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Computing the spike-triggered average (STA) is a simple method to estimate linear receptive fields (RFs) in sensory neurons. For random, uncorrelated stimuli, the STA provides an unbiased RF estimate, but in practice, white noise at high resolution is not an optimal stimulus choice as it usually evokes only weak responses. Therefore, for a visual stimulus, images of randomly modulated blocks of pixels are often used. This solution naturally limits the resolution at which an RF can be measured. Here, we present a simple super-resolution technique that can overcome these limitations. We define a novel stimulus type, the shifted white noise (SWN), by introducing random spatial shifts in the usual stimulus to increase the resolution of the measurements. In simulated data, we show that the average error using the SWN was 1.7 times smaller than when using the classical stimulus, with successful mapping of 2.3 times more neurons, covering a broader range of RF sizes. Moreover, successful RF mapping was achieved with brief recordings of light responses, lasting only about 1 min of activity, which is more than 10 times more efficient than the classical white noise stimulus. In recordings from mouse retinal ganglion cells with large scale multielectrode arrays, we successfully mapped 21 times more RFs than when using the traditional white noise stimuli. In summary, randomly shifting the usual white noise stimulus significantly improves RFs estimation, and requires only short recordings.NEW & NOTEWORTHY We present a novel approach to measure receptive fields in large and heterogeneous populations of sensory neurons recorded with large-scale, high-density multielectrode arrays. Our approach leverages super-resolution principles to improve the yield of the spike-triggered average method. By simply designing a new stimulus, we provide experimentalists with a new and fast technique to simultaneously detect more receptive fields at higher resolution in population of hundreds to thousands of neurons.

Developing neurons become spontaneously active while growing blood vessels begin to irrigate their surroundings. However, surprising little is known about early interactions between neural activity and angiogenesis. In the neonatal mouse retina, spontaneous waves of impulses sweep across the ganglion cell layer (GCL), just underneath the growing superficial vascular plexus. We discovered clusters of transient auto-fluorescent cells in the GCL, forming an annulus that co-localizes with the frontline of the growing plexus. Blood vessel density is highest within cluster areas, suggesting their involvement in angiogenesis. Once the clusters and blood vessels reach the retinal periphery by the end of the first postnatal week, the clusters disappear, eliminated by microglial phagocytosis. Electrical imaging suggests that they have their own electrophysiological signature. Blocking Pannexin1 (PANX1) hemi-channels with probenecid blocks the waves and the fluorescent clusters disappear following prolonged exposure to the drug. Spontaneous waves’ initiation points follow a developmental center-to-periphery progression similar to the cluster cells. We suggest that these transient cells are specialized, hyperactive neurons residing in the GCL. They generate spontaneous activity hotspots, thereby triggering waves through purinergic paracrine signaling via PANX1 hemi-channels. The strong activity generated around these hotspots triggers angiogenesis, attracting new blood vessels that provide local oxygen supply. Signaling through PANX-1 attracts microglia that establish contact with these cells, eventually leading to their elimination by phagocytosis. These cluster cells may provide the first evidence that specialized transient neuronal populations guide angiogenesis in the developing CNS through neural activity.

Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies.

Retinal dystrophies often lead to blindness. Developing therapeutic interventions to restore vision is therefore of paramount importance. Here we demonstrate the ability of pluripotent stem cell-derived cone precursors to engraft and restore light responses in the Pde6brd1 mouse, an end-stage photoreceptor degeneration model. Our data show that up to 1.5% of precursors integrate into the host retina, differentiate into cones, and engraft in close apposition to the host bipolar cells. Half of the transplanted mice exhibited visual behavior and of these 33% showed binocular light sensitivity. The majority of retinal ganglion cells exhibited contrast-sensitive ON, OFF or ON-OFF light responses and even motion sensitivity; however, quite a few exhibited unusual responses (eg, light-induced suppression), presumably reflecting remodeling of the neural retina. Our data indicate that despite relatively low engraftment yield, pluripotent stem cell-derived cone precursors can elicit light responsiveness even at advanced degeneration stages. Further work is needed to improve engraftment yield and counteract retinal remodeling to achieve useful clinical applications.

