Sexual dimorphism in cortical excitability: repetitive nitroglycerin unmasks maladaptive spreading depolarization in...

Cortical excitability is maintained by a finely regulated balance between excitatory and inhibitory neurotransmission. This balance is sexually dimorphic and shapes vulnerability to pathological events such as cortical spreading depolarization (CSD), the electrophysiological correlate of migraine aura and a key phenomenon in stroke, traumatic brain injury, and seizures. Although migraine and several CSD-related disorders disproportionately affect women, most preclinical studies rely on acute, male-only paradigms, limiting understanding of how biological sex shapes cortical responses to recurrent migraine-relevant stimuli. Here, we examined whether repetitive exposure to the migraine trigger nitroglycerin (NTG) unmasks sex-specific alterations in cortical excitability and CSD recurrence dynamics.

Male and female C57BL/6J mice received five systemic NTG or vehicle injections every other day to induce sustained sensitization. After the development of mechanical orofacial allodynia, in vivo electrophysiological recordings were performed to quantify CSD frequency, induction latency, and waveform characteristics. Generalized Poisson and mixed-effects models were used to assess the effects of sex, treatment, and their interaction, with model selection based on likelihood ratio tests and residual diagnostics performed using the DHARMa package.

Repetitive NTG induced progressive mechanical allodynia in both sexes (χ²(2) = 110.05, p < 0.001). In contrast, CSD metrics revealed marked sexual dimorphism in the adaptive response to repeated NTG stimulation. NTG increased CSD frequency by 41% in females (IRR [95% CI] = 1.41 [1.15-1.73], p = 0.001) but not in males (sex × group: p = 0.043). NTG also reduced first CSD induction latency (χ²(1) = 4.76, p = 0.029) and attenuated the prolongation of latency across successive CSD events (χ²(1) = 4.54, p = 0.033) selectively in females indicating sustained cortical hyperexcitability. CSD amplitude and duration remained unchanged across groups.

These findings demonstrate that repetitive NTG exposure unmasks a female-specific failure of cortical homeostatic adaptation to recurrent noxious stimuli in the context of CSD-related readouts. This maladaptive response is consistent with the involvement of sex-dependent regulatory pathways, potentially involving hormonal or epigenetic mechanisms, as candidate determinants of the cortical threshold for depolarization; however, causal mechanistic attribution awaits direct experimental testing. Our results highlight sex as a fundamental determinant of cortical excitability and underscore the need for sex-inclusive, repetitive paradigms to better understand female-biased vulnerability and chronification in migraine.