Günther Zeck

Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip

A hybrid circuit of a semiconductor chip and synaptically connected neurons was implemented and characterized. Individual nerve cells from the snail Lymnaea stagnalis were immobilized on a silicon chip by microscopic picket fences of polyimide. The cells formed a network with electrical synapses after outgrowth in brain conditioned medium. Pairs of neurons were electronically interfaced for noninvasive stimulation and recording. Voltage pulses were applied to a capacitive stimulator on the chip to excite the attached neuron. Signals were transmitted in the neuronal net and elicited an action potential in a second neuron. The postsynaptic excitation modulated the current of a transistor on the chip. The implementation of the silicon-neuron-neuron-silicon circuit constitutes a proof-of-principle experiment for the development of neuroelectronic systems to be used in studies on neuronal signal processing, neurocomputation, and neuroprosthetics.

Network Oscillations in Rod-Degenerated Mouse Retinas

In the mammalian retina, excitatory and inhibitory circuitries enable retinal ganglion cells (RGCs) to signal the occurrence of visual features to higher brain areas. This functionality disappears in certain diseases of retinal degeneration because of the progressive loss of photoreceptors. Recent work in a mouse model of retinal degeneration (rd1) found that, although some intraretinal circuitry is preserved and RGCs maintain characteristic physiological properties, they exhibit increased and aberrant rhythmic activity. Here, extracellular recordings were made to assess the degree of aberrant activity in adult rd1 retinas and to investigate the mechanism underlying such behavior. A multi-transistor array with thousands of densely packed sensors allowed for simultaneous recordings of spiking activity in populations of RGCs and of local field potentials (LFPs). The majority of identified RGCs displayed rhythmic (7–10 Hz) but asynchronous activity. The spiking activity correlated with the LFPs, which reflect an average synchronized excitatory input to the RGCs. LFPs initiated from random positions and propagated across the retina. They disappeared when ionotrophic glutamate receptors or electrical synapses were blocked. They persisted in the presence of other pharmacological blockers, including TTX and inhibitory receptor antagonists. Our results suggest that excitation—transmitted laterally through a network of electrically coupled interneurons—leads to large-scale retinal network oscillations, reflected in the rhythmic spiking of most rd1 RGCs. This result may explain forms of photopsias reported by blind patients, while the mechanism involved should be considered in future treatment strategies targeting the disease of retinitis pigmentosa.

Electrical stimulation of retinal neurons in epiretinal and subretinal configuration using a multicapacitor array.

Electrical stimulation of retinal neurons offers the possibility of partial restoration of visual function. Challenges in neuroprosthetic applications are the long-term stability of the metal-based devices and the physiological activation of retinal circuitry. In this study, we demonstrate electrical stimulation of different classes of retinal neurons with a multicapacitor array. The array--insulated by an inert oxide--allows for safe stimulation with monophasic anodal or cathodal current pulses of low amplitude. Ex vivo rabbit retinas were interfaced in either epiretinal or subretinal configuration to the multicapacitor array. The evoked activity was recorded from ganglion cells that respond to light increments by an extracellular tungsten electrode. First, a monophasic epiretinal cathodal or a subretinal anodal current pulse evokes a complex burst of action potentials in ganglion cells. The first action potential occurs within 1 ms and is attributed to direct stimulation. Within the next milliseconds additional spikes are evoked through bipolar cell or photoreceptor depolarization, as confirmed by pharmacological blockers. Second, monophasic epiretinal anodal or subretinal cathodal currents elicit spikes in ganglion cells by hyperpolarization of photoreceptor terminals. These stimuli mimic the photoreceptor response to light increments. Third, the stimulation symmetry between current polarities (anodal/cathodal) and retina-array configuration (epi/sub) is confirmed in an experiment in which stimuli presented at different positions reveal the center-surround organization of the ganglion cell. A simple biophysical model that relies on voltage changes of cell terminals in the transretinal electric field above the stimulation capacitor explains our results. This study provides a comprehensive guide for efficient stimulation of different retinal neuronal classes with low-amplitude capacitive currents.

