetime tests in 80°C saline for 2 weeks. We demonstrate a basic proof-of-concept using stimulation with tens of microamps of current delivered to the sciatic nerve in rat to produce a motor response.

Saturation of the right-leg drive amplifier in low-voltage ECG monitors.

Electrocardiogram (ECG) monitoring is a critical tool in patient care, but its utility is often balanced with frustration from clinicians who are constantly distracted by false alarms. This has motivated the need to readdress the major factors that contribute to ECG noise with the goal of reducing false alarms. In this study, we describe a previously unreported phenomenon in which ECG noise can result from an unintended interaction between two systems: 1) the dc lead-off circuitry that is used to detect whether electrodes fall off the patient; and 2) the right-leg drive (RLD) system that is responsible for reducing ac common-mode noise that couples into the body. Using a circuit model to study this interaction, we found that in the presence of a dc lead-off system, even moderate increases in the right-leg skin-electrode resistance can cause the RLD amplifier to saturate. Such saturation can produce ECG noise because the RLD amplifier will no longer be capable of attenuating ac common-mode noise on the body. RLD saturation is particularly a problem for modern ECG monitors that use low-voltage supply levels. For example, for a 12-lead ECG and a 2 V power supply, saturation will occur when the right-leg electrode resistance reaches only 2 MΩ. We discuss several design solutions that can be used in low-voltage monitors to avoid RLD saturation.

Neural substrates of tactile imagery: a functional MRI study.

fMRI investigation on the neural substrates involved in tactile imagery is reported. Healthy subjects performed mental imagery of tactile stimulation on the dorsal aspect of the right hand. The results were compared with the regions of activation during the actual tactile stimulation. During imagery, contralateral primary and secondary somatosensory areas were activated along with activation in the left parietal lobe. Activations in left inferior frontal gyri (Brodmann's area 44), left dorsolateral prefrontal area, left precentral gyrus, left insula, and medial frontal gyrus were also observed. In the basal ganglia, activation in the left thalamus (ventral posteromedial nucleus) and putamen was found. Our results suggest that the primary and secondary somatosensory areas are recruited during tactile imagery, and have partially overlapping neural substrates for the perception of tactile stimulation.

Microscopic magnetic stimulation of neural tissue.

Electrical stimulation is currently used to treat a wide range of cardiovascular, sensory and neurological diseases. Despite its success, there are significant limitations to its application, including incompatibility with magnetic resonance imaging, limited control of electric fields and decreased performance associated with tissue inflammation. Magnetic stimulation overcomes these limitations but existing devices (that is, transcranial magnetic stimulation) are large, reducing their translation to chronic applications. In addition, existing devices are not effective for deeper, sub-cortical targets. Here we demonstrate that sub-millimeter coils can activate neuronal tissue. Interestingly, the results of both modelling and physiological experiments suggest that different spatial orientations of the coils relative to the neuronal tissue can be used to generate specific neural responses. These results raise the possibility that micro-magnetic stimulation coils, small enough to be implanted within the brain parenchyma, may prove to be an effective alternative to existing stimulation devices.

Multiple components of ganglion cell desensitization in response to prosthetic stimulation.

Retinal prostheses aim to restore functional vision to those blinded by outer retinal diseases using electric stimulation of surviving neurons. Previous work indicates that repetitive stimulation with stimuli that activate the synaptic network reduces the sensitivity of retinal neurons to further stimulation. Such desensitization may contribute to the fading of visual percepts over time reported by human subjects. Here, we show that desensitization may be more complex than previously considered. We recorded spike trains from rabbit retinal ganglion cells and found that desensitization persists in the presence of inhibitory blockers (strychnine and picrotoxin), indicating amacrine cell inhibition is not solely responsible for reducing sensitivity in response to electric stimulation. The threshold for direct activation of the ganglion cell changes little during the simultaneous desensitization of the synaptically mediated response, indicating that desensitization likely occurs upstream of the spike generator. In addition to rapid desensitization acting over hundreds of milliseconds (τ = 176.4 ± 8.8 ms), we report the presence of slow acting desensitization with a time course of seconds (τ = 14.0 ± 1.1 s). The time courses of the two components of desensitization that we found are similar to the two phases of brightness fading seen in human subjects. This suggests that the reduction in ganglion cell firing due to desensitization may be responsible for the fading of visual percepts over time in response to prosthetic stimulation.

