Arousal Promoting Effects of Basal Forebrain are Mediated through Prefrontal Cortex in Rat

Background We recently showed that cholinergic stimulation of prefrontal cortex (PFC), via local carbachol delivery, increased local acetylcholine (ACh) levels and restored wakefulness in sevoflurane-anesthetized rats.[1] PFC receives cholinergic projections from basal forebrain (BF), and activation of cholinergic BF neurons has been shown to promote behavioral arousal.[2] Increase in prefrontal ACh during carbachol-induced wake state reflects BF activation, but it is not known if BF acts via PFC to promote behavioral arousal. The objective of the current study is to test the hypothesis that PFC gates the arousal promoting effects of BF. Methods Under isoflurane anesthesia, Sprague Dawley rats (n=3) were implanted with bipolar wire electrodes (500um diameter) bilaterally into BF (Bregma: posterior 0.48mm, mediolateral 2.0mm, ventral 8.2mm), screw electrodes across the cortex to record electroencephalogram (EEG), and a bilateral guide cannula aimed at PFC (Bregma: anterior 3.0mm, mediolateral 0.5mm, ventral 4.0mm). Another group of Sprague Dawley rats (n=2) was implanted with the electrical stimulation electrodes into piriform cortex (Bregma: posterior 0.48mm, mediolateral 5.0mm, ventral 9.0mm), as a control anatomical site. All coordinates are as per the stereotaxic atlas by Paxinos and Watson.[3] After 7-10 days of post-surgical recovery, we conducted bilateral constant current electrical stimulation of BF (200Hz, 30s ON and 30s OFF - three cycles at 60uA and three cycles at 100uA) in sevoflurane-anesthetized rats (1.9-2.4%) with or without concurrent inactivation of PFC via local bilateral infusion of tetrodotoxin (156µM, 500nL). The same paradigm was used for electrical stimulation of piriform cortex. Results Electrical stimulation of BF in sevoflurane-anesthetized rats induced signs of behavioral arousal (whisker, orofacial and limb movements, and attempts at/regaining of the righting reflex), activated the EEG, and increased the heart (~13%) and respiration (~67%) rate. By contrast, electrical stimulation of BF in the presence of tetrodotoxin in PFC failed to induce behavioral arousal and EEG activation. Electrical stimulation of the control anatomical site (piriform cortex) in sevoflurane-anesthetized rat did not induce behavioral arousal. Conclusions These results suggest that the arousal promoting effects of BF are gated through PFC, potentially through a cholinergic circuit. References 1. Pal D et al. Differential Role of Prefrontal and Parietal Cortices in Controlling Level of Consciousness. Curr Biol 2018; 28:2145-2152 e5.2. Luo TY et al. Basal Forebrain Cholinergic Activity Modulates Isoflurane and Propofol Anesthesia. Front Neurosci 2020; 14:559077.3. Paxinos G. & Watson C. The Rat Brain in Stereotaxic Coordinates. 2007. Academic Press.