Daylight Vision Repair by Cell Transplantation

Human daylight vision depends on cone photoreceptors and their degeneration results in visual impairment and blindness as observed in several eye diseases including age‐related macular degeneration, cone‐rod dystrophies, or late stage retinitis pigmentosa, with no cure available. Preclinical cell replacement approaches in mouse retina have been focusing on rod dystrophies, due to the availability of sufficient donor material from the rod‐dominated mouse retina, leaving the development of treatment options for cone degenerations not well studied. Thus, an abundant and traceable source for donor cone‐like photoreceptors was generated by crossing neural retina leucine zipper‐deficient (Nrl−/−) mice with an ubiquitous green fluorescent protein (GFP) reporter line resulting in double transgenic tg(Nrl−/−; aGFP) mice. In Nrl−/− retinas, all rods are converted into cone‐like photoreceptors that express CD73 allowing their enrichment by CD73‐based magnetic activated cell sorting prior transplantation into the subretinal space of adult wild‐type, cone‐only (Nrl−/−), or cone photoreceptor function loss 1 (Cpfl1) mice. Donor cells correctly integrated into host retinas, acquired mature photoreceptor morphology, expressed cone‐specific markers, and survived for up to 6 months, with significantly increased integration rates in the cone‐only Nrl−/− retina. Individual retinal ganglion cell recordings demonstrated the restoration of photopic responses in cone degeneration mice following transplantation suggesting, for the first time, the feasibility of daylight vision repair by cell replacement in the adult mammalian retina. Stem Cells 2015;33:79–90

Spike train signatures of retinal ganglion cell types

The mammalian retina deconstructs the visual world using parallel neural channels, embodied in the morphological and physiological types of ganglion cells. We sought distinguishing features of each cell type in the temporal pattern of their spikes. As a first step, conventional physiological properties were used to cluster cells in eight types by a statistical analysis. We then adapted a method of P. Reinagel et al. (1999: J. Neurophysiol., 81, 2558–2569) to define epochs within the spike train of each cell. The spike trains of many cells were found to contain robust patterns that are defined by the (averaged) timing of successive interspike intervals in brief activity epochs. The patterns were robust across four different types of visual stimulus. Although the patterns are conserved in different visual environments, they do not prevent the cell from signaling the strength of its response to a particular stimulus, which is expressed in the number of spikes contained in each coding epoch. Clustering based on the spike train patterns alone showed that the spike train patterns correspond, in most but not all cases, to cell types pre‐defined by traditional criteria. That the congruence is less than perfect suggests that the typing of rabbit ganglion cells may need further refinement. Analysis of the spike train patterns may be useful in this regard and for distinguishing the many unidentified ganglion cell types that exist in other mammalian retinas.

Organotypic Culture of Physiologically Functional Adult Mammalian Retinas

Background The adult mammalian retina is an important model in research on the central nervous system. Many experiments require the combined use of genetic manipulation, imaging, and electrophysiological recording, which make it desirable to use an in vitro preparation. Unfortunately, the tissue culture of the adult mammalian retina is difficult, mainly because of the high energy consumption of photoreceptors. Methods and Findings We describe an interphase culture system for adult mammalian retina that allows for the expression of genes delivered to retinal neurons by particle-mediated transfer. The retinas retain their morphology and function for up to six days— long enough for the expression of many genes of interest—so that effects upon responses to light and receptive fields could be measured by patch recording or multielectrode array recording. We show that a variety of genes encoding pre- and post-synaptic marker proteins are localized correctly in ganglion and amacrine cells. Conclusions In this system the effects on neuronal function of one or several introduced exogenous genes can be studied within intact neural circuitry of adult mammalian retina. This system is flexible enough to be compatible with genetic manipulation, imaging, cell transfection, pharmacological assay, and electrophysiological recordings.

The spatial filtering properties of local edge detectors and brisk–sustained retinal ganglion cells

We compared image computation in the rabbit retina by two different cell types: the so‐called ‘local edge detecting’ ganglion cells and the well‐known brisk–sustained ganglion cells. From both anatomical and physiological evidence, these cells are present in nearly equal numbers and thus overlap to sample the same regions of visual space. We recorded simultaneously from overlapping cells on a dense microelectrode array. The results were analysed using an anatomically realistic simulation of the retinas processing levels. The ‘local edge detecting’ cell was found to be tuned to higher spatial frequencies and to have a narrower spatial frequency bandpass than the brisk–sustained cells. Simulation revealed that this is due primarily to the ‘zero‐crossing’ detector implied by the definition of the local edge detector. The outputs of the simulations in response to complex images were analysed quantitatively. The results showed the population of local edge detectors to transmit a sparser code than the brisk–sustained cells.

