The goal of this study was to define the organization of the ECS in neural regions associated with pain in the context of sex. Levels of the primary eCBs, AEA and 2-AG, differed between cortical and brainstem (i.e., lPAG), but not trigeminal components (Vc or TG), in a sex-dependent manner. Unbiased, quantitative proteomic analysis confirmed sex differences in the PAG proteome such that detection of MAGL and ABHD6 was statistically higher in female than in male samples. Immunohistochemical analysis confirmed the female prevalence of NAPE-PLD, DAGL, and MAGL in the PAG. Moreover, the distribution of CB1R and CB2R were significantly different between the sexes as well, with CB1R lower in males than females in the V1M cortex and PAG. Together, these results suggest differences between the physiology of the male and female ECS in brain regions associated with migraine aura and descending pain modulation. These differences may underscore the sex differences in certain pain etiologies, as well as pharmacological responsivity.
AEA:
Dysregulation of AEA signaling has been implicated in migraine including chronic migraine and medication overuse [39,40], though contested [41]. Peripheral actions of AEA at CB1R receptors inhibit trigeminal afferent release of neurotransmitters to mitigate headache pain and regulate vascular tone [42]. Levels of AEA are reduced in the plasma of chronic migraine and MOH patients [39]. In addition, female migraineurs, but not male, were reported to have increased activity of AEA transporters and FAAH in platelets, suggesting sex differences [43]. Measurement of AEA in the cerebrospinal fluid revealed that chronic migraineurs and patients with MOH had significantly lower levels as compared to controls or episodic migraineurs [44] suggesting widespread dysfunction of the AEA in the CNS without regional information.
Notably, preclinical study of AEA and its modulation have yielded promising results as a strategy to mitigate migraine. In male rats, following administration of nitroglycerin, an NO-donor used to induce headache pain [45,46], AEA administration blocked changes in expression of TRPV1, NFKb, CRP, and NO [47] and reduced trigeminal activation [48]. Further, FAAH inhibitors were effective as both preventative and reversal agents against NO-induced headache [47,49,50]. Of importance, though NO-induced headache has a reported sexual dimorphism [51], the studies using this model were performed in males [47-50]. Current findings suggest that NAPE-PLD and FAAH are distributed differently amongst neuron, astrocyte, and glial populations as well. These observations combined with clinical reports, indicate that sexual dimorphic responses of restoring physiological AEA tone with FAAH or AMT inhibitors warrant additional investigation with attention to female subjects [52].
Our data support sex differences in the levels of AEA in the V1M cortex, but not PAG. As the above studies have demonstrated the anti-migraine effects of AEA, this may offer males increased protection against trigeminal nociception.
2-AG:
The role of 2-AG has been understudied in migraine and pain states. Clinically, levels of 2-AG in platelets of chronic migraineurs and MOH patients were lower than controls subjects [53]. However, CSF levels were below the limit of detection in other studies [43,44]. 2-AG acts on both cannabinoid receptors, leading to reduction in neurotransmitter release at glutamatergic and GABA-ergic synapses [11-15]. Thus, alterations in levels of 2-AG are pertinent to pain sensation in the V1M cortex and descending pain modulation in the PAG and V1M cortex.
Our results show that 2-AG levels are significantly higher in the female PAG as compared to male PAG and V1M cortex. Thus, investigations of platelets or CSF in headache patients may not reflect important differences within discrete brain circuits relevant to pain transmission. Given the differences in PAG 2-AG levels between male and female rats and clinical inconsistencies in reporting of 2-AG levels, it is possible that sex differences in the expression or activity of synthetic (DAGL) or degradative enzymes (MAGL, ABHD6) for 2-AG underlie the observed sex difference in 2-AG levels.
DAGL:
Preclinically, DAGLα expression is reported in the perisynaptic region of the dendritic spines of glutamatergic synapses [54], although cytosolic and nuclear DAGLα are also described in cortical neurons [55]. Studies above observed DAGLα in neuronal soma and fibers, astrocytes, and microglia in both the V1M cortex and PAG. Recently, mutations in DAGLA, the gene for DAGLα, within the CNS, were identified and implicated in neurological disorders in humans [56]. Furthermore, there is evidence that depletion of 2-AG via inhibition of DAGLα may be sufficient to trigger migraine-like pain in animals [57]. Thus, pathological reductions in the functional expression of DAGLα in females during pain states, including headache, may have a pronounced impact via decreased 2-AG signaling.
Our studies revealed that DAGLα expression was higher in female rat PAG as compared to male using immunofluorescence; similar patterns were observed in the V1M cortex. The significantly greater amount of DAGLα detected in the female PAG offers one explanation for the greater levels of 2-AG observed in this region; however, sex differences in the levels of 2-AG hydrolyzing enzymes must be considered as well.
