Adult mouse sensory neurons on microelectrode arrays exhibit increased spontaneous and stimulus-evoked activity in the presence of interleukin-6

Abstract

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.

Assessment of Spontaneous Neuronal ActivityIn VitroUsing Multi-Well Multi-Electrode Arrays: Implications for Assay Development

“…Assessing several methodological aspects of MEA experiments, many of the commonly reported methods appear adequate for estimating firing activity within in vitro neuronal cultures, while other elements of MEA experimental design and analysis have either been underreported in the literature or investigators have employed techniques that may be inappropriate for the task. Specifically, recording duration of 20 min or longer as reported by several studies (Kuperstein et al, 2010;McConnell et al, 2012;Vincent et al, 2013;Slomowitz et al, 2015;Black et al, 2018;Feng et al, 2018), appear sufficiently long to capture the activity in individual arrays with a high degree of reproducibility. Similarly, the common practice of performing experiments with primary cultures that have been aged two to three weeks is reasonable, since the primary rat cortical cultures examined here showed a high degree of spontaneous firing by the end of the first week that plateaued through these time frames.…”

“…Similarly, the common practice of performing experiments with primary cultures that have been aged two to three weeks is reasonable, since the primary rat cortical cultures examined here showed a high degree of spontaneous firing by the end of the first week that plateaued through these time frames. Conversely, the highly skewed distribution of firing frequencies across both individual electrodes and entire arrays has been largely omitted from reports or accounted for in presentation of results, with a limited number of noted exceptions (Biffi et al, 2013;Vincent et al, 2013;Slomowitz et al, 2015;Wainger et al, 2015;Black et al, 2018). Despite this observation, several of the reviewed publications used parametric statistical tests such as Student's t test and ANOVA for assessing differ- Table indicates number of spike clusters, and spike events available for analysis in each condition.…”

“…The distribution of array firing frequencies does form a symmetrical, approximately normal distribution following transformation of the values by log 10 , as shown in Figure 2B. Other studies have used log 10 transformation in the reporting of MEA data, including Wainger et al (2014) and Black et al (2018), who did so to compare firing frequencies of individual electrodes, as well as Slomowitz et al (2015), to compare the activity of individual neurons following spike sorting. Given the log-normal behavior of the observed frequencies, log 10 Hz rather than Hz appears to be a more appropriate metric to describe the spontaneous firing activity of neuronal cultures, especially to enable analysis using parametric methods.…”

Herpesviral Latency—Common Themes

“…Changes in core body temperature, such as hyperthermia caused by fever, as well as hypothermia also facilitate the reactivation of HSV [241]. Prostaglandin PGE2 and cytokines such as IL-6 that are produced during high fever can directly stimulate neurons with latent HSV reservoirs [242][243][244]. Persistent psychological stress and fatigue are often responsible for re-emerging herpes symptoms, which partially correlates with reduced CD8+ T cell surveillance of latently infected neurons (Figure 1) [245].…”

Strength in diversity: Understanding the pathways to herpes simplex virus reactivation

“…Additional cytokines and prostaglandins produced during fever also have the capability of acting directly on neurons (Poon et al, 2015). IL-6 and prostaglandins can both trigger sensory neuron hyperstimulation (Black et al, 2018), which, at least for prostaglandins, appears to occur as a result of increased levels of cAMP (Emery et al, 2011). As mentioned previously, elevated cAMP levels can trigger HSV reactivation.…”