Cultured Vagal Afferent Neurons as Sensors for Intestinal Effector Molecules

Abstract

The gut–brain axis embodies the bi-directional communication between the gastrointestinal tract and the central nervous system (CNS), where vagal afferent neurons (VANs) serve as sensors for a variety of gut-derived signals. The gut is colonized by a large and diverse population of microorganisms that communicate via small (effector) molecules, which also act on the VAN terminals situated in the gut viscera and consequently influence many CNS processes. However, the convoluted in vivo environment makes it difficult to study the causative impact of the effector molecules on VAN activation or desensitization. Here, we report on a VAN culture and its proof-of-principle demonstration as a cell-based sensor to monitor the influence of gastrointestinal effector molecules on neuronal behavior. We initially compared the effect of surface coatings (poly-L-lysine vs. Matrigel) and culture media composition (serum vs. growth factor supplement) on neurite growth as a surrogate of VAN regeneration following tissue harvesting, where the Matrigel coating, but not the media composition, played a significant role in the increased neurite growth. We then used both live-cell calcium imaging and extracellular electrophysiological recordings to show that the VANs responded to classical effector molecules of endogenous and exogenous origin (cholecystokinin serotonin and capsaicin) in a complex fashion. We expect this study to enable platforms for screening various effector molecules and their influence on VAN activity, assessed by their information-rich electrophysiological fingerprints.

Microfluidic compartmentalization of rat vagal afferent neurons to model gut-brain axis

“…In addition to transport studies, microfluidic VAN compartmentalization allows for studying electrophysiological response due to independent stimulation at the soma and neurite terminals. VANs readily exhibit increased intracellular calcium, captured by fluorescence microscopy, in response to capsaicin and elevated extracellular potassium chloride, as previously reported (Girardi et al 2023 ; Riley et al 2013 ; Rienecker et al 2020 ). Here, we investigated whether isolated exposure on the neurite terminals to these molecules result in an increase in intracellular calcium activity at the soma.…”

“…We have previously shown that acute primary VAN cultures exhibit elevated electrophysiological activity (confirmed by intra-cellular calcium activity and extracellular recordings) when exposed to physiologically-relevant gastrointestinal effector molecules, including capsaicin, serotonin, and cholecystokinin (Girardi et al 2023 ). Here, we expanded on that work to demonstrate implementation of VAN cultures within microfluidic devices, where anatomically-relevant cases can be tested (e.g., retrograde transport or neurite terminal activation).…”

“…S 5 ), suggesting that the internalized CTB diffuses throughout the cell. VANs exhibit significant cellular heterogeneity, which is observed as varying cellular functions such as differential electrophysiological response to distinct effector molecules (Girardi et al 2023 ; Simasko et al 2002 ). It is plausible that the heterogeneity also manifests itself in uptake and transport of proteins, including CTB, thereby resulting in a fraction of cells exhibiting transport of CTB, as also observed in in vivo tracing studies (Wang et al 1998 ; Cailotto et al 2012 ; Cui et al 2022 ).…”

“…Elevated extracellular potassium chloride (KCl, 20 mM) with an equimolar reduction of NaCl was used as a positive control (Rienecker et al 2020 ). Ethanol (same working concentration with capsaicin) was used as the vehicle since other diluents (e.g., DPBS +) do not generally affect cell viability and function (Girardi et al 2023 ).…”

“…Here, we combine microfluidic approaches and vagal afferent neuron culture (Goshi et al 2022 ; Girardi et al 2023 ), to create a tool for studying the role of vagal afferent neurons in GBA while excluding the confounding signals from the circulatory and endocrine systems that are present in vivo. Specifically, we report on a microfluidic device that physically separates the VAN soma and neurite terminals to independently expose them to various stimuli, allowing us to probe the two independently.…”