Optogenetic cortical spreading depression originating from the primary visual cortex induces migraine-like pain and anxiety behaviors in freely moving C57BL/6 J mice

Abstract

Background: Migraine is the second disabling neurological disorder with a high prevalence. Aura occurs in one-third of migraineurs and visual aura accounts for over 90%. Cortical spreading depression (CSD) underlies aura and might trigger migraine headaches. Compared with CSD induction by invasive electrical, chemical, or mechanical stimulation, optogenetics avoids direct influences on meninges in the stimulation process. However, previous optogenetic CSD models mainly use Thy1-ChR2-YFP or CaMKIIα-cre transgenic mice. They are limited when the pathogenesis study requires transgenic mice to express other specific promotor, such as the dopamine or serotonin transporter promotor. In addition, reported behavioral paradigms were based on CSD induction under anesthesia. This study aimed to establish an optogenetic CSD-induced migraine model originating in the primary visual cortex (VISp) in C57BL/6 J mice and presented the behavioral paradigm when CSD induction was under awake condition.

Methods: We performed viral transduction for the expression of light-sensitive channelrhodopsin-2 in pyramidal neurons of VISp in C57BL/6 J mice. Regional cerebral blood flow (rCBF) was measured by laser speckle flowmetry to confirm CSD induction. The von Frey, light-dark box, elevated plus maze, and open field test were conducted to verify migraine-related behaviors in freely moving mice.

Results: An optogenetic stimulus induced typical spreading triphasic rCBF change with a reduction of over 20%, confirming CSD induction. A single unilateral CSD in freely moving C57BL/6 J mice triggered bilateral periorbital and hind-paw allodynia lasting for 4-24 h. Notably, the ipsilateral periorbital mechanical threshold was significantly lower than the contralateral at 1 h. It also generated photophobia and anxiety behaviors persisting for 24-48 h. Furthermore, cutaneous allodynia and anxiety behaviors were alleviated by sumatriptan.

Conclusions: This study proposes an optogenetic CSD-induced migraine model originating from VISp in awake and freely moving C57BL/6 J mice and presents the behavioral paradigm in detail. The CSD model in wild-type mice is promising to be wildly used to study the pathogenesis of MwA.

Keywords: Allodynia; Anxiety; Cerebral blood flow; Cortical spreading depression; Migraine; Optogenetic; Primary visual cortex.

Fig. 1

The experimental flow graph. A A right occipital craniotomy over the primary visual cortex (3.5 mm posterior and 2 mm lateral to the bregma) and virus injection 0.5 mm below the dura mater via a glass microelectrode. B After at least 5 weeks, a fiber optic cannula was implanted 0.1 mm over the dura mater at the injection site. Regional cerebral blood flow (rCBF) was recorded to confirm the occurrence of cortical spreading depression (CSD). C The time course of von Frey tests on bilateral periorbital skins and hind paws, light–dark test, elevated plus maze (EPM), and open field test (OFT) after a single light stimulus (465 nm, 4 mW, 10 s). In each mouse, optogenetic stimulation was implemented under awake and freely moving condition. Eleven mice in each of VEH and CSD group were used for periorbital von Frey tests, and twelve mice for hind paw von Frey tests. Twenty-seven mice in each group were used for light–dark box, EPM, and OFT, and each mouse was conducted different tests at different time points (2.5 h, 24 h, and 48 h). D After a two-week washout period, sixteen mice in the CSD group were evenly divided into CSD + NS and CSD + Suma group, suffering an illumination (465 nm, 4 mW, 10 s) immediately followed by 0.9% normal saline and sumatriptan injection, respectively. The hind-paw and periorbital withdrawal thresholds were assessed at 2.5 h and 3 h and the EPM test at 4 h. Figure 1 was created in BioRender.com with permission

Fig. 2

An LED light stimulation induces spreading triphasic rCBF changes detected by laser speckle flowmetry. A The positions of ROI 1 (the motor cortex, blue) and ROI 2 (the somatosensory cortex, red) were manually marked. ROIs were amplified for display. A and B After recording the stable baseline over 5 min (I), optogenetic stimulus in the right hemisphere evoked initial hypoperfusion (II, III), transient normalization (IV), and post-CSD oligemia (V) at the same side. Triphasic rCBF changes spread from the somatosensory cortex (ROI 2, red) towards the motor cortex (ROI 1, blue). C The VEH group had little changes in the rCBF after optogenetic stimulus

