Abstract

Sleep is an essential bodily process that hopefully, we all partake in every night. It aids in repairing and restoring the body and overall, is paramount to our physical and mental well-being. 

Although, the benefits of sleep are widely known, there is still much to learn about how sleep works, especially at the cellular level. Research investigating sleep at the cellular level has primarily focused on how neurons contribute to sleep and sleep processes. However, recent research has looked into how the most abundant cell type in the brain, astrocytes, are involved with sleep regulation (Haydon, 2017). One study conducted by Vaidyanathan and colleagues at the University of California San Francisco specifically aimed to investigate how activation of G-protein pathways coupled to receptors astrocytes can express aid in mediating sleep depth and duration. Their findings support that activation of astrocytic G-protein pathways independently mediate NREM sleep depth and duration (Vaidyanathan, 2021). 

Background

Sleep is comprised of three non-rapid eye movement (NREM) and one rapid eye movement (REM) sleep stage, and all occur multiple times a night in succession. The NREM sleep stages one, two, and three consist of slowing breathing, stopping eye movements, and during NREM sleep stage three, slow brain waves or slow wave activity (SWA) begins. Following NREM sleep stage three, the body transitions into REM sleep where rapid eye movements and dreaming occurs (Patel, 2021). 

As mentioned, astrocytes are the most abundant cell type in the brain (Miller, 2018). Astrocytes are specialized star-shaped glial cells that aid in extracellular maintenance (Allen, 2009). These cells have recently been implicated to participate in chemical transmission by expressing neurochemical receptors and releasing neurotransmitters which, when released by a glial cell, are denoted as gliotransmitters. Some of the neurochemical receptors that astrocytes can express include G-protein coupled receptors (GPCRs). These receptors bind neurotransmitters and upon binding, activate the G-protein coupled to the receptor. The activation of these G-proteins leads to downstream intracellular events that include but are not limited to calcium movement and protein translation (Rosenbaum, 2014). While there are many types of GPCRs that exist, there are only four types of G-proteins that are coupled to these receptors. Two G-proteins of importance that can be coupled to astrocytic GPCRs include inhibitory Gi and inhibitory or stimulatory Gq proteins (Nagai, 2021). 

Upon G-protein pathway activation one downstream action that can take place is calcium movement. Previous to the work conducted by Vaidyanathan and colleagues on specific astrocytic G-protein pathways, another study generally investigated whether astrocytic G-protein pathway activated calcium movement contributes to NREM SWA. It was found that astrocytic calcium movement mediates transitions from high to low SWA during NREM sleep stage three (Bojarskaite, 2020). The study conducted by Vaidyanathan and colleagues compounded upon this finding to investigate what specific type of astrocytic G-protein pathway is mediating these transitions and which, if at all, other types of G-proteins contribute to NREM sleep mediation. 

Methods

The study first targeted the Gi-coupled IP3 receptor 2 (IP3R2) expressed in mice cortical astrocytes to assess if calcium movement by IP3R2 pathway activation was responsible for transitions from low to high SWA. To do this, the astrocytic IP3R2 was experimentally removed in one adult mice group and compared the results to a control group. Prior to analysis, both mice groups were anesthetized and a headplate to measure brainwave activity was surgically implanted. Following implantation, the mice received an injection that fluorescently tagged astrocytic calcium to monitor calcium movement. After this, SWA activity and calcium events were monitored for both mice groups and compared against each other.   

The second part of this study targeted astrocytic Gq protein pathways in mice cortical astrocytes. These Gq proteins were targeted by genetically tagging them with Gq-DREADD. DREADDs can be experimentally activated with application of a chemical called CNO (Smith, 2016). The Gq-DREADD positive mice were compared to a control group and both groups were hooked up to electrodes to monitor brain wave activity. Then, the Gq-DREADD positive mice received an injection of CNO, and the control group received an injection of saline. The CNO injection activated the Gq-DREADDs and therefore mice cortical astrocyte Gq-protein pathways. For both groups the time spent in NREM sleep and time spent awake was measured and compared against each other. 

Results

Upon experimentally removing the IP3R2 Gi-coupled GPCR in mice cortical astrocytes there was no IP3R2 pathway mediated calcium movement and little to no SWA compared to controls. With the experimental removal of astrocytic IP3R2, the relationship between transitions from high to low SWA and calcium movement was completely diminished. These results support that the calcium events driving these SWA transitions are mediated by activation of the Gi-protein pathway coupled to the astrocyte expressed IP3R2. Due to this IP3R2 pathway mechanism, it can also be said that IP3R2 pathway activation mediates sleep depth as sleep depth can be denoted as the amount of SWA (Kurth, 2016) . In the second part of the experiment mice cortical astrocyte Gq-protein pathways were targeted through the use of Gq-DREADD. When CNO was applied to the mice cortical astrocytes to activate the Gq-DREADD and therefore the Gq-protein pathway, a significant increase in time spent in NREM sleep and a significant decrease in time spent awake was observed. This finding supports that cortical astrocytic Gq-protein pathway activation contributes to sleep duration. Overall, these findings support that cortical astrocytes play a key role in mediating sleep depth and duration via activation of different G-protein pathways.

Conclusion

Sleep research of the past has primarily focused on how neurons contribute to this process. However, new research, including the study discussed in this article, has focused on how astrocytes participate in sleep and sleep-related processes. In the study conducted by Vaidyanathan and colleagues, mice cortical astrocytes were used to investigate how astrocytic G-protein pathway activation contributes to sleep. Upon investigation it was found that independent activation of astrocytic Gi and Gq protein pathways lead to NREM sleep depth and duration mediation. This research provides greater insight into sleep at the cellular level. Although exploring sleep at the cellular level is a complex process, the work done by Vaidyanathan and colleagues unmask key pieces to the puzzle that is sleep. To further explore the relationship between astrocytes and sleep, future research could look into how astrocytic G-proteins in brain areas associated with sleep contribute to sleep processes. 

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