SummaryRetinal dystrophies often lead to blindness. Developing therapeutic interventions to restore vision is therefore of paramount importance. Here we demonstrate the ability of pluripotent stem cell-derived cone precursors to engraft and restore light responses in thePde6brd1mouse, an end-stage photoreceptor degeneration model. Up to 1.5% of precursors integrated into the host retina, differentiated into cones and formed synapses with bipolar cells. Half of the transplanted mice exhibited visual behaviour and 33% showed binocular light sensitivity. The majority of ganglion cells exhibited contrast-sensitive ON, OFF or ON-OFF light responses and even motion sensitivity. Many cells also exhibited unusual responses (e.g. light-induced suppression), presumably reflecting remodelling of the neural retina. Our data indicate that despite relatively low engraftment yield, engrafted pluripotent stem cell-derived cone precursors can elicit light responsiveness even at advanced degeneration stages. Further work is needed to improve engraftment yield and counteract retinal remodelling to achieve useful clinical applications.

Chronic pain is a global healthcare problem with a huge societal impact. Its management remains unsatisfactory, with no single treatment clinically approved in most cases. In this study we use an in vitro experimental model of erythromelalgia consisting of sensory neurons derived from human induced pluripotent stem cells obtained from a patient (carrying the mutation F1449V) and a control subject. We combine neurophysiology and computational modelling to focus on the Nav1.7 voltage gated sodium channel, which acts as an amplifier of the receptor potential in nociceptive neurons and plays a critical role in erythromelalgia due to gain of function mutations causing the channel to open with smaller depolarisations.Using multi-electrode array (extracellular) recordings, we found that the scorpion toxin OD1 increases the excitability of sensory neurons in cultures obtained from the control donor, evidenced by increased spontaneous spike rate and amplitude. In erythromelalgia cultures, the application of the Nav1.7 blocker PF-05089771 effectively stopped spontaneous firing. These results, which are in accordance with current clamp and voltage clamp recordings reported in the literature, are explained with a conductance-based computational model of a single human nociceptive neuron. The disease was simulated through a decrease of the Nav1.7 half activation voltage, which decreased the rheobase and increased the response to supra threshold depolarizing currents. This enhanced response could be successfully supressed by blocking the Nav1.7 channels. The painful effects of OD1 were simulated through a slower establishment and a quicker removal of Nav1.7 inactivation, reproducing the effects of the toxin on the spike frequency and amplitude. Our model simulations suggest that the increase in extracellular spike amplitude observed in the MEA after OD1 treatment can be due mainly to a slope increase in the ascending phase of the intracellular spike caused by impaired inactivation gating.

Chronic pain is a global healthcare problem with a huge societal impact. Its management remains generally unsatisfactory, with no single treatment clinically approved in most cases. In this study we use an in vitro model of erythromelalgia consisting of dorsal root ganglion neurons derived from human induced pluripotent stem cells obtained from a patient (carrying the mutation F1449V) and a control subject. We combine neurophysiology and computational modelling to focus on the Nav1.7 voltage gated sodium channel, which acts as an amplifier of the receptor potential in nociceptive neurons and plays a critical role in erythromelalgia due to gain of function mutations causing the channel to open with smaller depolarisations. Using extracellular recordings, we found that the scorpion toxin OD1 (a Nav1.7 channel opener) increases dorsal root ganglion cell excitability in cultures obtained from the control donor, evidenced by an increase in spontaneous discharges, firing rate and spike amplitude. In addition, we confirmed previous reports of voltage clamp experiments concerning an increase in spontaneous discharge in the patient cell cultures and the analgesic effects of the Nav1.7 blocker PF-05089771. Our findings are explained with a conductance-based model of the dorsal root ganglion neuron, exploring its behaviour for different values of half activation voltage and inactivation removal rate of the Nav1.7 current. Erythromelalgia was simulated through a decrease of the Nav1.7 half activation voltage, turning previously subthreshold stimuli to pain-inducing, and successfully counteracted with the channel blocker. The painful effects of OD1 were simulated through a quicker removal of Nav1.7 inactivation that reproduced the effects of the toxin not only on the spike frequency but also on its amplitude. New & Noteworthy We investigate Nav1.7 channel gating mechanisms in human iPSC-derived dorsal root ganglion cell cultures using multielectrode array recordings. The scorpion toxin OD1 increases firing frequency and spike amplitudes whilst the analgesic PF-05089771 decreases or even abolishes spontaneous activity. The antagonistic effect of these compounds is explained with a computational model to reach deeper understanding of changes in channel kinetics in erythromelalgia, a chronic pain disorder.

