Optogenetic Spreading Depression Elicits Trigeminal Pain and Anxiety Behavior

Abstract

Objective: Cortical spreading depression (SD) is an intense depolarization underlying migraine aura. Despite the weight of evidence linking SD to the pain phase of migraine, controversy remains over a causal role of SD in cephalgia because of the invasive nature of previous SD induction methods. To overcome this problem, we used a novel minimally invasive optogenetic SD induction method and examined the effect of SD on behavior.

Methods: Optogenetic SD was induced as a single event or repeatedly every other day for 2 weeks. End points, including periorbital and hindpaw mechanical allodynia, mouse grimace, anxiety, and working memory, were examined in male and female mice.

Results: A single SD produced bilateral periorbital mechanical allodynia that developed within 1 hour and resolved within 2 days. Sumatriptan prevented periorbital allodynia when administered immediately after SD. Repeated SDs also produced bilateral periorbital allodynia that lasted 4 days and resolved within 2 weeks after the last SD. In contrast, the hindpaw withdrawal thresholds did not change after repeated SDs suggesting that SD-induced allodynia was limited to the trigeminal region. Moreover, repeated SDs increased mouse grimace scores 2 days after the last SD, whereas a single SD did not. Repeated SDs also increased thigmotaxis scores as a measure of anxiety. In contrast, neither single nor repeated SDs affected visuospatial working memory. We did not detect sexual dimorphism in any end point.

Interpretation: Altogether, these data show a clinically congruent causal relationship among SD, trigeminal pain, and anxiety behavior, possibly reflecting SD modulation of hypothalamic, thalamic, and limbic mechanisms. ANN NEUROL 2021;89:99-110.

Figure 1.. Optogenetic SD and behavioral testing.

(A) Motor cortex was stimulated with 470 nM wavelength light using an optical fiber for 10 seconds at 1mW power increments every 2 minutes until an SD occurred (max 10mW). SD was visualized through a glass coverslip using intrinsic optical signal imaging acquired at 0.5 Hz (*, bregma). Each image was subtracted from the baseline image to demonstrate SD propagation over the dorsal cortex. (B) In the single SD paradigm, animals were assessed 1 hour, 2 days or 4 days after SD. Individual readouts are shown on the right, including periorbital and hindpaw mechanical allodynia using von Frey monofilaments, and anxiety-like behavior and working memory using open field and Y maze, respectively. (C) In the repeated SD paradigm, SDs were induced once every other day for 14 days and animals were assessed 2 days, 4 days or 14 days after the last SD.

Figure 2.. Single SD produces acute periorbital mechanical allodynia.

Experimental timeline is shown on top. Left top panel shows individual log force data (grams) from females (triangle) and males (circles) along with the median. A single SD (1SD) reduced periorbital mechanical thresholds 1 hour after SD as compared with sham controls. This effect was lost 2 and 4 days after SD (two-way ANOVA on ranks). Right top panel shows group averages of the same dataset to illustrate the time course of periorbital allodynia (mean ± SEM). Left bottom panel shows normalization of the reduced mechanical thresholds 1 hour after a single SD by sumatriptan (600 μg/kg, intraperitoneal immediately after SD) compared with vehicle (Mann-Whitney U test). Right bottom panel shows single 10 sec, 7mW 470nM optogenetic light stimulation in wild type mice did not produce periorbital mechanical allodynia (Mann-Whitney U test p=0.7714).

Figure 3.. Repeated SDs induce lasting periorbital mechanical allodynia.

Left panel shows individual log force data (grams) from females (triangle) and males (circles) along with the median. Seven SDs once every other day (7SD) reduced periorbital mechanical thresholds 2 and 4 but not 14 days after the last SD compared with sham controls (two-way ANOVA on ranks). Middle panel shows group averages of the same dataset to better illustrate the time course of periorbital allodynia (mean ± SEM). Right panel shows unchanged mechanical hindpaw thresholds in repeated SD and sham groups tested 2 days following the last SD (Mann-Whitney U test).

Figure 4.. SD induced facial grimace.

(A) Facial features of animals with mild, moderate and severe discomfort are illustrated. (B) Single SD (1SD) produced no change in mean mouse grimace score as compared to sham animals when assessed 1 hour later. (C) Repeated SDs (7SD) increased mean mouse grimace score. Individual data points from females (triangle) and males (circles) as well as mean ± SEM are shown (t-test).

Figure 5.. Sexual dimorphism in periorbital mechanical allodynia in sham and SD groups.

Individual log force data (grams) in previous figures are reorganized to display males (circles) and females (triangle) separately in sham and single (1SD) or repeated SD (7SD) arms. There were no significant differences in pooled periorbital mechanical thresholds between males and females in pooled sham (left panel, Mann-Whitney U test) or SD (right panel, two-way ANOVA on ranks) groups.

Figure 6.. Repeated SDs induce anxiety-like behavior but do not impact working memory.

(A) Anxiety-like behavior and working memory were tested using open field thigmotaxis scores and Y-maze alternations, respectively, 2 days after a single (1SD) or repeated SDs (7SD). (B) Upper panel shows representative exploratory paths and heatmaps from a sham and a repeated SD animal. Individual data for total distance traveled and thigmotaxis scores from females (triangle) and males (circles) are shown along with mean ± SEM after a single (middle panel) or repeated SDs (lower panel). Total distance traveled was not affected by SD. Repeated, but not a single, SD increased thigmotaxis scores. (C) Upper panel shows the calculation of percent correct alternations. Individual data for total arm entries and correct alternations from females (triangle) and males (circles) are shown along with mean ± SEM after a single (middle panel) or repeated SDs (lower panel). Sham and SD groups were compared using t-tests.