Dysfunctions of the paraventricular hypothalamic nucleus induce hypersomnia in human and mice

Hypersomnolence disorder (HD) is characterized by excessive sleep, which is a common sequela following stroke, infections or tumorigenesis. HD was traditionally thought to be associated with lesions of wake-promoting nuclei. However, lesion of a single, even two or more wake-promoting nucleuses simultaneously did not exert serious HD. The specific nucleus and neural circuitry for HD remain unknown. Here, we observed that three patients with lesions around the paraventricular nucleus of the hypothalamus (PVH) showed hypersomnolence lasting more than 20 h per day and their excessive sleep decreased with the recovery of the PVH area. Therefore, we hypothesized that the PVH might play an essential role in the occurrence of HD. Using multichannel electrophysiological recording and fiber photometry, we found that PVHvglut2 neurons were preferentially active during wakefulness. Chemogenetic activation of PVHvglut2 neurons potently induced 9-h wakefulness, and PVHCRH, PVHPDYN and PVHOT neuronal activation also exerted wakefulness. Most importantly, ablation of PVHvglut2 neurons drastically induced hypersomnia-like behaviors (30.6% reduction in wakefulness). These results indicate that dysfunctions of the PVH is crucial for physiological arousal and pathogenesis underlying HD.


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Hypersomnolence disorder (HD) is characterized by an irresistible need for sleep and an 36 inability to stay awake during major waking episodes, which results in reduced function 37 and overall worse quality of life and even induces mental diseases, highlighting its public 38 health importance 1 . However, few dysfunctional wake-promoting nuclei have been 39 identified to induce hypersomnia. Therefore, further identification of key hypersomnia 40 control nuclei and neural circuitry represents a common goal for clinicians and researchers. 41 In the last 100 years, more than 16 wake-promoting nuclei have been identified. Von 42 Economo first proposed a hypersomnia-controlling region located in the posterior 43 hypothalamus from observations of marked somnolence in patients with epidemic 44 encephalitis lethargic [1]. Furthermore, Moruzzi et al. and other studies have revealed that 45 and after treatments, we found that the lesioned area in FLAIR sequences became smaller 114 and that TST was decreased (case c, Figures 1A and 1D), suggesting that dysfunctions of 115 the PVH area might act as a central node for the occurrence of HD.  [9, 15-17, 26, 27], suggesting that the PVH might act as a key central node 128 for sleep-wake regulation.  approximately 9 h and concomitantly decreased both NREM and REM sleep ( Figure 4C).

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No sleep rebound followed the long-lasting wakefulness, as indicated by no change in the 169 time spent in NREM sleep during the following dark period (19:00-07:00; Figure 4E).

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Besides, there is no significant difference in the EEG power density of NREM sleep during 171 the day (7:00-18:00) before/after the day of CNO injection ( Figure 4G). Similarly, CNO 172 injection during the dark period also significantly increased wakefulness ( Figure   173 supplement 4), further demonstrating that activation of PVH vglut2 neurons prolonged 174 arousal even during the dark (active) period.

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Considering the potent wake-promoting effect of PVH vglut2 neurons and the 176 millisecond-scale control of neuronal activity through optogenetic manipulation, we next 177 employed optogenetic methods to elucidate the causal role of the PVHVglut2 neurons in 178 controlling wakefulness. We stereotaxically injected AAVs expressing channelrhodopsin-       However, bidirectional chemogenetic manipulations that inhibit the PPT or activate SLD 247 neurons have been shown to have little influence on baseline sleep [29,30]. In our present 248 study, three patients with lesions mostly around the PVH showed hypersomnolence lasting 249 above 20 h per day. PSG recordings from these patients showed that stage-two NREM was 250 strikingly dominant, indicating that these patients slept stably and were not easily 251 awakened. Importantly, we found that following recovery from injury around the PVH in 252 one of these patients, the proportion of stage-two NREM sleep decreased, and this patient 253 was concomitantly better able to stay awake. Furthermore, ablation of PVH vglut2 neurons in 254 mice induced a 30.6% reduction in wakefulness across the 24-h light/dark cycle,

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Taken together, our present findings provide evidence of the sufficient and necessary wake-262 promoting action of PVH vglut2 neurons in preventing hypersomnia.

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The PVH is composed of abundant, diverse, and functionally distinct groups of 264 neuroendocrine neurons, including CRH, OT,and PDYN neurons[23,24,33,34]     Shenzhen, China). The skin above the skull was cut, a burr hole was made, and a small 337 craniotomy was performed above the PVH. AAV constructs were slowly injected (30 338 nL/min) into the bilateral PVH (70 nL for each position; AP = -0.5 mm; ML = ±0.2 mm; 339 DV = -4.2 mm) for PSG recordings and brain-slice electrophysiology, or were unilaterally 340 injected into the PVH for neuronal tracing. The glass pipette was left in the brain for an 341 additional 10 min following injections and was then slowly withdrawn. All mice were 342 implanted with electrodes for EEG and EMG recordings that were used for in-vivo tests at 343 four weeks after injections under anesthesia of chloral hydrate (intraperitoneal, 360 mg/kg).

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The implant consisted of two stainless steel screws (1 mm in diameter), and EEG electrodes 345 were inserted through the skull (+1.5 mm anteroposterior; -2.0 mm mediolateral from 346 bregma or lambda), while two flexible silver wires were inserted into the neck muscles.

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The electrodes were attached to a mini-connector and were fixed to the skull with dental 348 cement. The scalp wound was sutured, and the mouse was when kept in a warm 349 environment until it resumed normal activity.  low-frequency (0.5-4 Hz) EEG signals in the absence of motor activity; and REM sleep 365 was considered to have pronounced theta-like (4-9 Hz) EEG activity and muscle atonia.