Max B. Kelz

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Introduction: Here, we create a conditional Adra2a line and use it to show that sedative, hypnotic, and hypothermic effects of α ₂ -agonists are neuronally mediated via the α _2A adrenergic receptor. Methods: We generated mice with loxP sites flanking Adra2a using CRISPR/Cas9 gene targeting. This line was crossed with lines encoding Cre recombinase (Cre) under the control of the Vgat, Snap25, and Dbh promoters. Cell-specific knockout was confirmed using fluorescent in-situ hybridization demonstrating targeted reduction in Adra2a mRNA in the appropriate cell types. Mice were given intraperitoneal dexmedetomidine (0.3 or 1 mg/kg) or saline, and 20 minutes later righting reflex was assessed, followed by 3 rounds of rotarod testing, with fall time and end temperature recorded. Spontaneous activity was recorded using beam break for an hour after. Mice of each genotype were implanted with EEG leads and recorded while given 0.3 mg/kg IP dexmedetomidine. Results: We created a conditional knockout and demonstrated cell-type specific reduction of Adra2a mRNA in crossed lines with cell-specific Cre. The pan-neuronal Adra2a knockout showed resistance to all temperature, sedative, and hypnotic effect endpoints in response to the α ₂ -agonist dexmedetomidine. Adrenergic knockout demonstrated resistance to α ₂ -agonist hypnosis and moderate resistance to hypothermia and impaired coordination with forced movement. GABAergic knockout showed resistance only to impairment of spontaneous movement by α ₂ -agonists. Spectral analysis of the EEG showed an increase in proportion of delta power with a sedative dose of dexmedetomidine in all lines except the pan-neuronal Adra2a knockout. Discussion: Future studies will pursue both the specific subtype(s) and location of neuronal populations responsible for sedative, hypnotic, and hypothermic effects of α ₂ -agonists.

The cortical mechanisms that actively suppress consciousness remain poorly understood. Here, we identify a specific subset of prefrontal cortical (PFC) neurons preferentially active under anesthesia while most neurons are suppressed. Chemogenetic activation of these excitatory neurons enhances anesthetic potency and deepens NREM sleep, whereas their inhibition blunts anesthetic effects. We identify these NREM and Anesthesia Promoting (NAP) neurons as PFC Layer 5 extratelencephalic (L5 ET) neurons. Remarkably, NAPs have sparse cortical projections and predominately communicate with subcortical nuclei including anterior and reticular thalamic nuclei, hypothalamus, and claustrum. We identify a transgenic mouse line that labels L5 ET neurons and verify that PFC L5 ET neurons are uniquely activated under anesthesia. Furthermore, we show that activation of PFC L5 ET neurons promotes deep NREM sleep. These findings identify a unique excitatory PFC circuit that promotes both naturally-occurring and drug-induced unconsciousness, with implications for both sleep regulation and anesthetic action.

Despite the widespread use of clinical anesthesia, the process of emergence from general anesthesia remains primarily driven by anesthetic elimination. Although emergence from general anesthesia is typically safe, prolonged delays strain resource-intensive settings and contribute to increased healthcare costs. In addition to improving access to care, providing clinicians with more precise control over emergence could offer diagnostic potential and improve patient outcomes. For decades, this unmet need has motivated research into the mechanisms underlying anesthetic emergence. Now, the first agents for facilitating emergence are entering the market, with more in development. This narrative review critically evaluates advancements in the development of emergence-promoting therapies, examining insights from preclinical research to clinical trials. This study categorizes prospective emergence agents/strategies into one of three primary approaches: (1) strategies that primarily manipulate anesthetic pharmacokinetics, (2) agents designed to directly target anesthetic receptor–binding sites, and (3) strategies that leverage arousal-promoting neural pathways. The parallel development of these approaches, each with their distinct strengths and limitations, holds promise for paving the way for a tailored approach to facilitate emergence.

Understanding the neurophysiological changes underlying conscious-unconscious transitions is a key goal in neuroscience. Using magnetic resonance neuroimaging, we investigate the network connectivity and neurovascular changes occurring as the human brain transitions from wakefulness to dexmedetomidine-induced hypnosis, and recovery. Hypnosis led to widespread decreases in functional connectivity strength and increased structure-function coupling, indicating functional patterns more constrained by the underlying anatomical connectivity. As individuals began to regain consciousness, both connectivity markers returned towards awake levels, with particularly prominent coupling changes across the cerebellum. Neurovascular dynamics were disrupted during hypnosis as well: cerebral blood flow decreased globally—most notably in the brainstem, thalamus, and cerebellum—and continued decreasing even as recovery commenced, except within the cerebellum. Notably, regions with higher functional connectivity strength during wakefulness exhibited greater blood flow reductions during hypnosis. Hypnosis also heightened the amplitude of low-frequency fluctuations in the hemodynamic signal, especially in visual and somatomotor regions. Critically, individuals who regained consciousness faster displayed higher baseline levels of both neurovascular, but not connectivity, markers. Together, these results reveal that the induction of, and emergence from, dexmedetomidine-induced unconsciousness involve widespread, coordinated changes in brain connectivity and neurovascular function; across our findings, we also highlight the recurrent role of cerebellum in conscious-unconscious transitions.

Nitrous oxide (N₂O) induces rapid and durable antidepressant effects. The cellular and circuit mechanisms mediating this process are not known. Here we find that a single dose of inhaled N₂O induces rapid and specific activation of layer V (L5) pyramidal neurons in the cingulate cortex of rodents exposed to chronic stress conditions. N₂O-induced L5 activation rescues a stress-associated hypoactivity state, persists following exposure, and is necessary for its antidepressant-like activity. Although NMDA-receptor antagonism is believed to be a primary mechanism of action for N₂O, L5 neurons activate even when NMDA-receptor function is attenuated through both pharmacological and genetic approaches. By examining different molecular and circuit targets, we identify N₂O-induced inhibition of calcium-sensitive potassium (SK2) channels as a key molecular interaction responsible for driving specific L5 activity along with ensuing antidepressant-like effects. These results suggest that N₂O-induced L5 activation is crucial for its fast antidepressant action and this effect involves novel and specific molecular actions in distinct cortical cell types.