MAGL and ABHD6:
In contrast to increases in DAGL synthesis of 2-AG, higher levels of 2-AG in female PAG may reflect lower expression of functional degradative enzymes, MAGL and ABHD6. Both MAGL and ABHD6 degrade many monoacylglycerol species, including 2-AG [7,8,58-62]. MAGL and ABHD6 inhibitors have shown promise as pain therapeutics [57, 62-66] and in treating neurological disorders [7,8,62], respectively. One study investigated the contribution of MAGL to NO-induced headache, but results were not obtained in both sexes [50]. Mechanistically, the effect of the inhibitors has correlated to increasing 2-AG levels and CBR signaling while attenuating eicosanoid signaling [12,68].
Present proteomic and immunohistochemical data indicate that 2-AG degradation may be higher in females as compared to males in the PAG. Notably, ABHD6 has been shown to have DAGL activity in addition to the breakdown of monoacylglycerols [69], and this may contribute to the sex differences in 2-AG levels within PAG. Our findings suggest that sex differences in the prevalence of disorders where the PAG is implicated, including migraine and other pain states, [70-78], may result from inherent differences in synthesis and degradation of 2-AG. Sex differences in signaling of 2-AG at the eCB receptors within pain modulatory pathways may influence the frequency and intensity of pain signals sent to the somatosensory cortex.
CB Receptors:
In the context of pain, preclinical data support sex differences in responsivity to cannabinoid receptor agonists including ∆9-tetrahydrocannabinol (THC), cannabidiol, beta-caryophyllene (BCP), WIN55-212-2, and CP55-940 [78-81]. In support of the theory of CED, Kandasamy et al. demonstrated THC, a CB1/2 agonist, ameliorated the effects of induced migraine in rats, and that this effect was attenuated on administration of a CB1 antagonist [79]. CB2R expression in the CNS has been controversial, and current clinical literature supports a role for it during inflammation [35,83-86].
Agonism of the CB receptors is being pursued for several neurological disorders, including migraine [6,86-91]. In addition, more than 300 publications report sex differences in pathologies where the ECS plays a regulatory role. Our data now reveal that these sex differences are discernable in discrete brain regions and cell types within pain relevant regions of the CNS. Thus, cannabinoid pharmacology may have anatomical and circuit-based differences between the sexes in basal and pathological states.
Female cortical and lPAG sections showed significantly higher CB1R immunoreactivity as compared to the respective male tissue. Male CB2R expression was largely in neurons, whereas female colocalization was primarily in glial cells. These data suggest that differences in the literature reporting CNS expression of CB2R would benefit from analysis by sex and that CB2R in the CNS may play different roles between male and female subjects. Given the interest in developing CB2R targeting therapeutics for pain and neurological disorders, these sex differences in the biological role of CB2R are important [92,93].
ECS distribution in Neurons, Glia, and Immune Cells:
Throughout the immunohistochemical analyses, it was observed that for all proteins in the ECS, colocalization with Nissl (neuronal soma), GFAP (astrocyte marker), and Iba1 (microglial marker) was limited to 40-70%. Thus, 30-60% of the ECS proteins were observed independent of these markers. It is possible that a portion of the observed proteins that were non-localized were due to aberrant pixel detection, either from background noise on the image or from image artifacts. However, there is evidence within the literature for the role of these proteins at synaptic terminals, which may have eluded the selected cell markers [6-8,38]. Proteins present in the neurovasculature and extracellular spaces likely would not be co-localized with our cellular markers. For all immunohistochemical analyses, the overall staining/immunoreactivity with these biomarkers of cellular identity (neuron, astrocyte, or microglia) were the same between regions and each sex, confirming that regional comparisons were appropriately controlled for cell types and number (Additional Fig 1).
Limitations:
Though carefully designed, this study does have some limitations. First, immunohistochemistry with antibodies targeting proteins of interest was used. Antibodies, particularly those against the cannabinoid receptors, are reported to be non-specific and unreliable [94,95]. To combat this, we paired imaging with an unbiased quantitative proteomics approach. This unbiased method allowed for determining presence of proteins in each region by sex, but itself is limited in spatial resolution by cell type. This lack of detection may reflect masking by proteins with higher abundance (i.e. low signal to noise). Second, all tissue was obtained from Sprague-Dawley rats between 7-8 weeks old. As the ECS is implicated in neural development [96,97], it is possible that sex differences identified here become more/less prominent during aging. Finally, endocannabinoid levels were obtained from uncycled female animals. Several reports indicate that both 2-AG and AEA levels change with the estrus cycle including in the brain [80,98]. Therefore, sex differences noted here of ECS functional expression may fluctuate in magnitude across the estrus cycle. Future studies should incorporate newly developed fluorescent chemical probes for labeling these CBRs to confirm the present observations [99,100] and investigate the sex difference in the ECS with aging and in cycled females.