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In this report, we describe the development of a modified adeno‐associated virus (AAV) capsid and promoter for transduction of retinal ON‐bipolar cells. The bipolar cells, which are post‐synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON‐bipolar cells light‐sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON‐bipolar cells. This evokes high‐frequency (~100 Hz) spiking responses in ganglion cells of previously blind, rd1 , mice. Our vector is a promising vehicle for further development toward potential clinical use. Synopsis An engineered genetically modified adeno‐associated virus is shown here to efficiently and specifically drive the optogenetic molecule channelrhodopsin‐2 in ON‐bipolar cells, rendering them light sensitive and restoring retinal function in blind rd1 mice. A synthetic AAV capsid and modified bipolar‐cell specific promoter were developed to enhance transgene expression in retinal bipolar cells. The new virus transduced at least 59% of ON‐bipolar cells in mouse retina. In the blind rd1 mouse the virus was used to drive expression of optogenetic channels at levels high enough to elicit strong and robust spiking responses from the ganglion cells. This new virus‐promoter combination is thus presented as a candidate vector for clinical intervention in advanced forms of retinal degeneration. Graphical Abstract An engineered genetically modified adeno‐associated virus is shown here to efficiently and specifically drive the optogenetic molecule channelrhodopsin‐2 in ON‐bipolar cells, rendering them light sensitive and restoring retinal function in blind rd1 mice.

Bioelectronic medicine is emerging as a powerful approach for restoring lost endogenous functions and addressing life‐altering maladies such as cardiac disorders. Systems that incorporate both modulation of cellular function and recording capabilities can enhance the utility of these approaches and their customization to the needs of each patient. Here we report an integrated optogenetic and bioelectronic platform for stable and long‐term stimulation and monitoring of cardiomyocyte function in vitro. Optical inputs are achieved through the expression of a photoactivatable adenylyl cyclase, that when irradiated with blue light causes a dose‐dependent and time‐limited increase in the secondary messenger cyclic adenosine monophosphate with subsequent rise in autonomous cardiomyocyte beating rate. Bioelectronic readouts are obtained through a multi‐electrode array that measures real‐time electrophysiological responses at 32 spatially‐distinct locations. Irradiation at 27 µW mm−2 results in a 14% elevation of the beating rate within 20–25 min, which remains stable for at least 2 h. The beating rate can be cycled through “on” and “off” light states, and its magnitude is a monotonic function of irradiation intensity. The integrated platform can be extended to stretchable and flexible substrates, and can open new avenues in bioelectronic medicine, including closed‐loop systems for cardiac regulation and intervention, for example, in the context of arrythmias. This study reports an integrated platform for simultaneous modulation and monitoring of cardiomyocyte function in vitro. Optical inputs are achieved through expression of a photoactivatable adenylyl cyclase, that when irradiated causes a reversible increase in beating rate. Bioelectronic outputs are achieved with a multi‐electrode array, which can record the beating rate and wavefront propagation characteristics with high spatiotemporal resolution.

Paired associative stimulation is a relatively new non-invasive brain stimulation technique that combines transcranial magnetic stimulation and peripheral nerve stimulation. The effects of paired associative stimulation on the excitability of the cerebral cortex can vary according to the time interval between the transcranial magnetic stimulation and peripheral nerve stimulation. We established a model of cerebral ischemia in rats via transient middle cerebral artery occlusion. We administered paired associative stimulation with a frequency of 0.05 Hz 90 times over 4 weeks. We then evaluated spatial learning and memory using the Morris water maze. Changes in the cerebral ultra-structure and synaptic plasticity were assessed via transmission electron microscopy and a 64-channel multi-electrode array. We measured mRNA and protein expression levels of brain-derived neurotrophic factor and N-methyl-D-aspartate receptor 1 in the hippocampus using a real-time polymerase chain reaction and western blot assay. Paired associative stimulation treatment significantly improved learning and memory in rats subjected to cerebral ischemia. The ultra-structures of synapses in the CA1 area of the hippocampus in rats subjected to cerebral ischemia were restored by paired associative stimulation. Long-term potentiation at synapses in the CA3 and CA1 regions of the hippocampus was enhanced as well. The protein and mRNA expression of brain-derived neurotrophic factor and N-methyl-D-aspartate receptor 1 increased after paired associative stimulation treatment. These data indicate that paired associative stimulation can protect cognition after cerebral ischemia. The observed effect may be mediated by increases in the mRNA and protein expression of brain-derived neurotrophic factor and N-methyl-D-aspartate receptor 1, and by enhanced synaptic plasticity in the CA1 area of the hippocampus. The animal experiments were approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science & Technology, China (approval No. TJ-A20151102) on July 11, 2015.