Fig. 3

A single optogenetic CSD triggers prolonged allodynia and photophobia. Time sequential variation of bilateral periorbital (A, n = 11 per group) and hind paw (C, n = 12 per group) withdrawal pain thresholds after a single optogenetic stimulus. The data were shown as mean ± SEM. The difference was analyzed by two-way repeated measures ANOVA with post hoc analysis. A Left (contralateral): F = 2.657, p = 0.116 for group; F = 10.106, p < 0.001 for time; F = 4.505, p = 0.003 for group × time interaction. Right (ipsilateral): F = 11.234, p = 0.003 for group; F = 13.541, p < 0.001 for time; F = 8.375, p < 0.001 for group × time interaction. C Left (contralateral): F = 16.062, p = 0.001 for group; F = 19.656, p < 0.001 for time; F = 5.353, p < 0.001 for group × time interaction. Right (ipsilateral): F = 21.002, p = 0.001 for group; F = 31.214, p < 0.001 for time; F = 9.168, p < 0.001 for group × time interaction. Solid lines for VEH, dotted lines for CSD. VEH_left versus CSD_left, # p < 0.05, ## p < 0.01, ### p < 0.001; VEH_right versus CSD_right, * p < 0.05, ** p < 0.01, *** p < 0.001; CSD_left versus CSD_right, ▲▲ p < 0.01. Individual time-sequential changes of bilateral periorbital (B) and hind-paw (D) withdrawal thresholds in the CSD group (blue, left side; red, right side). The significance between post-stimulation time points and the baseline was assessed by the post-hoc pairwise comparison. * p < 0.05, *** p < 0.001, ns = no significance. E Sample trajectories of 10-min light–dark box test at 2.5 h (VEH, upper panel; CSD, lower panel) after a single light stimulus. The differences in total distance (F), speed of movement (G), distance in the light (H), and time in the light (I) were presented as bar graphs (mean ± SEM) and were assessed by the unpaired t-test between the VEH and CSD group (n = 9 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significance

Fig. 4

A single optogenetic CSD produces anxiety behaviors in EPM. A Sample trajectories of 10-min EPM test at 2.5 h, 24 h, and 48 h (VEH, upper panel; CSD, lower panel) after a single light stimulus. The differences in total distance (B), speed of movement (C), time in open arms (D), and OT/(OT + CT)% (E) were presented as bar graphs (mean ± SEM) and were assessed by unpaired t-test between the VEH and CSD group (n = 9 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significance

Fig. 5

A single optogenetic CSD generates anxiety behaviors in OFT. A Sample trajectories of 10-min OFT at 2.5 h, 24 h, and 48 h (VEH, upper panel; CSD, lower panel) after a single light stimulus. The differences in total distance (B), speed of movement (C), time in the center (D), and percent time in the center (E) were presented as bar graphs (mean ± SEM) and were assessed by unpaired t-test between the VEH and CSD group (n = 9 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significance

Fig. 6

Sumatriptan alleviates cutaneous allodynia and anxiety behaviors induced by a single optogenetic CSD. The hind-paw (A) and periorbital (B) withdrawal thresholds before and after normal saline and sumatriptan injection following optogenetic CSD induction in the CSD + NS (n = 8) and CSD + Suma (n = 8) group. The data were shown as box-whisker plots (median, the first quartile, and the third quartile). The difference was assessed by unpaired t-test (CSD + NS versus CSD + Suma) or paired t-test (post-stimulation time points versus baseline). C Sample trajectories of 10-min EPM test at 4 h (CSD + NS, upper panel; CSD + Suma, lower panel). The differences in total distance (D), speed of movements (E), time in open arms (F), and OT/(OT + CT)% (G) were presented as bar graphs (mean ± SEM) and were assessed by unpaired t-test between the CSD + NS and CSD + Suma group (n = 8 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significance