The choice of electrode material is of paramount importance in neural prosthetic devices. Electrodes must be biocompatible yet able to sustain repetitive current injections in a highly corrosive environment. We explored the suitability of carbon nanotube (CNT) electrodes to stimulate retinal ganglion cells (RGCs) in a mouse model of outer retinal degeneration. We investigated morphological changes at the bio-hybrid interface and changes in RGC responses to electrical stimulation following prolonged in vitro coupling to CNT electrodes. We observed gradual remodelling of the inner retina to incorporate CNT assemblies. Electrophysiological recordings demonstrate a progressive increase in coupling between RGCs and the CNT electrodes over three days, characterized by a gradual decrease in stimulation thresholds and increase in cellular recruitment. These results provide novel evidence for time-dependent formation of viable bio-hybrids between CNTs and the retina, demonstrating that CNTs are a promising material for inclusion in retinal prosthetic devices.

Retinal ganglion cells, the sole output neurons of the retina, exhibit surprising diversity. A recent study reported over 30 distinct types in the mouse retina, indicating that the processing of visual information is highly parallelised in the brain. The advent of high density multi-electrode arrays now enables recording from many hundreds to thousands of neurons from a single retina. Here we describe a method for the automatic classification of large-scale retinal recordings using a simple stimulus paradigm and a spike train distance measure as a clustering metric. We evaluate our approach using synthetic spike trains, and demonstrate that major known cell types are identified in high-density recording sessions from the mouse retina with around 1000 retinal ganglion cells. A comparison across different retinas reveals substantial variability between preparations, suggesting pooling data across retinas should be approached with caution. As a parameter-free method, our approach is broadly applicable for cellular physiological classification in all sensory modalities.

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Objective. Our main objective is to demonstrate that compact high radiance gallium nitride displays can be used with conventional virtual reality optics to stimulate an optogenetic retina. Hence, we aim to introduce a non-invasive approach to restore vision for people with conditions such as retinitis pigmentosa where there is a remaining viable communication link between the retina and the visual cortex. Approach. We design and implement the headset using a high-density µLED matrix, Raspberry Pi, microcontroller from NXP and virtual reality lens. Then, a test platform is developed to evaluate the performance of the headset and the optical system. Furthermore, image simplification algorithms are used to simplify the scene to be sent to the retina. Moreover, in vivo evaluation of the genetically modified retina response at different light intensity is discussed to prove the reliability of the proposed system. Main results. We demonstrate that in keeping with regulatory guidance, the headset displays need to limit their luminance to 90 kcd m−2. We demonstrate an optical system with 5.75% efficiency which allows for 0.16 mW mm−2 irradiance on the retina within the regulatory guidance, but which is capable of an average peak irradiance of 1.35 mW mm−2. As this is lower than the commonly accepted threshold for channelrhodopsin-2, we demonstrate efficacy through an optical model of an eye onto a biological retina. Significance. We demonstrate a fully functional 8100-pixel headset system including software/hardware which can operate on a standard consumer battery for periods exceeding a 24 h recharge cycle. The headset is capable of delivering enough light to stimulate the genetically modified retina cells and also keeping the amount of light below the regulation threshold for safety.

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light‐responsive retinal organoids from renewable and patient specific sources. We investigated five different human‐induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC‐derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC‐derived retinal organoids exhibited at this time a well‐formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal‐pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC‐derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. stem cells 2018;36:1535–1551

Automatic detection and segmentation of neurons from microscopy acquisition is essential for statistically characterizing neuron morphology that can be related to their functional role. In this paper, we propose a combined pipeline that starts from the automatic detection of the soma through a new multiscale blob enhancement filtering. Then, a precise segmentation of the detected cell body is obtained by an active contour approach. The resulted segmentation is used as initial seed for the second part of the approach that proposes a dendrite arborization tracing method.

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

The immature retina generates spontaneous waves of spiking activity that sweep across the ganglion cell layer during a limited period of development before the onset of visual experience. The spatiotemporal patterns encoded in the waves are believed to be instructive for the wiring of functional connections throughout the visual system. However, the ontogeny of retinal waves is still poorly documented as a result of the relatively low resolution of conventional recording techniques. Here, we characterize the spatiotemporal features of mouse retinal waves from birth until eye opening in unprecedented detail using a large-scale, dense, 4096-channel multielectrode array that allowed us to record from the entire neonatal retina at near cellular resolution. We found that early cholinergic waves propagate with random trajectories over large areas with low ganglion cell recruitment. They become slower, smaller and denser when GABAA signalling matures, as occurs beyond postnatal day (P) 7. Glutamatergic influences dominate from P10, coinciding with profound changes in activity dynamics. At this time, waves cease to be random and begin to show repetitive trajectories confined to a few localized hotspots. These hotspots gradually tile the retina with time, and disappear after eye opening. Our observations demonstrate that retinal waves undergo major spatiotemporal changes during ontogeny. Our results support the hypotheses that cholinergic waves guide the refinement of retinal targets and that glutamatergic waves may also support the wiring of retinal receptive fields.