Axonal Transmission in the Retina Introduces a Small Dispersion of Relative Timing in the Ganglion Cell Population Response

Background Visual stimuli elicit action potentials in tens of different retinal ganglion cells. Each ganglion cell type responds with a different latency to a given stimulus, thus transforming the high-dimensional input into a temporal neural code. The timing of the first spikes between different retinal projection neurons cells may further change along axonal transmission. The purpose of this study is to investigate if intraretinal conduction velocity leads to a synchronization or dispersion of the population signal leaving the eye. Methodology/Principal Findings We ‘imaged’ the initiation and transmission of light-evoked action potentials along individual axons in the rabbit retina at micron-scale resolution using a high-density multi-transistor array. We measured unimodal conduction velocity distributions (1.3±0.3 m/sec, mean ± SD) for axonal populations at all retinal eccentricities with the exception of the central part that contains myelinated axons. The velocity variance within each piece of retina is caused by ganglion cell types that show narrower and slightly different average velocity tuning. Ganglion cells of the same type respond with similar latency to spatially homogenous stimuli and conduct with similar velocity. For ganglion cells of different type intraretinal conduction velocity and response latency to flashed stimuli are negatively correlated, indicating that differences in first spike timing increase (up to 10 msec). Similarly, the analysis of pair-wise correlated activity in response to white-noise stimuli reveals that conduction velocity and response latency are negatively correlated. Conclusion/Significance Intraretinal conduction does not change the relative spike timing between ganglion cells of the same type but increases spike timing differences among ganglion cells of different type. The fastest retinal ganglion cells therefore act as indicators of new stimuli for postsynaptic neurons. The intraretinal dispersion of the population activity will not be compensated by variability in extraretinal conduction times, estimated from data in the literature.

A CMOS-based sensor array for in-vitro neural tissue interfacing with 4225 recording sites and 1024 stimulation sites

A CMOS-based microelectrode array (MEA) with 4225 recording sites and 1024 stimulation sites and a related data acquisition system are presented. The chip provides high spatiotemporal resolution on an active area of 1 mm × 1 mm or 2 mm × 2 mm, respectively, and allows in-vitro neural tissue interfacing experiments with full imaging capability. The entire chip surface is covered by a thin high-k dielectric layer so that electric coupling between biological tissue and solid-state chip is purely capacitive. Biological experiments reveal proper functionality of the system.

A Comprehensive Small Interfering RNA Screen Identifies Signaling Pathways Required for Gephyrin Clustering

The postsynaptic scaffold protein gephyrin is clustered at inhibitory synapses and serves for the stabilization of GABAA receptors. Here, a comprehensive kinome-wide siRNA screen in a human HeLa cell-based model for gephyrin clustering was used to identify candidate protein kinases implicated in the stabilization of gephyrin clusters. As a result, 12 hits were identified including FGFR1 (FGF receptor 1), TrkB, and TrkC as well as components of the MAPK and mammalian target of rapamycin (mTOR) pathways. For confirmation, the impact of these hits on gephyrin clustering was analyzed in rat primary hippocampal neurons. We found that brain-derived neurotrophic factor (BDNF) acts on gephyrin clustering through MAPK signaling, and this process may be controlled by the MAPK signaling antagonist sprouty2. BDNF signaling through phosphatidylinositol 3-kinase (PI3K)–Akt also activates mTOR and represses GSK3β, which was previously shown to reduce gephyrin clustering. Gephyrin is associated with inactive mTOR and becomes released upon BDNF-dependent mTOR activation. In primary neurons, a reduction in the number of gephyrin clusters due to manipulation of the BDNF–mTOR signaling is associated with reduced GABAA receptor clustering, suggesting functional impairment of GABA signaling. Accordingly, application of the mTOR antagonist rapamycin leads to disinhibition of neuronal networks as measured on microelectrode arrays. In conclusion, we provide evidence that BDNF regulates gephyrin clustering via MAPK as well as PI3K–Akt–mTOR signaling.