BACKGROUND: Cholinergic stimulation of prefrontal cortex (PFC) can reverse anesthesia. Conversely, inactivation of PFC can delay emergence from anesthesia. PFC receives cholinergic projections from basal forebrain, which contains wake-promoting neurons. However, the role of basal forebrain cholinergic neurons in arousal from the anesthetized state requires refinement, and it is currently unknown whether the arousal-promoting effect of basal forebrain is mediated through PFC. To address these gaps in knowledge, we implemented a novel approach to the use of chemogenetic stimulation and tested the role of basal forebrain cholinergic neurons in behavioral arousal during sevoflurane anesthesia. Next, we investigated the effect of tetrodotoxin-mediated inactivation of PFC on behavioral arousal produced by electrical stimulation of basal forebrain during sevoflurane anesthesia. METHODS: Adult male and female transgenic rats ( Long-Evans-Tg [ ChAT-Cre ] 5.1 Deis ; n = 22) were surgically prepared for expression of excitatory hM3D(Gq) receptors or mCherry in basal forebrain cholinergic neurons, and activation of these neurons by local delivery of compound 21, an agonist for hM3D(Gq) receptors. The transgenic rats were fitted with microdialysis probes for agonist delivery into basal forebrain and simultaneous prefrontal acetylcholine measurement. Adult male and female Sprague Dawley rats were surgically prepared for bilateral electrical stimulation of basal forebrain and tetrodotoxin infusion (156 μM and 500 nL) into PFC (n = 9) or bilateral electrical stimulation of piriform cortex (n = 9) as an anatomical control. All rats were implanted with electrodes to monitor the electroencephalogram. Heart and respiration rates were monitored using noninvasive sensors. A 6-point scale was used to score behavioral arousal (0 = no arousal and 5 = return of righting reflex). RESULTS: Compound 21 delivery into basal forebrain of rats with hM3D(Gq) receptors during sevoflurane anesthesia produced increases in arousal score ( P < .001; confidence interval [CI], 1.80–4.35), heart rate ( P < .001; CI, 36.19–85.32), respiration rate ( P < .001; CI, 22.81–58.78), theta/delta ratio ( P = .008; CI, 0.028–0.16), and prefrontal acetylcholine ( P < .001; CI, 1.73–7.46). Electrical stimulation of basal forebrain also produced increases in arousal score ( P < .001; CI, 1.85–4.08), heart rate ( P = .018; CI, 9.38–98.04), respiration rate ( P < .001; CI, 24.15–53.82), and theta/delta ratio ( P = .020; CI, 0.019–0.22), which were attenuated by tetrodotoxin-mediated inactivation of PFC. CONCLUSIONS: This study validates the role of basal...

No abstract

Studies aimed at investigating brain regions involved in arousal state control have been traditionally limited to subcortical structures. In the current study, we tested the hypothesis that inactivation of prefrontal cortex, but not two subregions within parietal cortex—somatosensory barrel field and medial/lateral parietal association cortex—would suppress arousal, as measured by an increase in anesthetic sensitivity. Male and female Sprague Dawley rats were surgically prepared for recording electroencephalogram and bilateral infusion into prefrontal cortex (N = 13), somatosensory barrel field (N = 10), or medial/lateral parietal association cortex (N = 9). After at least 10 days of post-surgical recovery, 156 μM tetrodotoxin or saline was microinjected into one of the cortical sites. Ninety minutes after injection, rats were anesthetized with 2.5% sevoflurane and the time to loss of righting reflex, a surrogate for loss of consciousness, was measured. Sevoflurane was stopped after 45 min and the time to return of righting reflex, a surrogate for return of consciousness, was measured. Tetrodotoxin-mediated inactivation of all three cortical sites decreased (p &lt; 0.05) the time to loss of righting reflex. By contrast, only inactivation of prefrontal cortex, but not somatosensory barrel field or medial/lateral parietal association cortex, increased (p &lt; 0.001) the time to return of righting reflex. Burst suppression ratio was not altered following inactivation of any of the cortical sites, suggesting that there was no global effect due to pharmacologic lesion. These findings demonstrate that prefrontal cortex plays a causal role in emergence from anesthesia and behavioral arousal.

BACKGROUND: Neurophysiologic complexity has been shown to decrease during states characterized by a depressed level of consciousness, such as sleep or anesthesia. Conversely, neurophysiologic complexity is increased during exposure to serotonergic psychedelics or subanesthetic doses of dissociative anesthetics. However, the neurochemical substrates underlying changes in neurophysiologic complexity are poorly characterized. Cortical acetylcholine appears to relate to cortical activation and changes in states of consciousness, but the relationship between cortical acetylcholine and complexity has not been formally studied. We addressed this gap by analyzing simultaneous changes in cortical acetylcholine (prefrontal and parietal) and neurophysiologic complexity before, during, and after subanesthetic ketamine (10 mg/kg/h) or 50% nitrous oxide. METHODS: Under isoflurane anesthesia, adult Sprague Dawley rats (n = 24, 12 male and 12 female) were implanted with stainless-steel electrodes across the cortex to record monopolar electroencephalogram (0.5–175 Hz; 30 channels) and guide canulae in prefrontal and parietal cortices for local microdialysis quantification of acetylcholine levels. One subgroup of these rats was instrumented with a chronic catheter in jugular vein for ketamine infusion (n = 12, 6 male and 6 female). The electroencephalographic data were analyzed to determine subanesthetic ketamine or nitrous oxide–induced changes in Lempel-Ziv complexity and directed frontoparietal connectivity. Changes in complexity and connectivity were analyzed for correlation with concurrent changes in prefrontal and parietal acetylcholine. RESULTS: Subanesthetic ketamine produced sustained increases in normalized Lempel-Ziv complexity (0.5–175 Hz; P < .001) and high gamma frontoparietal connectivity (125–175 Hz; P < .001). This was accompanied by progressive increases in prefrontal (104%; P < .001) and parietal (159%; P < .001) acetylcholine levels that peaked after 50 minutes of infusion. Nitrous oxide induction produced a transient increase in complexity ( P < .05) and high gamma connectivity ( P < .001), which was accompanied by increases ( P < .001) in prefrontal (56%) and parietal (43%) acetylcholine levels. In contrast, the final 50 minutes of nitrous oxide administration were characterized by a decrease in prefrontal (38%; P < .001) and parietal (45%; P < .001) acetylcholine levels, reduced complexity ( P < .001), and comparatively weaker frontoparietal high gamma connectivity ( P < .001). Cortical acetylcholine and complexity were correlated with both subanesthetic ketamine (prefrontal: cluster-weighted marginal correlation [CW r] [144] = 0.42, ...

No abstract

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.