Retinitis pigmentosa is a progressive retinal dystrophy that causes irreversible visual impairment and blindness. Retinal prostheses currently represent the only clinically available vision-restoring treatment, but the quality of vision returned remains poor. Recently, it has been suggested that the pathological spontaneous hyperactivity present in dystrophic retinas may contribute to the poor quality of vision returned by retinal prosthetics by reducing the signal-to-noise ratio of prosthetic responses. Here, we investigated to what extent blocking this hyperactivity can improve optogenetic retinal prosthetic responses. We recorded activity from channelrhodopsin-expressing retinal ganglion cells in retinal wholemounts in a mouse model of retinitis pigmentosa. Sophisticated stimuli, inspired by those used in clinical visual assessment, were used to assess light sensitivity, contrast sensitivity and spatial acuity of optogenetic responses; in all cases these were improved after blocking spontaneous hyperactivity using meclofenamic acid, a gap junction blocker. Our results suggest that this approach significantly improves the quality of vision returned by retinal prosthetics, paving the way to novel clinical applications. Moreover, the improvements in sensitivity achieved by blocking spontaneous hyperactivity may extend the dynamic range of optogenetic retinal prostheses, allowing them to be used at lower light intensities such as those encountered in everyday life.

We have investigated the ontogeny of light-driven responses in mouse retinal ganglion cells (RGCs). Using a large-scale, high-density multielectrode array, we recorded from hundreds to thousands of RGCs simultaneously at pan-retinal level, including dorsal and ventral locations. Responses to different contrasts not only revealed a complex developmental profile for ON, OFF and ON-OFF RGC types, but also unveiled differences between dorsal and ventral RGCs. At eye-opening, dorsal RGCs of all types were more responsive to light, perhaps indicating an environmental priority to nest viewing for pre-weaning pups. The developmental profile of ON and OFF RGCs exhibited antagonistic behavior, with the strongest ON responses shortly after eye-opening, followed by an increase in the strength of OFF responses later on. Further, we found that with maturation receptive field (RF) center sizes decrease, responses to light get stronger, and centers become more circular while seeing differences in all of them between RGC types. These findings show that retinal functionality is not spatially homogeneous, likely reflecting ecological requirements that favour the early development of dorsal retina, and reflecting different roles in vision in the mature animal.

The choice of electrode material is of paramount importance in neural prosthetic devices. Electrodes must be biocompatible yet able to sustain repetitive current injections in a highly corrosive environment. We explored the suitability of carbon nanotube (CNT) electrodes to stimulate retinal ganglion cells (RGCs) in a mouse model of outer retinal degeneration. We investigated morphological changes at the bio-hybrid interface and changes in RGC responses to electrical stimulation following prolonged in vitro coupling to CNT electrodes. We observed gradual remodelling of the inner retina to incorporate CNT assemblies. Electrophysiological recordings demonstrate a progressive increase in coupling between RGCs and the CNT electrodes over three days, characterized by a gradual decrease in stimulation thresholds and increase in cellular recruitment. These results provide novel evidence for time-dependent formation of viable bio-hybrids between CNTs and the retina, demonstrating that CNTs are a promising material for inclusion in retinal prosthetic devices.

A new method for automated spike sorting for recordings with high density, large scale multielectrode arrays is presented. Exploiting the dense sampling of single neurons by multiple electrodes, we obtain an efficient, low-dimensional representation of detected spikes consisting of estimated spatial spike locations and dominant spike shape features, which enables fast and reliable clustering into single units. Millions of events can be sorted in minutes, and the method is parallelized and scales better than quadratically with the number of detected spikes. We demonstrate this method using recordings with a 4,096 channel array, and present validation based on anatomical imaging, optogenetic stimulation and model-based quality control. A comparison with semi-automated, shape-based spike sorting exposes significant limitations of conventional methods. Our analysis shows that it is feasible to reliably isolate the activity of hundreds to thousands of neurons in a single recording, and that dense, multi-channel probes substantially aid reliable spike sorting.

