Investigating the role of the endogenous cannabinoid system in emotional modulation of pain: neurochemical and molecular mechanisms

Abstract

Abstract Pain represents a major unmet clinical need and affects a large number of individuals worldwide. The modulation of pain by cognitive and emotional factors is now widely recognised. Increased understanding of the endogenous mechanisms of pain modulation could lead to new or improved pain therapies. While evidence suggests that intense fear induces a potent form of endogenous analgesia termed fear-conditioned analgesia (FCA) which is adaptive and evolutionarily preserved, more sustained but less intense stress/anxiety is often associated with enhanced pain termed anxiety-related hyperalgesia (ARH). Evidence exists for a role of the endogenous cannabinoid (endocannabinoid) system in FCA; however, its role in ARH is unknown. Activation of the signal transduction molecule extracellular signal regulated kinase (Erk) and plasticity-related genes zif268 and sgk1 plays a role in pain, fear and CB1 receptor-mediated events. The aims of the work presented in this thesis are (1) to further examine the role of the endocannabinoid system in key neural substrates in FCA and investigate molecular mechanisms underpinning endocannabinoid-mediated FCA and (2) to characterize a genetic model of ARH using two rat strains which can then be used to investigate the role of the endocannabinoid system in ARH. FCA was modeled by combining the formalin test of persistent pain with classical Pavlovian fear conditioning in male Lister-Hooded rats. Rats received 10 footshocks followed 23.5 hrs later by an intra-right hind paw injection of formalin (2.5%) and then were re-exposed to the contextually aversive footshock arena 30min later. In the studies performed, behaviours were recorded for 3min, 15min or 30min following arena exposure. In some of these experiments, rats were implanted with cannulae into the right dorsolateral periaqueductal grey (dlPAG) approximately one week before testing and then received intracerebral injections of the fatty acid amide hydrolase (FAAH) inhibitor, URB597, or the CB1 receptor antagonist/inverse agonist, rimonabant, 15min prior to re-exposure to the context. In other experiments, rats received systemic (intraperitoneal) injection of the CB1 receptor antagonist, AM251, 30min prior to re-exposure to the context. Post-mortem analysis involved measurement of endocannabinoids and N-acylethanolamines (NAEs) using liquid chromatography coupled with tandem mass spectrophotometry (LC/MS-MS), measurement of Erk activation using western immunoblotting or measurement of the expression of zif268 and sgk1 mRNA in discrete brain regions or dorsal horn of the spinal cord (DHSC) by RT-qPCR. Additional work consisted of characterising a model of ARH by comparing nociceptive responding in Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats, two strains with different baseline emotionality. Anxiety-related behaviour in the open field and elevated plus maze tests and nociceptive behaviour in the hot plate and the formalin tests were assessed and compared in the two strains. In one of these experiments, rats received systemic (intraperitoneal) injection of URB597 or AM251 60min and 30min prior to the behavioural tests, respectively. Post mortem work included measurement of endocannabinoids and NAEs using LC/MS-MS, measurement of the expression of CB1, FAAH, MAGL, zif268 and sgk1 mRNA in discrete brain regions or DHSC. The results demonstrated a differential response of endocannabinoids and related lipids during exposure to conditioned stress, noxious inflammatory stimulus or during expression of FCA in discrete brain regions in rats including the PAG, BLA, hippocampus, insular cortex, PFC and RVM. FCA was accompanied by a strong trend towards increased levels of pErk1/2 in the right dlPAG and increased pErk1 in the right BLA. The present study demonstrated that direct administration of the CB1 receptor antagonist, rimonabant, into the right dlPAG prevented FCA. In addition, intra-dlPAG URB597 was antinociceptive and showed a strong trend to enhance FCA. FCA was accompanied by attenuation of the formalin-evoked increase in the expression of zif268 in the ipsilateral DHSC. Pharmacological blockade of the CB1 receptor using systemic administration of AM251 attenuated FCA and prevented the fear-induced suppression of zif268 expression in the ipsilateral DHSC in formalin-treated rats. WKY rats exhibited enhanced anxiety-related behaviour and showed enhanced nociceptive responding to acute and persistent noxious stimuli compared to SD, confirming ARH. The two rat strains differed with respect to baseline levels of endocannabinoids, NAEs and expression of CB1, FAAH and MAGL mRNA in discrete brain regions. Moreover, systemic AM251 enhanced and URB597 attenuated ARH in WKY rats. The data provide further evidence for an important physiological role of the endocannabinoid system within the BLA-PAG-RVM pathway in FCA, conditioned fear and pain. These data provide strong evidence for zif268 expression in the DHSC as a molecular correlate of endocannabinoid-mediated FCA. The work presented here also suggests that alterations in central endocannabinoid function may, at least in part, underlie ARH. These results enhance our understanding of the fundamental physiology of pain and fear/anxiety and facilitate the development of new therapeutic approaches to the treatment of pain- and anxiety-related disorders and their co-morbidity.