Development of an ex Vivo Method for Multi-unit Recording of Microbiota-Colonic-Neural Signaling in Real Time

Abstract

Background and Objectives: Bidirectional signaling between the gastrointestinal tract and the brain is vital for maintaining whole-body homeostasis. Moreover, emerging evidence implicates vagal afferent signaling in the modulation of host physiology by microbes, which are most abundant in the colon. This study aims to optimize and advance dissection and recording techniques to facilitate real-time recordings of afferent neural signals originating in the distal colon.New Protocol: This paper describes a dissection technique, which facilitates extracellular electrophysiological recordings from visceral pelvic, spinal and vagal afferent neurons in response to stimulation of the distal colon.Examples of Application: Focal application of 75 mM KCl to a section of distal colon with exposed submucosal or myenteric nerve cell bodies and sensory nerve endings evoked activity in the superior mesenteric plexus and the vagal nerve. Noradrenaline stimulated nerve activity in the superior mesenteric plexus, whereas application of carbachol stimulated vagal nerve activity. Exposure of an ex vivo section of distal colon with an intact colonic mucosa to peptidoglycan, but not lipopolysaccharide, evoked vagal nerve firing.Discussion: Previous studies have recorded vagal signaling evoked by bacteria in the small intestine. The technical advances of this dissection and recording technique facilitates recording of afferent nerve signals evoked in extrinsic sensory pathways by neuromodulatory reagents applied to the distal colon. Moreover, we have demonstrated vagal afferent activation evoked by bacterial products applied to the distal colonic mucosa. This protocol may contribute to our understanding of functional bowel disorders where gut-brain communication is dysfunctional, and facilitate real-time interrogation of microbiota-gut-brain signaling.

GHSR‐1 agonist sensitizes rat colonic intrinsic and extrinsic neurons to exendin‐4: A role in the manifestation of postprandial gastrointestinal symptoms in irritable bowel syndrome?

“…This dissection has been described previously in detail . In brief, a vertical abdominal incision was made below the sternum to expose the intestine with intact peripheral nerves.…”

Opportunities and Challenges for Single-Unit Recordings from Enteric Neurons in Awake Animals

“…However, several limitations exist for all excised tissue preparations, including, most notably, the lack of peripheral innervation and extrinsic circuitry. In some ex vivo preparations, peripheral fiber recordings are possible, but they lack extrinsic circuits in the central nervous system [ 26 , 27 ]. The limitations of ex vivo preparations can be addressed by studying the enteric nervous system in live animal models.…”

Microbiota-gut-brain axis: enteroendocrine cells and the enteric nervous system form an interface between the microbiota and the central nervous system

“…In the previous section, we have discussed the contribution of TLRs in the microbiota-gut-brain axis. Recently, electrophysiological recordings from the vagal afferent pathway stimulated by TLRs in the intestine have been described; the application of peptidoglycan (a ligand for TLR2), a major component of the wall of gram-positive bacteria, to rat distal colonic mucosa resulted in increased nerve firing in the vagus (Buckley et al 2018). However, LPS (a ligand for TLR4) had no effect on vagal nerve activity, although TLR4, a selective receptor for LPS, was expressed in the vagal afferent neurons in the NG (Hosoi et al 2005;Reardon et al 2018).…”