Microelectrode electrophysiology has become a widespread technique for the extracellular recording of bioelectrical signals. To date, electrodes are made of metals or inorganic semiconductors, or hybrids thereof. We demonstrate that these traditional conductors can be completely substituted by highly flexible electroconductive polymers. Pursuing a two-level replica-forming strategy, conductive areas for electrodes, leads and contact pads are defined as microchannels in poly(dimethylsiloxane) (PDMS) as a plastic carrier and track insulation material. These channels are coated by films of organic conductors such as polystyrenesulfonate-doped poly(3,4-ethylenedioxy-thiophene) (PEDOT:PSS) or filled with a graphite-PDMS (gPDMS) composite, either alone or in combination. The bendable, somewhat stretchable, non-cytotoxic and biostable all-polymer microelectrode arrays (polyMEAs) with a thickness below 500 μm and up to 60 electrodes reliably capture action potentials (APs) and local field potentials (LFPs) from acute preparations of heart muscle cells and retinal whole mounts, in vivo epicortical and epidural recordings as well as during long-term in vitro recordings from cortico-hippocampal co-cultures.

Over the last few years, simultaneous recordings of multiple spike trains have become widely used by neuroscientists. Therefore, it is important to develop new tools for analysing multiple spike trains in order to gain new insight into the function of neural systems. This paper describes how techniques from the field of visual analytics can be used to reveal specific patterns of neural activity. An interactive raster plot called iRaster has been developed. This software incorporates a selection of statistical procedures for visualization and flexible manipulations with multiple spike trains. For example, there are several procedures for the re-ordering of spike trains which can be used to unmask activity propagation, spiking synchronization, and many other important features of multiple spike train activity. Additionally, iRaster includes a rate representation of neural activity, a combined representation of rate and spikes, spike train removal and time interval removal. Furthermore, it provides multiple coordinated views, time and spike train zooming windows, a fisheye lens distortion, and dissemination facilities. iRaster is a user friendly, interactive, flexible tool which supports a broad range of visual representations. This tool has been successfully used to analyse both synthetic and experimentally recorded datasets. In this paper, the main features of iRaster are described and its performance and effectiveness are demonstrated using various types of data including experimental multi-electrode array recordings from the ganglion cell layer in mouse retina. iRaster is part of an ongoing research project called VISA (Visualization of Inter-Spike Associations) at the Visualization Lab in the University of Plymouth. The overall aim of the VISA project is to provide neuroscientists with the ability to freely explore and analyse their data. The software is freely available from the Visualization Lab website (see www.plymouth.ac.uk/infovis).

Visual Abstract, How a population of retinal ganglion cells (RGCs) encodes the visual scene remains an open question. Going beyond individual RGC coding strategies, results in salamander suggest that the relative latencies of a RGC pair encode spatial information. Thus, a population code based on this concerted spiking could be a powerful mechanism to transmit visual information rapidly and efficiently., How a population of retinal ganglion cells (RGCs) encodes the visual scene remains an open question. Going beyond individual RGC coding strategies, results in salamander suggest that the relative latencies of a RGC pair encode spatial information. Thus, a population code based on this concerted spiking could be a powerful mechanism to transmit visual information rapidly and efficiently. Here, we tested this hypothesis in mouse by recording simultaneous light-evoked responses from hundreds of RGCs, at pan-retinal level, using a new generation of large-scale, high-density multielectrode array consisting of 4096 electrodes. Interestingly, we did not find any RGCs exhibiting a clear latency tuning to the stimuli, suggesting that in mouse, individual RGC pairs may not provide sufficient information. We show that a significant amount of information is encoded synergistically in the concerted spiking of large RGC populations. Thus, the RGC population response described with relative activities, or ranks, provides more relevant information than classical independent spike count- or latency- based codes. In particular, we report for the first time that when considering the relative activities across the whole population, the wave of first stimulus-evoked spikes is an accurate indicator of stimulus content. We show that this coding strategy coexists with classical neural codes, and that it is more efficient and faster. Overall, these novel observations suggest that already at the level of the retina, concerted spiking provides a reliable and fast strategy to rapidly transmit new visual scenes.

