Systematic comparison of GTN- and CGRP-induced migraine-like behaviors across sex and strain in mice

Preclinical migraine research relies on a number of experimental triggers and behavioral assays, often applied under heterogeneous conditions that hinder direct comparison across studies. In addition, small effect sizes frequently require large group sizes, reducing feasibility for pharmacological screening and conflicting with the 3Rs (Replace, Reduce, Refine) principle. Here, we performed a systematic and parallel comparison of two widely used migraine triggers, glyceryl trinitrate (GTN) and calcitonin gene–related peptide (CGRP), across sex and strain in mice. Male and female CD-1 and C57BL/6 J mice were assessed using three commonly employed migraine-relevant readouts: periorbital mechanical allodynia (PMA), light aversion, and facial grimace scoring. All behavioral assessments were conducted sequentially in the same animals using a standardized protocol, with effect sizes deliberately set to be large enough to allow detection with moderate group sizes and to preserve suitability for pharmacological reversal. GTN and CGRP produced partially overlapping behavioral profiles that were assay-, strain-, and sex-dependent. While both triggers reliably induced PMA, their effects on light aversion and grimace score diverged across strains and sexes. Together, this study provides a side-by-side evaluation of migraine-like behavioral endpoints under controlled experimental conditions, highlighting how trigger selection, strain, sex, and behavioral assay critically influence outcomes. The data support a standardized, multi-endpoint approach for preclinical migraine studies aimed at pharmacological screening while maximizing information yield and adhering to the 3Rs principle.