Because of their close interaction with neuronal physiology, astrocytes can modulate brain function in multiple ways. Here, we demonstrate a yet unknown astrocytic phenomenon: Astrocytes cultured on microelectrode arrays (MEAs) exhibited extracellular voltage fluctuations in a broad frequency spectrum (100–600 Hz) after electrical stimulation. These aperiodic high‐frequency oscillations (HFOs) could last several seconds and did not spread across the MEA. The voltage‐gated calcium channel antagonist cilnidipine dose‐dependently decreased the power of the oscillations. While intracellular calcium was pivotal, incubation with bafilomycin A1 showed that vesicular release of transmitters played only a minor role in the emergence of HFOs. Gap junctions and volume‐regulated anionic channels had just as little functional impact, which was demonstrated by the addition of carbenoxolone (100 μmol/L) and NPPB (100 μmol/L). Hyperpolarization with low potassium in the extracellular solution (2 mmol/L) dramatically raised oscillation power. A similar effect was seen when we added extra sodium (+50 mmol/L) or if we replaced it with NMDG+ (50 mmol/L). The purinergic receptor antagonist PPADS suppressed the oscillation power, while the agonist ATP (100 μmol/L) had only an increasing effect when the bath solution pH was slightly lowered to pH 7.2. From these observations, we conclude that astrocytic voltage oscillations are triggered by activation of voltage‐gated calcium channels and driven by a downstream influx of cations through channels that are permeable for large ions such as NMDG+. Most likely candidates are subtypes of pore‐forming P2X channels with a low affinity for ATP. Astrocytes cultured on microelectrode arrays exhibited robust high‐frequency (100–600 Hz) extracellular voltage fluctuations after electrical stimulation. These voltage oscillations had amplitudes in the range of extracellularly recorded action potentials (~50 μV), were locally restricted to the site of the stimulus, lasted several seconds, and were highly susceptible to changes in the extracellular medium, e.g., [Ca2+]e and pH. The oscillations were triggered by activation of voltage‐gated Ca2+‐channels and driven by cation influx through channels with large membrane pores, presumably P2X channels.

[This corrects the article DOI: 10.3389/fncel.2023.1287089.].

Brain organoids, or cerebral organoids, have become widely used to study the human brain in vitro . As pluripotent stem cell-derived structures capable of self-organization and recapitulation of physiological cell types and architecture, brain organoids bridge the gap between relatively simple two-dimensional human cell cultures and non-human animal models. This allows for high complexity and physiological relevance in a controlled in vitro setting, opening the door for a variety of applications including development and disease modeling and high-throughput screening. While technologies such as single cell sequencing have led to significant advances in brain organoid characterization and understanding, improved functional analysis (especially electrophysiology) is needed to realize the full potential of brain organoids. In this review, we highlight key technologies for brain organoid development and characterization, then discuss current electrophysiological methods for brain organoid analysis. While electrophysiological approaches have improved rapidly for two-dimensional cultures, only in the past several years have advances been made to overcome limitations posed by the three-dimensionality of brain organoids. Here, we review major advances in electrophysiological technologies and analytical methods with a focus on advances with applicability for brain organoid analysis.

Neural lamination is a common feature of the CNS, with several subcellular structures, such as adherens junctions (AJs), playing a role in this process. The retina is also heavily laminated, but it remains unclear how laminar formation impacts retinal cell morphology, synapse integrity, and overall retinal function. In this study, we demonstrate that the loss of afadin, a key component of AJs, in mice leads to significant pathological changes. These include the disruption of outer retinal lamination and a notable decrease as well as mislocalization of photoreceptors, their outer segments, and photoreceptor synapses. Interestingly, despite these severe impairments, we recorded small local field potentials, including the a- and b-waves. We also classified retinal ganglion cells (RGCs) into ON, ON-OFF, and OFF types based on their firing patterns in response to light stimuli. Additionally, we successfully characterized the receptive fields of certain RGCs. Overall, these findings provide evidence that retinal circuit function can be partially preserved even when there are significant disruptions in both retinal lamination and photoreceptor synapses. Our results indicate that retinas with severely altered morphology still retain some capacity to process light stimuli.

The development of in vitro networks composed of distinct but interacting neuronal sub-populations (clusters) has advanced the study of emergent behaviors in neural networks as individual functional units. In a previous work, we developed an in vitro model of a network formed by four mutually interconnected clusters of rat embryonic cortical neurons cultured on multi-electrode arrays (MEA), where we observed recurring, spatially and temporally structured activation sequences. In the present study, we examined the effects of NMDAR blockade (MK-801) to modulate such temporal patterns. We found that MK-801 reduced the overall excitability of the network and disrupted the diversity of repeated activation patterns, while paradoxically increasing their temporal persistence. This led the network to transition from a dynamic regime characterized by frequent and flexible repetitions to one dominated by fewer, more stable and enduring activation motifs. Functional connectivity analysis further revealed a selective weakening of inter-cluster links alongside a strengthening of intra-cluster connections. This reorganization likely explains the observed reduction in activity propagation between clusters and the simultaneous emergence of more persistent activation sequences among clusters. Data suggest that clustered neural networks serve as semi-autonomous modules, capable of sustaining internal dynamics even under diminished excitatory drive. The stable repetition of activation patterns may reflect a functional “closure” within clusters, forming self-sustained loops that enable the reactivation of previously formed motifs. From a neuroengineering perspective, this model provides a versatile platform to explore how spatiotemporal neural dynamics underpin inter-network communication, information encoding, and complex cortical functions.

While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity in vivo , functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a “bottom-up” 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.

At least 20 distinct retinal ganglion cell (RGC) types have been identified morphologically in the primate retina, but our understanding of the distinctive visual messages they send to various targets in the brain remains limited, particularly for naturalistic stimuli. Here, we use large-scale multi-electrode recordings to examine how multiple functionally distinct RGC types in the macaque retina respond to flashed natural images. Responses to white noise visual stimulation were used to functionally identify 936 RGCs of 12 types in three recordings. Each cell type was confirmed by the mosaic organization of receptive fields, and seven cell types were cross-identified between recordings. Responses to thousands of natural images were used to examine the average firing rate kinetics in each RGC type as well as the repertoire of distinct firing patterns that each type produced. The average response across images was highly stereotyped for cells of each type and distinct for cells of different types. The responses to natural images more clearly distinguished certain cell types than did the response to white noise stimulation. Moreover, the full repertoires of firing patterns produced by different cell types, assessed by their latency and duration, were largely distinct in most cases and in some cases non-overlapping. Together these data provide an overview of the diversity of RGC signals transmitted from the primate retina to the brain in natural viewing conditions.