PhD Candidate Oregon Health & Science University Portland, Oregon, United States
Courtney Bouchet (Oregon Health & Science University)| Amy Eshleman (Oregon Health & Science University, Oregon Health & Science University)| Aaron Janowsky (Oregon Health & Science University, Oregon Health & Science University)| Susan Ingram (Oregon Health & Science University)
Cannabinoid receptors (CBRs) are sensitive to environmental perturbations and we have previously observed that presynaptic cannabinoid 1 receptor (CB1R) inhibition of GABA release is reduced but that presynaptic cannabinoid 2 receptor (CB2R) function is increased by persistent inflammation in the rostral ventromedial medulla (RVM). The goal of this study is to determine the effect of persistent inflammation on CBRs within the ventrolateral periaqueductal gray (vlPAG), a region upstream of the RVM in the descending pain modulatory pathway. CB1R-mediated inhibition of GABA release was assessed with whole cell patch clamp electrophysiology measuring spontaneous miniature inhibitory postsynaptic currents (mIPSC) and electrically evoked inhibitory postsynaptic currents (eIPSCs). CBR binding was measured from microdissected vlPAG tissue with radioligand binding saturation curves using [3H]CP-55,940.
In brain slices from naïve rats, the CBR agonist WIN55,212-2 (3 µM) suppressed presynaptic GABA release and this effect was reversed by application of the CB1R-specific antagonist rimonabant (3 µM). After persistent inflammation (5-7 days) induced by subcutaneous injection of Complete Freund’s Adjuvent (CFA) into the hindpaw, WIN55,212 no longer suppressed spontaneous or evoked GABA release. Further, this effect was selective to CBRs as persistent inflammation did not impact presynaptic mu-opioid receptor inhibition of GABA release. To determine if CFA-induced neural activity elevates endocannabinoids that desensitize CB1Rs, we tested CB1R function 24h after CFA injection. WIN55,212 inhibition of GABA release was normal, similar to vlPAG slices from naïve rats at this timepoint, indicating that the mechanism underlying the loss of CB1R function takes longer than 24h and is likely not due to the increased spontaneous neural activity immediately following CFA injection. Radioligand saturation curves in vlPAG tissue indicated no effect of 5-7d CFA treatment on CBR density or kinetics. Since CP-55,940 is a nonselective CBR agonist, the binding results could be due to increased CB2R expression. However, electrophysiological experiments indicated no increase in CB2R receptor function in the vlPAG after persistent inflammation. These results are in stark contrast to the CBR regulation observed previously in the RVM. Together these data indicate that persistent inflammation reduced CB1R function in the vlPAG through a mechanism that requires more than 24h. The discrepancy in CB2R activity between the vlPAG and RVM after CFA indicates that CBRs in these brain regions are differentially impacted by persistent inflammation- but in both regions CB1R function is significantly reduced. Future experiments are focused on the underlying mechanisms resulting in decreased CB1R function in presynaptic terminals following persistent inflammation.