αCGRP and βCGRP are co-expressed in the trigeminal ganglia

Calcitonin gene-related peptide (CGRP) is a neuropeptide involved in pain transmission and migraine pathogenesis. CGRP has two major isoforms, αCGRP and βCGRP, which differ by three amino acids in humans and mice. αCGRP is highly expressed in the trigeminal ganglia (TG) and has a well-established role in pain sensitisation and migraine. In contrast, βCGRP expression is poorly defined and is often reported to be primarily expressed in the enteric nervous system, with limited relevance to migraine. Therefore, this study aimed to compare the relative distribution of αCGRP and βCGRP in the mouse TG.

Detection of the mouse CGRP isoforms by anti-CGRP antibodies was tested by dot blot. Wildtype (WT) and αCGRP knockout (KO) adult mouse TG (C57BL/6J background, WT 3M/1F, KO 4F) were immunostained for CGRP relative to neuronal markers (β tubulin III and NF200) and the calcitonin receptor (CTR). RNA-fluorescent in situ hybridisation (RNA-FISH) probes against αCGRP (Calca) and βCGRP (Calcb) mRNA were validated in transfected HEK293S cells. These probes were then used to examine isoform expression in WT adult mouse TG (C57BL/6J, 2F/3M).

All anti-CGRP antibodies tested detected both isoforms of CGRP. CGRP-like immunoreactivity was present in WT and αCGRP KO mouse TG in neuronal cell bodies, often colocalising with CTR. However, compared to the WT mice, CGRP immunoreactivity in the αCGRP KO TG was of a lower fluorescence intensity and lacked pearl-like fibre immunoreactivity. The RNA-FISH probes were specific to each isoform and indicated that αCGRP and βCGRP mRNA were often present in the same neuronal cell bodies, although some neuronal cell bodies only expressed one CGRP isoform.

βCGRP is expressed in mouse TG neurons, suggesting it plays a role in neuromodulation and nociception, alongside αCGRP. Both isoforms may be important for mediating CGRP's pathogenic activity in migraine.