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Microglia are highly adaptable innate immune cells that rapidly respond to damage signals in the brain through adoption of a reactive phenotype and production of defensive inflammatory cytokines. Microglia express a distinct transcriptome, encoding receptors that allow them to dynamically respond to pathogens, damage signals, and cellular debris. Expression of one such receptor, the microglia-specific purinergic receptor P2ry12, is known to be downregulated in reactive microglia. Here, we explore the microglial response to purinergic damage signals in reactive microglia in the TMEV mouse model of viral brain infection and temporal lobe epilepsy. Using two-photon calcium imaging in acute hippocampal brain slices, we found that the ability of microglia to detect damage signals, engage calcium signaling pathways, and chemoattract towards laser-induced tissue damage was dramatically reduced during the peak period of seizures, cytokine production, and infection. Using combined RNAscope in situ hybridization and immunohistochemistry, we found that during this same stage of heightened infection and seizures, microglial P2ry12 expression was reduced, while the pro-inflammatory cytokine TNF-a expression was upregulated in microglia, suggesting that the depressed ability of microglia to respond to new damage signals via P2ry12 occurs during the time when local elevated cytokine production contributes to seizure generation following infection. Therefore, changes in microglial purinergic receptors during infection likely limit the ability of reactive microglia to respond to new threats in the CNS and locally contain the scale of the innate immune response in the brain.

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Electrical activity recordings are critical for evaluating and understanding brain function. We present a novel wireless, implantable, and battery-free device, namely the Wireless Neurosensing System (WiNS), and for the first time, we evaluate multichannel recording capabilities in vivo. For a preliminary evaluation, we performed a benchtop experiment with emulated sinusoidal signals of varying amplitude and frequency, representative of neuronal activity. We later performed and analyzed electrocortical recordings in rats of evoked somatosensory activity in response to three paradigms: hind/fore limb and whisker stimulation. Wired recordings were used for comparison and validation of WiNS. We found that through the channel multiplexing element of WiNS, it is possible to perform multichannel recordings with a maximum sampling rate of ∼10 kHz for a total of eight channels. This sampling rate is appropriate for monitoring the full range of neuronal signals of interest, from low-frequency population recordings of electrocorticography and local field potentials to high-frequency individual neuronal spike recordings. These in vivo experiments demonstrated that the evoked neuronal activity recorded with WiNS is comparable to that recorded with a wired system under identical circumstances. Analysis of critical parameters for interpreting the somatosensory evoked activity showed no statistically significant difference between the parameters obtained by a wired system versus those obtained using WiNS. Therefore, WiNS can match the performance of more invasive recording systems. WiNS is a groundbreaking technology with potential applications throughout neuroscience as it offers a simple alternative to address the pitfalls of battery-powered neuronal implants.

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Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.

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Optogenetic targeting of astrocytes provides a robust experimental model to differentially induce Ca2+ signals in astrocytes in vivo. However, a systematic study quantifying the response of optogenetically modified astrocytes to light is yet to be performed. Here, we propose a novel stochastic model of Ca2+ dynamics in astrocytes that incorporates a light sensitive component-channelrhodopsin 2 (ChR2). Utilizing this model, we investigated the effect of different light stimulation paradigms on cells expressing select variants of ChR2 (wild type, ChETA, and ChRET/TC). Results predict that depending on paradigm specification, astrocytes might undergo drastic changes in their basal Ca2+ level and spiking probability. Furthermore, we performed a global sensitivity analysis to assess the effect of variation in parameters pertinent to the shape of the ChR2 photocurrent on astrocytic Ca2+ dynamics. Results suggest that directing variants towards the first open state of the ChR2 photocycle (o1) enhances spiking activity in astrocytes during optical stimulation. Evaluation of the effect of Ca2+ buffering and coupling coefficient in a network of ChR2-expressing astrocytes demonstrated basal level elevations in the stimulated region and propagation of calcium activity to unstimulated cells. Buffering reduced the diffusion range of Ca2+ within the network, thereby limiting propagation and influencing the activity of astrocytes. Collectively, the framework presented in this study provides valuable information for the selection of light stimulation paradigms that elicit desired astrocytic activity using existing ChR2 constructs, as well as aids in the engineering of future application-oriented optogenetic variants.

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Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+ ) oscillations by incorporating light-sensitive cation channels like Channelrhodopsin-2 (ChR2) offers a promising avenue in influencing the long-term progression of these disorders. However, using adult animals for Ca2+ imaging poses major challenges, including accelerated deterioration of in situ slice health and age- related changes. Additionally, optogenetic preparations necessitate usage of a red-shifted Ca2+ indicator like Rhod-2 AM to avoid overlapping light issues between ChR2 and the Ca2+ indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2-5 months) from a knock-in model expressing Channelrhodopsin-2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod-2 AM to elicit robust Ca2+ response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod-2 AM loading, maintenance of slice health, and Ca2+ imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod-2 AM staining Basic Protocol 2: Image acquisition and analysis.

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Wireless implantable neural interfaces can record high-resolution neuropotentials without constraining patient movement. Existing wireless systems often require intracranial wires to connect implanted electrodes to an external head stage or/and deploy an application-specific integrated circuit (ASIC), which is battery-powered or externally power-transferred, raising safety concerns such as infection, electronics failure, or heat-induced tissue damage. This work presents a biocompatible, flexible, implantable neural recorder capable of wireless acquisition of neuropotentials without wires, batteries, energy harvesting units, or active electronics. The recorder, fabricated on a thin polyimide substrate, features a small footprint of 9 mm × 8 mm × 0.3 mm and is composed of passive electronic components. The absence of active electronics on the device leads to near zero power consumption, inherently avoiding the catastrophic failure of active electronics. We performed both in vitro validation in a tissue-simulating phantom and in vivo validation in an epileptic rat. The fully passive wireless recorder was implanted under rat scalp to measure neuropotentials from its contact electrodes. The implanted wireless recorder demonstrated its capability to capture low voltage neuropotentials, including somatosensory evoked potentials (SSEPs), and interictal epileptiform discharges (IEDs). Wirelessly recorded SSEP and IED signals were directly compared to those from wired electrodes to demonstrate the efficacy of the wireless data. In addition, a convoluted neural network-based machine learning algorithm successfully achieved IED signal recognition accuracy as high as 100 and 91% in wired and wireless IED data, respectively. These results strongly support the fully passive wireless neural recorder's capability to measure neuropotentials as low as tens of microvolts. With further improvement, the recorder system presented in this work may find wide applications in future brain machine interface systems.

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