Immunohistochemical analysis of tyrosine hydroxylase, CGRP and TRPV1 in mouse colon myenteric plexus

In the gastrointestinal tract, spinal afferent and sympathetic efferent neurons are key regulators of motility, secretion, and sensation. Strong colocalization of the afferent markers calcitonin gene-related peptide (CGRP) and TRPV1 is established in mouse gut. However, the expression of these markers in sympathetic axons-identifiable by immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme for noradrenaline synthesis- has not yet been characterized in the colon. Using multiple-label immunohistochemistry, we quantified the coexistence of CGRP, TRPV1, and TH in varicose axons in the myenteric plexus of the mouse proximal and mid colon. Approximately half of CGRP-immunoreactive (IR) varicosities were also TRPV1-IR in the proximal (48.5 ± 3.5) and mid colon (50.3 ± 3.5) and the great majority of TRPV1-IR varicosities were CGRP-IR in the proximal (87 ± 1.1) and mid colon (90 ± 3.3). In the proximal and mid colon, TH-IR varicosities were neither CGRP- or TRPV1-IR, and the converse was also true; CGRP- and TRPV1-IR varicosities were not TH-IR. Our observations reveal that while CGRP and TRPV1 expression largely occurs together in axons in the proximal and mid colon, there is a population of CGRP axons that do not express TRPV1. In varicose axons, TH expression is mutually exclusive from expression of CGRP and TRPV1. Together, the results support the notion that CGRP together with TRPV1 and TH serve as respective "neurochemical markers" to distinguish spinal afferents from sympathetic efferents in the murine GI tract.