Oxytocin shortens spreading depolarization-induced periorbital allodynia

Abstract

Background: Migraine is among the most prevalent and burdensome neurological disorders in the United States based on disability-adjusted life years. Cortical spreading depolarization (SD) is the most likely electrophysiological cause of migraine aura and may be linked to trigeminal nociception. We previously demonstrated, using a minimally invasive optogenetic approach of SD induction (opto-SD), that opto-SD triggers acute periorbital mechanical allodynia that is reversed by 5HT_1B/1D receptor agonists, supporting SD-induced activation of migraine-relevant trigeminal pain pathways in mice. Recent data highlight hypothalamic neural circuits in migraine, and SD may activate hypothalamic neurons. Furthermore, neuroanatomical, electrophysiological, and behavioral data suggest a homeostatic analgesic function of hypothalamic neuropeptide hormone, oxytocin. We, therefore, examined the role of hypothalamic paraventricular nucleus (PVN) and oxytocinergic (OXT) signaling in opto-SD-induced trigeminal pain behavior.

Methods: We induced a single opto-SD in adult male and female Thy1-ChR2-YFP transgenic mice and quantified fos immunolabeling in the PVN and supraoptic nucleus (SON) compared with sham controls. Oxytocin expression was also measured in fos-positive neurons in the PVN. Periorbital mechanical allodynia was tested after treatment with selective OXT receptor antagonist L-368,899 (5 to 25 mg/kg i.p.) or vehicle at 1, 2, and 4 h after opto-SD or sham stimulation using von Frey monofilaments.

Results: Opto-SD significantly increased the number of fos immunoreactive cells in the PVN and SON as compared to sham stimulation (p < 0.001, p = 0.018, respectively). A subpopulation of fos-positive neurons also stained positive for oxytocin. Opto-SD evoked periorbital mechanical allodynia 1 h after SD (p = 0.001 vs. sham), which recovered quickly within 2 h (p = 0.638). OXT receptor antagonist L-368,899 dose-dependently prolonged SD-induced periorbital allodynia (p < 0.001). L-368,899 did not affect mechanical thresholds in the absence of opto-SD.

Conclusions: These data support an SD-induced activation of PVN neurons and a role for endogenous OXT in alleviating acute SD-induced trigeminal allodynia by shortening its duration.

Keywords: Migraine with aura; Oxytocin; Spreading depolarization.

Fig. 1

Opto-SD increases fos-IR in PVN and SON and co-localizes with oxytocin. A Single opto-SD was induced 1 h prior to removal of a tissue block containing the hypothalamus. 10 µM thick tissue sections were stained for fos. B SD (n = 9), significantly increased the number of fos postive cells in the PVN as compared to sham (n = 7). In tissue sections where SON was identified, SD (n = 7) also produced a significant increase in the number of fos positive cells as compared to sham (n = 5). Males are represented as circles and females as triangles. Because of the sample size, sex differences were not tested. *p < 0.0001, #p = 0.0182, unpaired t-test. C Left upper panel shows symmetric fos staining in the PVN following SD, and right upper panel following sham stimulation. Fos-IR in SON following SD and sham stimulation are shown in the left lower and right lower panels respectively. D In the right upper panels fos and oxytocin staining are shown following SD. The lower panel shows colocalization of fos and oxytocin in some PVN cells in an example of a merged image (arrows are shown in the magnified image to the right). E Demonstrates the same for SON following SD. Bar = 100 µM

Fig. 2

Oxytocin receptor antagonist (L-368,899, Oxy-R ⊣) prolongs opto-SD-induced acute periorbital allodynia (n = 8/group). A Following opto-SD, vehicle or oxytocin receptor antagonist (5, 10 and 25 mg/kg in 3 separate groups) was administered via intraperitoneal injection. Periorbital mechanical allodynia was tested 1, 2 and 4 h later. There was no significant effect of Oxy-R antagonist on periorbital thresholds in sham mice at any dose tested. Opto-SD produced periorbital allodynia at 1 h which recovered by 2 h. At low doses of Oxy-R antagonist (5 mg/kg), there was no effect on opto-SD induced periorbital allodynia. However, with increasing doses, mice displayed longer lasting periorbital allodynia. In the presence of Oxy-R antagonist 10 mg/kg, opto-SD produced periorbital allodynia that lasted 2 h and in the presence of Oxy-R antagonist 25 mg/kg, opto-SD produced periorbital allodynia that lasted at least 4 h. B Shows individual data for each animal tested for each dose of vehicle or drug per group. * p = 0.001, ** p = 0.003, # p = 0.0003 at 1 h, p = 0.005 at 2 h, ‡ p < 0.0001 at 1, 2 and 4 h; two-way ANOVA, Tukey’s post-hoc analysis