Following inflammation or injury, sensory neurons located in the dorsal root ganglia (DRG) may exhibit increased spontaneous and/or stimulus-evoked activity, contributing to chronic pain. Current treatment options for peripherally mediated chronic pain are highly limited, driving the development of cell- or tissue-based phenotypic (function-based) screening assays for peripheral analgesic and mechanistic lead discovery. Extant assays are often limited by throughput, content, use of tumorigenic cell lines, or tissue sources from immature developmental stages (i.e., embryonic or postnatal). Here, we describe a protocol for culturing adult mouse DRG neurons on substrate-integrated multiwell microelectrode arrays (MEAs). This approach enables multiplexed measurements of spontaneous as well as stimulus-evoked extracellular action potentials from large populations of cells. The DRG cultures exhibit stable spontaneous activity from 9 to 21 days in vitro. Activity is readily evoked by known chemical and physical agonists of sensory neuron activity such as capsaicin, bradykinin, PGE, heat, and electrical field stimulation. Most importantly, we demonstrate that both spontaneous and stimulus-evoked activity may be potentiated by incubation with the inflammatory cytokine interleukin-6 (IL-6). Acute responsiveness to IL-6 is inhibited by treatment with a MAPK-interacting kinase 1/2 inhibitor, cercosporamide. In total, these findings suggest that adult mouse DRG neurons on multiwell MEAs are applicable to ongoing efforts to discover peripheral analgesic and their mechanisms of action. NEW & NOTEWORTHY This work describes methodologies for culturing spontaneously active adult mouse dorsal root ganglia (DRG) sensory neurons on microelectrode arrays. We characterize spontaneous and stimulus-evoked adult DRG activity over durations consistent with pharmacological interventions. Furthermore, persistent hyperexcitability could be induced by incubation with inflammatory cytokine IL-6 and attenuated with cercosporamide, an inhibitor of the IL-6 sensitization pathway. This constitutes a more physiologically relevant, moderate-throughput in vitro model for peripheral analgesic screening as well as mechanistic lead discovery.

We have characterized the in vitro and in vivo extracellular neural recording and stimulation properties of ruthenium oxide (RuOx) based microelectrodes. Cytotoxicity and functional neurotoxicity assays were carried out to confirm the in vitro biocompatibility of RuOx. Material extract assays, in accordance to ISO protocol "10993-5: Biological evaluation of medical devices", revealed no significant effect on neuronal cell viability or the functional activity of cortical networks. In vitro microelectrode arrays (MEAs), with indium tin oxide (ITO) sites modified with sputtered iridium oxide (IrOx) and RuOx in a single array, were developed for a direct comparison of electrochemical and recording performance of RuOx to ITO and IrOx deposited microelectrode sites. The impedance of the RuOx-coated electrodes measured by electrochemical impedance spectroscopy was notably lower than that of ITO electrodes, resulting in robust extracellular recordings from cortical networks in vitro. We found comparable signal-to-noise ratios (SNRs) for RuOx and IrOx, both significantly higher than the SNR for ITO. RuOx-based MEAs were also fabricated and implanted in the rat motor cortex to demonstrate manufacturability of the RuOx processing and acute recording capabilities in vivo. We observed single-unit extracellular action potentials with a SNR >22, representing a first step for neurophysiological recordings in vivo with RuOx based microelectrodes.

Polymer-based biomedical electronics provide a tunable platform to interact with nervous tissue both in vitro and in vivo. Ultimately, the ability to control functional properties of neural interfaces may provide important advantages to study the nervous system or to restore function in patients with neurodegenerative disorders. Liquid crystal elastomers (LCEs) are a class of smart materials that reversibly change shape when exposed to a variety of stimuli. Our interest in LCEs is based on leveraging this shape change to deploy electrode sites beyond the tissue regions exhibiting inflammation associated with chronic implantation. As a first step, we demonstrate that LCEs are cellular compatible materials that can be used as substrates for fabricating microelectrode arrays (MEAs) capable of recording single unit activity in vitro. Extracts from LCEs are non-cytotoxic (>70% normalized percent viability), as determined in accordance to ISO protocol 10993-5 using fibroblasts and primary murine cortical neurons. LCEs are also not functionally neurotoxic as determined by exposing cortical neurons cultured on conventional microelectrode arrays to LCE extract for 48 h. Microelectrode arrays fabricated on LCEs are stable, as determined by electrochemical impedance spectroscopy. Examination of the impedance and phase at 1 kHz, a frequency associated with single unit recording, showed results well within range of electrophysiological recordings over 30 days of monitoring in phosphate-buffered saline (PBS). Moreover, the LCE arrays are shown to support viable cortical neuronal cultures over 27 days in vitro and to enable recording of prominent extracellular biopotentials comparable to those achieved with conventional commercially-available microelectrode arrays.

The tolerance, abuse, and potential exacerbation associated with classical chronic pain medications such as opioids creates a need for alternative therapeutics. Phenotypic screening provides a complementary approach to traditional target-based drug discovery. Profiling cellular phenotypes enables quantification of physiologically relevant traits central to a disease pathology without prior identification of a specific drug target. For complex disorders such as chronic pain, which likely involves many molecular targets, this approach may identify novel treatments. Sensory neurons, termed nociceptors, are derived from dorsal root ganglia (DRG) and can undergo changes in membrane excitability during chronic pain. In this review, we describe phenotypic screening paradigms that make use of nociceptor electrophysiology. The purpose of this paper is to review the bioelectrical behavior of DRG neurons, signaling complexity in sensory neurons, various sensory neuron models, assays for bioelectrical behavior, and emerging efforts to leverage microfabrication and microfluidics for assay development. We discuss limitations and advantages of these various approaches and offer perspectives on opportunities for future development.

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