SEX DIFFERENCES IN GENE EXPRESSION AND PAIN-RELATED BEHAVIORS IN A PRECLINICAL MODEL OF MIGRAINE

Abstract

Many laboratory animal studies of migraine have employed electrophysiological techniques to assess neuronal sensitization, but few have examined behaviors using International Headache Society criteria, which are based on behavioral changes including duration and intensity of pain and avoidance of routine activity. Fewer still have attempted to correlate the appearance of these diagnostic symptoms with changes in the activity of pain-related neurotransmitters and neuromodulators, such as calcitonin gene related peptide (CGRP). A vasoactive neuropeptide, CGRP might contribute to the vasodilatory component of migraine, and to the pain associated with this condition as it is present in nociceptors, including those in the trigeminal ganglion that innervate cerebral vasculature. Previous work has shown that CGRP levels are increased in animal models of inflammatory pain and in the external jugular vein of humans during migraine attacks. The CGRP receptor is comprised of three proteins: a G-protein coupled receptor called calcitonin-like receptor (CLR), a receptor activity-modifying protein (RAMP1), and a coupling protein, receptor component protein (RCP). Thus, the availability and sensitivity of this receptor is subject to regulation at numerous levels. The objectives of this study were to develop a preclinical behavioral model of chronic migraine, to test sensory and motor behaviors relevant to International Headache Society diagnostic criteria, to examine whether there are sex differences in these behaviors, and to assess whether alterations in the expression pattern of genes encoding CGRP and its receptor components are associated with sex differences or changes in pain-related behaviors. Male and female Sprague-Dawley rats were implanted with a dural cannula placed over the occipital cortex. Groups of rats were treated with 10 or 20 microliter volumes of an inflammatory soup containing 1 mM each of histamine, serotonin, and bradykinin, as well as 0.1 mM prostaglandin E2 (pH 5.5). A control group received sterile phosphate-buffered saline (pH 7.4) alone. Baseline behavioral testing was conducted on all eight groups of animals prior to surgery and seven days later. The inflammatory soup or control solution was administered supradurally 3 times/week for a total of eight applications. Locomotor activity was assessed using force plate actimetry during and following application of the inflammatory soup or vehicle. Total RNA was isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Real-time polymerase chain reaction was used to quantify the expression of amplified constructs using gene specific primers for CGRP, RAMP1, CLR, and RCP. The results reveal pronounced sex differences in behavior following application of the inflammatory soup. Female rats displayed dose-dependent migraine-related behaviors and a longer duration of these effect in measurements of distance traveled, bouts of low mobility, and spatial confinement. Both males and females experienced allodynia following exposure to the inflammatory mixture. Moreover, females displayed a higher baseline gene expression of CGRP and lower baseline gene expression of RAMP1, CLR, and RCP in the medulla than male animals. In addition to these baseline differences, gene expression of CLR and RCP was induced in the medulla of female rats but not in males. No sex difference in CGRP gene expression was noted in the trigeminal ganglia, although females do have a lower baseline expression of CGRP receptors, RAMP1, CLR, and RCP than males. It was also found that in the trigeminal ganglia RAMP1, CLR and RCP are inducible, especially in males, and that at least a portion of these responses are the result of volume effects associated with application to the dura of the inflammatory soup or vehicle. These findings indicate significant sex dependent changes in rat locomotor activity and CGRP-related gene expression in the brainstem and trigeminal ganglia associated with the application to the dura of an inflammatory soup. As the behavioral endpoints utilized in this study correspond to clinical signs considered by the International Headache Society as diagnostic for migraine, these data confirm that CGRP and its receptors are involved in migraine pathophysiology and reveal for the first time that alterations in the response to this peptide may be related to the increased prevalence of migraine in females. Such findings could be of value in devising new therapeutic strategies for the treatment of the debilitating condition.