Characterisation of sensory neurons innervating murine skeletal muscle, focusing on one class of Group III afferents

Author: Rochelle Peterson

Peterson, Rochelle, 2019 Characterisation of sensory neurons innervating murine skeletal muscle, focusing on one class of Group III afferents, Flinders University, College of Medicine and Public Health

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This study aimed to identify and characterise group III and IV sensory nerve endings within skeletal muscle. This population of neurons have terminals in the skeletal muscle and cell bodies in dorsal root ganglia (DRG). They are part of neural pathways that convey muscle work, pain, and fatigue. These sensory endings comprise a mixture of fine myelinated and unmyelinated axons with variable branching patterns. Until now it has been difficult to relate their morphology to their functional characteristics, with the exception of large group I and II afferent endings of muscle spindles and Golgi tendon organs.

An isolated preparation of mouse abdominal muscles was developed. A combination of techniques applied previously in preparations of visceral organs was used, allowing correlation of form and function of small diameter endings for the first time. We validated this preparation by using it to correlate the morphological and functional characteristic of the well-described muscle afferent endings in muscles spindles. This confirmed that these techniques could relate the morphological and functional properties a single class of well-characterised skeletal muscle afferents in detail.

Electrophysiological recordings from nerve trunks, innervating the muscles of the abdominal wall, enabled detailed studies of a distinctive class of group III muscle afferents. These afferents had discrete fields of innervation, were mechanically- sensitive, with distinctive saturating response to both punctate pressure and stretch. They were metabo-sensitive, activated by a known muscle metabolites mix but were insensitive to the TRPV1 agonist, capsaicin. Electrophysiological studies combined with anterograde labelling revealed, for the first time, that these group III muscle afferents have terminals located within the connective tissue between the muscle


layers. Additional immunohistochemical labelling for calcitonin gene related peptide (CGRP), demonstrated these were not CGRP immunoreactive.

Targeted extracellular recordings from these identified afferents, tentatively named CT3 afferents (group III afferents with endings in connective tissue), investigated responses to lactate. We showed lactate can directly excite CT3 afferents, but not all muscle sensory neurons. Real-time PCR demonstrated that mRNA for the G-protein coupled lactate receptor (hydroxyl carboxylic acid receptor 1 or HCAR1) was localised in DRGs containing afferents that innervated abdominal muscles. HCAR1 immunoreactivity was visualised in DRG sensory neurons retrogradely labelled from abdominal muscles. HCAR1 immunoreactivity was present in axons and nerve endings in abdominal muscles, often in CGRP-negative axons; consistent with belonging to CT3 afferents. This suggests that HCAR1 is expressed in a subset of primary afferents that innervate some skeletal muscles. These data are consistent with the novel proposal that lactate activates sensory afferent endings in skeletal muscle via its G-protein receptor, HCAR1.

This work has advanced knowledge of one class of group III muscle afferents (CT3s), revealing a distinctive class of mechanically and metabo-sensitve afferents with terminals in muscle connective tissue. We developed and validated an ex vivo preparation and a combination of techniques, in which different classes of group III and IV muscle afferents can be systematically characterised in detail, physiologically and morphologically. We propose that some muscle afferents, including CT3 afferents, can be directly activated by lactate via HCAR1.

Keywords: Sensory, chemosensitivity, mechanosensitivity, group III, muscle afferents, metabolites

Subject: Medical Science thesis

Thesis type: Doctor of Philosophy
Completed: 2019
School: College of Medicine and Public Health
Supervisor: Simon Brookes