Retinitis pigmentosa (RP) is a progressive retinal dystrophy that causes visual impairment and eventual blindness. Retinal prostheses are the best currently available vision-restoring treatment for RP, but only restore crude vision. One possible contributing factor to the poor quality of vision achieved with prosthetic devices is the pathological retinal ganglion cell (RGC) hyperactivity that occurs in photoreceptor dystrophic disorders. Gap junction blockade with meclofenamic acid (MFA) was recently shown to diminish RGC hyperactivity and improve the signal-to-noise ratio (SNR) of RGC responses to light flashes and electrical stimulation in the rd10 mouse model of RP. We sought to extend these results to spatiotemporally patterned optogenetic stimulation in the faster-degenerating rd1 model and compare the effectiveness of a number of drugs known to disrupt rd1 hyperactivity. We crossed rd1 mice with a transgenic mouse line expressing the light-sensitive cation channel channelrhodopsin2 (ChR2) in RGCs, allowing them to be stimulated directly using high-intensity blue light. We used 60-channel ITO multielectrode arrays to record ChR2-mediated RGC responses from wholemount, ex-vivo retinas to full-field and patterned stimuli before and after application of MFA, 18-β-glycyrrhetinic acid (18BGA, another gap junction blocker) or flupirtine (Flu, a Kv7 potassium channel opener). All three drugs decreased spontaneous RGC firing, but 18BGA and Flu also decreased the sensitivity of RGCs to optogenetic stimulation. Nevertheless, all three drugs improved the SNR of ChR2-mediated responses. MFA also made it easier to discern motion direction of a moving bar from RGC population responses. Our results support the hypothesis that reduction of pathological RGC spontaneous activity characteristic in retinal degenerative disorders may improve the quality of visual responses in retinal prostheses and they provide insights into how best to achieve this for optogenetic prostheses.

Mature retinal ganglion cells (RGCs) have distinct morphologies that often reflect specialized functional properties such as On and Off responses. But the structural correlates of many complex receptive field (RF) properties (e.g. responses to motion) remain to be deciphered. In this study, we have investigated whether motion anisotropies (non-homogeneities) characteristic of embryonic turtle RGCs arise from immature dendritic arborization in these cells. To test this hypothesis, we have looked at structure-function correlates of developing turtle RGCs from Stage 23 (S23) when light responses emerge, until 15 weeks post-hatching (PH). Using whole cell patch clamp recordings, RGCs were labelled with Lucifer Yellow (LY) while recording their responses to moving edges of light. Comparison of RF and dendritic arbor layouts revealed a weak correlation. To obtain a larger structural sample of developing RGCs, we have looked at dendritic morphology in RGCs retrogradely filled with the tracer horseradish peroxidase (HRP) from S22 (when RGCs become spontaneously active, shortly before they become sensitive to light) until two weeks PH. We found that there was intense dendritic growth from S22 onwards, reaching peak proliferation at S25 (a week before hatching), while RGCs are still exhibiting significant motion anisotropies. Based on these observations, we suggest that immature anisotropic RGC RFs must originate from sparse synaptic inputs onto RGCs rather than from the immaturity of their growing dendritic trees.

Early neural activity, both prenatal spontaneous bursts and early visual experience, is believed to be important for dendritic proliferation and for the maturation of neural circuitry in the developing retina. In this study, we have investigated the possible role of early neural activity in shaping developing turtle retinal ganglion cell (RGC) dendritic arbors. RGCs were back-labelled from the optic nerve with horseradish peroxidase (HRP). Changes in dendritic growth patterns were examined across development and following chronic blockade or modification of spontaneous activity and ⁄or visual experience. Dendrites reach peak proliferation at embryonic stage 25 (S25, one week before hatching), followed by pruning in large field RGCs around the time of hatching. When spontaneous activity is chronically blocked in vivo from early embryonic stages (S22) with curare, a cholinergic nicotinic antagonist, RGC dendritic growth is inhibited. On the other hand, enhancement of spontaneous activity by dark-rearing (Sernagor & Grzywacz (1996) Curr. Biol., 6, 1503‐1508) promotes dendritic proliferation in large-field RGCs, an effect that is counteracted by exposure to curare from hatching. We also recorded spontaneous activity from individual RGCs labelled with lucifer yellow (LY). We found a tendency of RGCs with large dendritic fields to be spontaneously more active than small-field cells. From all these observations, we conclude that immature spontaneous activity promotes dendritic growth in developing RGCs.

