Introduction: Visceral pain, originating from the body's internal organs, impacts up to one in four people at any time [1]. It is most reported within the abdominal area, where many patients are diagnosed with gastrointestinal disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), which together affect around 20% of the population [1]. The etiology of these disorders is poorly understood, but current hypotheses suggest abdominal pain may result from gut residing enteric neurons becoming hypersensitized or over-activated, leading to unnecessary pain signaling. Microelectrode Arrays (MEAs) are useful for measuring neuron response to various stimuli. Here, we utilize MEAs to measure activity of sensory TRPV1+ enteric neuron activation, the cells involved in pain transmission in the gut, in response to exogenous gamma-aminobutyric acid (GABA). GABA is of interest due to its role as an inhibitory neurotransmitter. It is also produced by the microbiome and has been found to be altered in patients with IBS [2]. We hypothesize that GABA may modulate TRPV1 channel activation of sensory neurons in the gut because of the close proximity of GABA and TRPV1 receptors, leading to a receptor complex and increased neuron firing [3].
Materials and Methods: Enteric neurons were isolated from the myenteric plexus of mixed-sex neonatal rat pups. After isolating, neurons were seeded onto MEAs (Multichannel Systems) at 500,000 cells per MEA. Before seeding, MEAs were plasma treated and coated with poly-d-lysine and laminin to support cell adhesion. The MEAs used contained 60, 30 micrometer diameter electrodes made of titanium nitride. Recordings were performed after two weeks of neuron culture to allow for maturation. Capsaicin (1 uM) and GABA (100 uM) were dosed in a bolus. Recordings were collected via MC_Rack software and analyzed using MATLAB and MEAnalyzer [4].
Results, Conclusions, and Discussions: Population firing patterns were obtained from complete enteric neuron cultures with glia. To identify if the cultures contained TRPV1+ cells, the TRPV1 agonist, capsaicin, was dosed at 1 uM, and a significant increase in overall mean firing rate was observed from 0.116 Hz to 0.216 Hz, as well as the number of active electrodes, defined as detecting at least one spike (Fig. 1A and 1B). After confirming sensory neuron presence, GABA (100 uM) was dosed into capsaicin stimulated cultures to assess the impact on the firing rate of sensory enteric neurons. The average firing frequency increased significantly 9-fold (0.085 Hz to 0.791 Hz), highlighting an excitatory role of GABA on enteric neuron populations (Fig. 1C). Additional confirmation of TRPV1+ cells was performed using immunocytochemistry (Fig. 1D). In progress work includes using GABAa and GABAb receptor subtype antagonists, bicuculline and CGP55845 respectively, in parallel with capsaicin to determine if blocking GABA modulates sensory neuron activation of TRPV1. Preliminary data indicates GABAb receptors may be serving a prominent role over GABAa in enteric neurons, causing an excitatory impact rather than the usual inhibitory role of GABA in the central nervous system. Assessing the role of the inflammatory compound, lipopolysaccharide (LPS) on enteric neuron firing, and GABA’s attenuation of that effect is also in progress.
This work contributes to a better understanding of enteric neuron function and exogenous neurotransmitters' role in neuron activation. Our findings suggest microbial metabolites like GABA may contribute to pain signaling in the gut, highlighting the need to understand these cellular-metabolite interactions better.
Acknowledgements (Optional): : We thank Northeastern University and the NIH and NSF CAREER for funding.
References (Optional): : [1] Rani, R.A., et al. (2016). Intestinal research. 14(4): p. 297. [2] Chen, M., et al. (2022). Frontiers in Endocrinology, 13, 23. [3] Hanack, CJ., et al. (2015), 160(4), 759-770. [4] Dastgheyb, R. M., et al. (2020). Neuroinformatics, 18, 163-179.