Encoding visual information in retinal ganglion cells with prosthetic stimulation.

Retinal prostheses aim to restore functional vision to those blinded by outer retinal diseases using electric stimulation of surviving retinal neurons. The ability to replicate the spatiotemporal pattern of ganglion cell spike trains present under normal viewing conditions is presumably an important factor for restoring high-quality vision. In order to replicate such activity with a retinal prosthesis, it is important to consider both how visual information is encoded in ganglion cell spike trains, and how retinal neurons respond to electric stimulation. The goal of the current review is to bring together these two concepts in order to guide the development of more effective stimulation strategies. We review the experiments to date that have studied how retinal neurons respond to electric stimulation and discuss these findings in the context of known retinal signaling strategies. The results from such in vitro studies reveal the advantages and disadvantages of activating the ganglion cell directly with the electric stimulus (direct activation) as compared to activation of neurons that are presynaptic to the ganglion cell (indirect activation). While direct activation allows high temporal but low spatial resolution, indirect activation yields improved spatial resolution but poor temporal resolution. Finally, we use knowledge gained from in vitro experiments to infer the patterns of elicited activity in ongoing human trials, providing insights into some of the factors limiting the quality of prosthetic vision.

Calcium channel dynamics limit synaptic release in response to prosthetic stimulation with sinusoidal waveforms.

Extracellular electric stimulation with sinusoidal waveforms has been shown to allow preferential activation of individual types of retinal neurons by varying stimulus frequency. It is important to understand the mechanisms underlying this frequency dependence as a step toward improving methods of preferential activation. In order to elucidate these mechanisms, we implemented a morphologically realistic model of a retinal bipolar cell and measured the response to extracellular stimulation with sinusoidal waveforms. We compared the frequency response of a passive membrane model to the kinetics of voltage-gated calcium channels that mediate synaptic release. The passive electrical properties of the membrane exhibited lowpass filtering with a relatively high cutoff frequency (nominal value = 717 Hz). This cutoff frequency was dependent on intra-axonal resistance, with shorter and wider axons yielding higher cutoff frequencies. However, we found that the cutoff frequency of bipolar cell synaptic release was primarily limited by the relatively slow opening kinetics of L- and T-type calcium channels. The cutoff frequency of calcium currents depended nonlinearly on stimulus amplitude, but remained lower than the cutoff frequency of the passive membrane model for a large range of membrane potential fluctuations. These results suggest that while it may be possible to modulate the membrane potential of bipolar cells over a wide range of stimulus frequencies, synaptic release will only be initiated at the lower end of this range.

Electric stimulation with sinusoids and white noise for neural prostheses.

We are investigating the use of novel stimulus waveforms in neural prostheses to determine whether they can provide more precise control over the temporal and spatial pattern of elicited activity as compared to conventional pulsatile stimulation. To study this, we measured the response of retinal ganglion cells to both sinusoidal and white noise waveforms. The use of cell-attached and whole cell patch clamp recordings allowed the responses to be observed without significant obstruction from the stimulus artifact. Electric stimulation with sinusoids elicited robust responses. White noise analysis was used to derive the linear kernel for the ganglion cell's spiking response as well as for the underlying excitatory currents. These results suggest that in response to electric stimulation, presynaptic retinal neurons exhibit bandpass filtering characteristics with a peak response that occurs 25 ms after onset. The experimental approach demonstrated here may be useful for studying the temporal response properties of other neurons in the CNS.

Single-unit in vivo recordings from the optic chiasm of rat.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.