We report the development of a semiconductor nanorod-carbon nanotube based platform for wire-free, light induced retina stimulation. A plasma polymerized acrylic acid midlayer was used to achieve covalent conjugation of semiconductor nanorods directly onto neuro-adhesive, three-dimensional carbon nanotube surfaces. Photocurrent, photovoltage, and fluorescence lifetime measurements validate efficient charge transfer between the nanorods and the carbon nanotube films. Successful stimulation of a light-insensitive chick retina suggests the potential use of this novel platform in future artificial retina applications.

Blindness represents an increasing global problem with significant social and economic impact upon affected patients and society as a whole. In Europe, approximately one in 30 individuals experience sight loss and 75% of those are unemployed, a social burden which is very likely to increase as the population of Europe ages. Diseases affecting the retina account for approximately 26% of blindness globally and 70% of blindness in the United Kingdom. To date, there are no treatments to restore lost retinal cells and improve visual function, highlighting an urgent need for new therapeutic approaches. A pioneering breakthrough has demonstrated the ability to generate synthetic retina from pluripotent stem cells under laboratory conditions, a finding with immense relevance for basic research,in vitrodisease modeling, drug discovery, and cell replacement therapies. This review summarizes the current achievements in pluripotent stem cell differentiation toward retinal cells and highlights the steps that need to be completed in order to generate human synthetic retinae with high efficiency and reproducibly from patient-specific pluripotent stem cells.

Hypoplastic left heart syndrome (HLHS) is a serious congenital cardiovascular malformation resulting in hypoplasia or atresia of the left ventricle, ascending aorta, and aortic and mitral valves. Diminished flow through the left side of the heart is clearly a key contributor to the condition, but any myocardial susceptibility component is as yet undefined. Using recent advances in the field of induced pluripotent stem cells (iPSCs), we have been able to generate an iPSC model of HLHS malformation and characterize the properties of cardiac myocytes (CMs) differentiated from these and control-iPSC lines. Differentiation of HLHS-iPSCs to cardiac lineages revealed changes in the expression of key cardiac markers and a lower ability to give rise to beating clusters when compared with control-iPSCs and human embryonic stem cells (hESCs). HLHS-iPSC-derived CMs show a lower level of myofibrillar organization, persistence of a fetal gene expression pattern, and changes in commitment to ventricular versus atrial lineages, and they display different calcium transient patterns and electrophysiological responses to caffeine and β-adrenergic antagonists when compared with hESC- and control-iPSC-derived CMs, suggesting that alternative mechanisms to release calcium from intracellular stores such as the inositol trisphosphate receptor may exist in HLHS in addition to the ryanodine receptor thought to function in control-iPSC-derived CMs. Together our findings demonstrate that CMs derived from an HLHS patient demonstrate a number of marker expression and functional differences to hESC/control iPSC-derived CMs, thus providing some evidence that cardiomyocyte-specific factors may influence the risk of HLHS.

In the developing vertebrate retina, ganglion cells fire spontaneous bursts of action potentials long before the eye becomes exposed to sensory experience at birth. These early bursts are synchronised between neighbouring retinal ganglion cells (RGCs), yielding unique spatiotemporal patterns: ‘waves’ of activity sweep across large retinal areas every few minutes. Both at retinal and extraretinal levels, these embryonic retinal waves are believed to guide the wiring of the visual system using hebbian mechanisms of synaptic strengthening. In the first part of this review, we recapitulate the evidence for a role of these embryonic spontaneous bursts of activity in shaping developing complex receptive field properties of RGCs in the turtle embryonic retina. We also discuss the role of visual experience in establishing RGC visual functions, and how spontaneous activity and visual experience interact to bring developing receptive fields to maturation. We have hypothesised that the physiological changes associated with development reflect modifications in the dendritic arbours of RGCs, the anatomical substrate of their receptive fields. We demonstrate that there is a temporal correlation between the period of receptive field expansion and that of dendritic growth. Moreover, the immature spontaneous activity contributes to dendritic growth in developing RGCs. Intracellular staining of RGCs reveals, however, that immature receptive fields only rarely show direct correlation with the layout of the corresponding dendritic tree. To investigate the possibility that not only the presence of the spontaneous activity, but even the precise spatiotemporal patterns encoded in retinal waves might contribute to the refinement of retinal neural circuitry, first we must clarify the mechanisms mediating the generation and propagation of these waves across development. In the second part of this review, we present evidence that turtle retinal waves, visualised using calcium imaging, exhibit profound changes in their spatiotemporal patterns during development. From fast waves sweeping across large retinal areas and recruiting many cells on their trajectory at early stages, waves become slower and eventually stop propagating towards hatching, when they become stationary patches of neighbouring coactive RGCs. A developmental switch from excitatory to inhibitory GABAA responses appears to mediate the modification in spontaneous activity patterns while the retina develops. Future chronic studies using specific spatiotemporal alterations of the waves will shed a new light on how the wave dynamics help in sculpting retinal receptive fields.

