The Ventral Subiculum Promotes Wakefulness Through Multiple Pathways in Male Mice

Background

The ventral subiculum (vsub) is a major output region of the hippocampus, playing a crucial role in motivation, stress integration, and anxiety-like behavior, all of which depend on a high state of wakefulness. However, the role of vsub in wakefulness and its underlying neural circuits are poorly understood. Using in vivo fiber photometry calcium imaging and multi-channel electrophysiological recordings, we found that glutamatergic neurons in the vsub exhibit high activity during wakefulness. Additionally, activation of vsub glutamatergic neurons increases wakefulness and anxiety-like behavior, and induces rapid transitions from sleep to wakefulness. Optogenetic stimulation of vsub glutamatergic terminals and retrograde chemogenetic activation of vsub glutamatergic neurons reveal that vsub regulates wakefulness through the lateral hypothalamus (LH), nucleus accumbens shell (NAc), and prefrontal cortex (PFC). Notably, local microinjection of dopamine D1 or D2/D3 receptor antagonists blocked the wakefulness effects induced by chemogenetic activation of the vsub pathways. Finally, chemogenetic inhibition of vsub glutamatergic neurons reduced wakefulness.

Source

This paper was authored by Xue-Fen Zhang, Yi-Dan Li, Yue Li, Ying Li, Dan Xu, Lin-Lin Bi, and Hai-Bo Xu, and published in the journal Neuropsychopharmacology. The research was conducted in collaboration with teams from the Radiology Department, Nuclear Medicine Department, Pathology Department of Wuhan University Zhongnan Hospital, and Taikang Medical School, among other institutions, and published in 2024.

Experimental Procedure

1. Subjects and Animal Handling: The study used 180 male C57BL/6 mice aged 8–20 weeks, weighing 20–30 grams. These mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were kept under appropriate temperature, humidity, and light cycle conditions. All animal experimental protocols were approved by the Ethical Committee on Animal Welfare of Zhongnan Hospital, Wuhan University (ZN2021159).

2. Virus Injection and Fiber Implantation: Under pentobarbital sodium anesthesia, mice underwent surgery for virus injection into the vsub using a stereotaxic apparatus. The injected viruses included AAV2/9-CaMKIIα-HM3Dq-mCherry, AAV2/9-CaMKIIα-HM4Di-mCherry, AAV2/9-CaMKIIα-ChR2-mCherry, etc. After virus injection, fibers were implanted to record the calcium activity of vsub glutamatergic neurons and EEG/EMG signals.

3. Fiber Photometry and Electrophysiological Recording: Using a fiber photometry system and a multi-channel electrophysiological recording system, neural activity was recorded during the spontaneous sleep-wake cycle in mice. The role of vsub glutamatergic neurons in wakefulness and anxiety-like behavior was studied by activating or inhibiting them using optogenetic and chemogenetic methods.

4. Behavioral Experiments: Open Field and Elevated Plus Maze experiments were used to assess anxiety-like behavior in mice. Additionally, real-time place preference experiments were employed to evaluate avoidance behavior.

5. Dopamine Receptor Blocking Experiments: To explore the role of DA signaling in sleep-wake regulation, dopamine D1 receptor antagonist SCH-23390 or D2/D3 receptor antagonist Raclopride were microinjected into different brain regions (NAc, LH, and PFC), and the wakefulness effects of chemogenetic activation of vsub pathways were assessed.

Experimental Results

1. High activity of vsub glutamatergic neurons during wakefulness: Real-time calcium activity recordings showed that the fluorescence signal of vsub glutamatergic neurons was significantly higher during wakefulness and REM sleep compared to non-REM (NREM) sleep. Multi-channel electrophysiological recordings also indicated that the firing frequency of vsub neurons was higher during wakefulness and REM sleep than during NREM sleep.

2. Activation of vsub glutamatergic neurons increases wakefulness and anxiety-like behavior: Chemogenetic (HM3Dq and CNO) and optogenetic (ChR2 and blue light) activation of vsub glutamatergic neurons significantly increased the wakefulness time and reduced NREM and REM sleep time in mice. Behavioral experiments showed that activation of vsub glutamatergic neurons led to changes in anxiety-like and avoidance behavior.

3. vsub regulates wakefulness through NAc, LH, and PFC pathways: Optogenetic activation of vsub projections to the NAc, LH, and PFC glutamatergic terminals each led to rapid transitions from NREM sleep to wakefulness. Specific chemogenetic activation of these projection paths further confirmed the role of vsub-glu-NAc, vsub-glu-LH, and vsub-glu-PFC pathways in wakefulness regulation.

4. Dopamine signaling is crucial in vsub pathway-induced wakefulness effects: Microinjection of dopamine receptor antagonists blocked the wakefulness induced by chemogenetic activation of vsub pathways, highlighting the importance of dopamine signaling in vsub wakefulness regulation.

5. vsub glutamatergic neurons are essential for maintaining wakefulness: Chemogenetic (HM4Di and CNO) inhibition of vsub glutamatergic neurons significantly reduced wakefulness time and increased NREM and REM sleep time in mice.

Conclusion and Significance

This study revealed the crucial role of vsub glutamatergic neurons in wakefulness regulation and identified neural circuit mechanisms that regulate wakefulness through different pathways (NAc, LH, and PFC). The results demonstrate the key role of dopamine signaling in vsub wakefulness regulation while also showing the essential nature of vsub glutamatergic neurons in maintaining wakefulness. This research provides new insights into sleep-wake regulation and offers potential targets for treating related sleep disorders.

Highlights and Value

1. Revealed new wakefulness-regulating regions and neural circuits: This study is the first to demonstrate the dominant role of vsub glutamatergic neurons in wakefulness and their mechanism of wakefulness regulation through different projection pathways (NAc, LH, and PFC).

2. Key role of dopamine signaling in wakefulness regulation: The study shows that dopamine receptor antagonists can block the wakefulness effects induced by activation of vsub pathways, proving the importance of dopamine signaling in wakefulness regulation.

3. Provides new targets for sleep disorder treatment: The findings offer a theoretical basis for developing intervention methods targeting vsub and its neural circuits, which have the potential to become new strategies for treating insomnia and other sleep disorders.

This study, completed by research teams from multiple institutions, is comprehensive and detailed, revealing the critical role and underlying mechanisms of the ventral subiculum in wakefulness and anxiety-like behavior. The research results have been stored in the FigShare database, ensuring transparency and enhancing the study’s credibility and reproducibility.