Migraine is caused by hyperactivity of the trigeminovascular system, where trigeminal ganglia (TG) play an important role. This hyperactivity might originate from an underfunctional GABAergic system in TG. To investigate this possibility, we adapted a mouse model of migraine by inducing migraine-like grimaces in male mice via repeated injections of nitroglycerin (NTG, 10 mg/kg, i.p.), once every 2 days, for up to 5 sessions. Migraine-like facial pain scores were measured using the mouse grimace scale. Repeated NTG treatments in mice caused significant increases in migraine-like grimaces that were aborted and prevented by two anti-migraine agents sumatriptan and topiramate, respectively. After 5 sessions of NTG injections, the GABAsynthesizing enzyme, 65-kDa glutamate decarboxylase (GAD65), but not the GABA transporter 1 (GAT1) or the α6 subunitcontaining GABA A receptors (α6GABA A Rs), was downregulated in mouse TG tissues. Taking advantage of the unaffected TG α6GABA A R expression in NTG-treated mice, we demonstrated that an α6GABA A R-selective positive allosteric modulator (PAM), DK-I-56-1, exhibited both abortive and prophylactic effects, comparable to those of sumatriptan and topiramate, respectively, in this migraine-mimicking mouse model. The brain-impermeable furosemide significantly prevented the effects of DK-I-56-1, suggesting its peripheral site of action, likely via preventing α6GABA A R modulation in TG. Results suggest that a decreased GABA synthesis caused by the reduced GAD65 expression in TG contributes to the trigeminovascular activation in this repeated NTG-induced migraine-mimicking model and that the unaltered α6GABA A Rs in TG are potential targets for migraine treatment. Thus, α6GABA A R-selective PAMs are potential anti-migraine agents for both abortive and preventive therapies.Key Words Migraine . α6 subunit-containing GABA A receptor . trigeminal ganglia . nitroglycerin . mouse grimacescale . 65-kDa glutamate decarboxylase . GABA transporter 1.Hung-Ruei Tzeng and Ming Tatt Lee contributed equally to this work.

Pain assessment is essential for preclinical and clinical studies on pain. The mouse grimace scale (MGS), consisting of five grimace action units, is a reliable measurement of spontaneous pain in mice. However, MGS scoring is labor-intensive and time-consuming. Deep learning can be applied for the automatic assessment of spontaneous pain. We developed a deep learning model, the DeepMGS, that automatically crops mouse face images, predicts action unit scores and total scores on the MGS, and finally infers whether pain exists. We then compared the performance of DeepMGS with that of experienced and apprentice human scorers. The DeepMGS achieved an accuracy of 70–90% in identifying the five action units of the MGS, and its performance (correlation coefficient = 0.83) highly correlated with that of an experienced human scorer in total MGS scores. In classifying pain and no pain conditions, the DeepMGS is comparable to the experienced human scorer and superior to the apprentice human scorers. Heatmaps generated by gradient-weighted class activation mapping indicate that the DeepMGS accurately focuses on MGS-relevant areas in mouse face images. These findings support that the DeepMGS can be applied for quantifying spontaneous pain in mice, implying its potential application for predicting other painful conditions from facial images.

Migraine, an unmet medical need, is due to an overactive trigeminovascular system (TGVS). The trigeminal ganglion (TG), a hub of the TGVS, transmits the pain signals arising from neurogenic inflammation in the meninges to the brainstem. Previously, we have revealed an unprecedented role of the α6 subunit‐containing GABAA receptor (the GABAA receptor α6 subtype, α6GABAAR) on TG in the neuropathogenesis of migraine using the α6GABAAR‐specific positive allosteric modulators (PAMs)1 we developed. In a rat model of migraine induced by intracisternal injection of capsaicin, we showed that Compound 6, a potent α6GABAAR‐selective PAM via acting on TG, attenuated TGVS activation‐induced peripheral and central responses, using immunohistochemical approaches.2 Here, we further investigated whether positive modulating TG α6GABAARs would attenuate migraine‐related nociceptive behaviors induced by repetitive injections of nitroglycerin (NTG), which induces headache in human due to dural vasodilatation. We gave NTG (10 mg/kg, i.p.) to mice (6–8 weeks, male ICR) once every two days for 5 times. The mouse grimace scale (MGS) was used to score, from 0–2, the headache‐like behaviors, including orbital tightening, nose bulge, cheek bulge, and the changes of ear position and whisker. The severity of migraine was assessed by the MGS on day 9 (the 5th session). To evaluate the abortive treatment efficacy of α6GABAAR PAMs, drug were administered 20 min after NTG injection on day 9. For their prevention efficacy, drugs were given 30 min before NTG injection in each session. Repeated NTG injections significantly increased MGS. Compound 6 and its deuterated derivative that has a longer half‐life, DK‐56‐I, significantly attenuated the elevated MGS induced by NTG. Importantly, the effect of the α6GABAAR‐selective PAM was antagonized by i.p. injection of furosemide (20 mg/kg), an α6GABAA R‐selective antagonist that is blood‐brain barrier‐impermeable. It was mimicked by sumatriptan, an effective drug for migraine abortion. Besides, we found that DK‐56‐I was effective in preventing repeated NTG‐induced elevation of MGS, like topiramate, an effective drug for migraine prevention. We also found that α6GABAARs are expressed in TG neurons and satellite glial cells of mice. These results suggest that the TG α6GABAAR is a novel therapeutic target for migraine and that the α6GABAAR‐selective PAM is a novel treatment for migraine abortion and prevention.Support or Funding InformationSupported grants: MOST 107‐2321‐B‐002‐010 and NHRI‐EX107‐10733NI.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.