The immature retina generates spontaneous waves of spiking activity that sweep across the ganglion cell layer during a limited period of development before the onset of visual experience. The spatiotemporal patterns encoded in the waves are believed to be instructive for the wiring of functional connections throughout the visual system. However, the ontogeny of retinal waves is still poorly documented as a result of the relatively low resolution of conventional recording techniques. Here, we characterize the spatiotemporal features of mouse retinal waves from birth until eye opening in unprecedented detail using a large-scale, dense, 4096-channel multielectrode array that allowed us to record from the entire neonatal retina at near cellular resolution. We found that early cholinergic waves propagate with random trajectories over large areas with low ganglion cell recruitment. They become slower, smaller and denser when GABAA signalling matures, as occurs beyond postnatal day (P) 7. Glutamatergic influences dominate from P10, coinciding with profound changes in activity dynamics. At this time, waves cease to be random and begin to show repetitive trajectories confined to a few localized hotspots. These hotspots gradually tile the retina with time, and disappear after eye opening. Our observations demonstrate that retinal waves undergo major spatiotemporal changes during ontogeny. Our results support the hypotheses that cholinergic waves guide the refinement of retinal targets and that glutamatergic waves may also support the wiring of retinal receptive fields.

We report on the temporal properties of the spontaneous bursts of activity in the developing turtle retina. Quantitative statistical criteria were used to detect, cluster, and analyze the temporal properties of the bursts. The interburst interval, duration, firing rate, and number of spikes per burst varied widely among cells and from burst to burst in a single cell. Part of this variability was due to the positive correlation between a burst's duration and the interburst interval preceding that burst. This correlation indicated the influence of a refractory period on the bursts' properties. Further evidence of such a refractoriness came from the bursts' auto-covariance function, which gives the tendency of a spike to occur a given amount of time after another spike. This function showed a positive phase (between ≈10 ms and 10 s) followed by a negative one (between 10 s and more than 100 s), suggestive of burst refractoriness. The bursts seemed to be propagating from cell to cell, because there was a long (symmetrically distributed) delay between the activation of two neighbor cells (median absolute delay = 2.3 s). However, the activity often failed to propagate from one cell to the other (median safety factor = 0.59). The number of spikes per burst in neighbor cells was statistically positively correlated, indicating that the activity in the two cells was driven by the same excitatory process. At least two factors contribute to the excitability during bursts, because the positive phase of the cross-covariance function (similar to auto-covariance but for two cells) had a temporally asymmetric fast component (1–3 ms) followed by a temporally symmetric slow component (1 ms to 10 s).

Extracellular recordings were obtained from the ganglion cell (GC) layer during correlated spontaneous bursting activity (SBA) in the immature turtle retina. Pharmacological agents were bath-applied, and their effects on burst and correlation parameters were determined.SBA requires synaptic transmission. It was blocked in the presence of curare and mecamylamine, two cholinergic nicotinic antagonists, and enhanced with neostigmine, a cholinesterase inhibitor. SBA was profoundly inhibited during blockade of glutamatergic receptors with the broad spectrum antagonist kynurenate and it vanished with 6,7-dinitroquinoxaline-2–3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), two AMPA/kainate receptor antagonists. Blockade of NMDA receptors withd(−)-2-amino-5-phosphonopentanoic acid (d-AP-5) led only to a modest reduction in SBA. Blockade of GABAAreceptors with bicuculline prolonged the duration of the bursts. Inhibition of GABA uptake with nipecotic acid led to a decrease in burst rate. Blockade of K+channels with cesium (Cs+) and tetraethylammonium (TEA) led to a dramatic decrease in excitability. Burst propagation between neighboring GCs was reduced by K+channel blockade. Gap junction blockade had no consistent effect on bursts or correlation parameters. None of these drugs had a strong effect on the refractory period between bursts.We conclude that correlated SBA in immature turtle GCs requires both cholinergic nicotinic and glutamatergic (mainly through AMPA/kainate receptors) synaptic transmission. GABAergic activity modulates the intensity and the duration of the bursts. Extracellular K+is involved in lateral activity propagation and increases retinal excitability, which may be required for burst generation.