0192 Brain Circuits That Regulate Awakenings to Pain Stimulus

Abstract Introduction Many neurons in the external lateral PB express the calcitonin gene-related peptide CGRP; PBelCGRP, and also receive spinal afferents carrying pain signals that are relayed to forebrain arousal promoting areas. Thus, PBelCGRP neurons form the critical relay node transmitting pain to induce cortical arousals. We tested this with genetic deletion or acute optogenetic inhibition in both inflammatory pain and opto-pain models, and found that blocking this node prevents pain-induced arousals. Our current focus is on understanding which terminal fields of the PBelCGRP neurons contribute to pain-induced wakefulness. Methods Using pharmacological antagonists and optogenetics, we blocked PBelCGRP neuron terminal fields located in the basal forebrain (BF), bed nucleus of stria terminalis (BNST), and central nucleus of Amygdala (CeA). For pharmacological blocking, wild type mice implanted with a guide cannula targeting one of the terminal fields received bilateral microinjections of NMDA antagonist (AP-5, 4.0 µg/200nl), or CGRP receptor antagonist (BIBN4096BS at 8 µg/200nl). For optogenetic (opto) blocking, CGRP-CreER mice injected bilaterally in the PB with AAV-Flex-Opn3-mscarlet (inhibitory opsin) and implanted with bilateral optical fibers in the terminal fields received laser light pulses for opto-inhibition. Both groups were implanted and recorded for sleep following acute inflammatory pain (AIP) of a 10 µl foot injection of 5% formalin in combination either with or without terminal field blocking. Results The effects on sleep-wake after pharmacological blocking or opto-inhibition were compared to saline and AIP groups. AIP induced a robust 54% decrease in sleep for 3h post-injection (F1, 17= 105; P 0.001) compared to saline. Preceding NMDA antagonist injections in the BNST and BF reversed AIP-induced sleep-loss by 95% (F4, 28= 17.1; P 0.001), followed by CeA (83% reversal). While blocking CGRP receptors (F4, 28= 12.2; P 0.001) followed the order BF BNST CeA for recovering AIP-induced sleep loss. A similar order of effect (F4, 23= 31.6; P 0.001) was produced by opto-inhibition of PBelCGRP neuron terminal fields at BF, BNST and CeA. Conclusion Our results show that PBelCGRP neurons likely target BF, BNST, and CeA by acting through both CGRP and NMDA receptors to promote awakenings in response to acute pain. Support (if any) NS112175