0045 Lateral Hypothalamic Neurotensin Neurons Are Necessary for Wake Maintenance

Abstract Introduction The lateral Hypothalamic area (LH) has been established as a critical site for regulating wakefulness. However, the exact neural elements within this region that promote and stabilize wakefulness are not completely understood. While the orexin neurons in the LH are well-known, other subpopulations within this area may also contribute to arousal control. In this regard, we have recently identified a subset of LH neurons expressing neurotensin (Nts) promoting wakefulness. Optogenetic activation of LH-Nts neurons rapidly induces arousal from non-rapid eye movement (NREM) sleep, while chemoactivation induces up to five hours of uninterrupted wakefulness. However, the precise role of these neurons in initiating and maintaining spontaneous wakefulness remains unknown. To address this question, we investigated 1) the activity dynamics of LH-Nts neurons across sleep-wake behavior and 2) the impact of their ablation on sleep-wake amounts and architecture. Methods Experiment 1 – LH-Nts neuron activity dynamics: Nts-Cre mice (expressing Cre recombinase specifically in Nts neurons) were injected with an adeno-associated viral (AAV) vector coding for a Cre-dependent GCaMPS (genetically encoded calcium sensors) into the LH. They were also implanted with optical fibers targeting 50µm above the injection site and EEG/EMG electrodes to assess sleep-wake. After three weeks, fiber photometry with concurrent sleep-wake recordings was performed for two hours during the light period in all mice. Experiment 2 – sleep-wake effects of LH-Nts neuron ablation: Nts-Cre mice were injected with an AAV coding for diphtheria toxin-A subunit (AAV-flex-DTA) or a control AAV coding for a red fluorescent protein (AAV-flex-mCherry) into the LH and were implanted with transmitters for the telemetric recording of EEG/EMG. Sleep-wake recordings were performed 24 hours at four weeks after surgery. All mice were then perfused, and injection/lesion sites were verified histologically. Results LH-Nts neurons were more active during Wakefulness and REM sleep when compared to NREM sleep. The loss of these neurons reduced the total amounts of Wake and the average duration of Wake bouts but increased the 0.5-4 Hz delta power in Wake EEG. Conclusion LH-Nts neurons are necessary for maintaining spontaneous wakefulness, and their loss leads to chronic sleepiness in mice. Support (if any) NIH R01-NS119223 (to R.V)