CGRPα-Expressing Sensory Neurons Respond to Stimuli that Evoke Sensations of Pain and Itch

Abstract

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10–50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP+ neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP+ cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid—reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP+ DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP+ neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα+ DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8+/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.

Identification of different functional types of spinal afferent neurons innervating the mouse large intestine using a novel CGRPα transgenic reporter mouse

“…This may represent cells that express low levels of CGRP␣, with little or no concomitant CGRP␤. A similar population of cells was reported by McCoy et al (38) in CGRP␣-GFP mice, suggesting that, in some cells, fluorescent protein expression is a more sensitive reporter than CGRP immunoreactivity.…”

“…A mouse expressing green fluorescent protein (GFP) under the CGRP␣ promoter has recently been generated (38). However, in order that intracellular carboxyfluorescein dye filling of sensory neurons could be visualized in live tissue from CGRP␣ reporter mice without overlapping emission spectra, we required a reporter different from GFP.…”

Validation of ion channel targets

“…By further crossing these mice with an Advillin-Cre strain, Advillin being a sensory neuron specific protein, this technique was used to define the specific contribution of CGRPa expressing sensory neurons in pain signaling. 16,17 In these mice peripheral responses to noxious heat but not cold or mechanical stimuli was attenuated. 18 The authors were then able to study the GFP labeled CGRPa neurons in ex vivo experiments to build a mechanistic understanding of this phenotype and understand the ion channels linked to the pathway and the phenotype.…”

TRP Channels and Pruritus

“…TRPV1+/CGRP+ DRG neurons very limitedly expressed TRPM8 and did not respond to icilin, although ca. 10-50% of them could be activated to capsaicin, mustard oil, menthol, acidic pH, ATP as well as by histamine and chloriquine [339]. Pruritogenic pro-inflammatory mediators such as e.g.…”