Introduction

Sleep is a regulatory process that is essential for everyone. It aids in overall bodily homeostasis by restoring, reenergizing, and repairing the body. Sleep is driven by two interacting processes: sleep homeostasis and circadian rhythms. Together these two processes create the sleep and wake cycle where sleep homeostasis drives sleep promotion and circadian rhythms serve as the body’s internal clock controlling the timing between sleep and wake (Achermann, 2004).

There are multiple stages within sleep that include three non-rapid eye movement (NREM) sleep stages and one rapid eye movement (REM) sleep stage. These stages comprise the sleep cycle and all four happen multiple times a night (Vaidyanathan, 2021). In the NREM sleep stages, the body transitions to wakefulness to sleep by dropping temperature, slowing breathing, slowing blood pressure, and stopping eye movements. During the third stage of NREM sleep, slow brain waves called delta waves begin to emerge before transitioning into REM sleep. Slow wave or delta wave activity can be used to measure sleep depth where an increase in slow wave activity indicates an increase in sleep depth. In the REM sleep stage, the eyes move rapidly and dreaming occurs (Vaidyanathan, 2021).  

There are multiple brain areas associated with sleep. One of high importance is the hypothalamus which houses the suprachiasmatic nucleus (SCN) and the ventrolateral preoptic area (VPLO). The SCN is the control center for circadian rhythms and receives light input from retinal ganglion cells via glutamatergic signaling. The SCN also receives input from the midbrain serotonin neuromodulatory center, the raphe nucleus. Serotonin input from the midbrain to the SCN helps control phase shifts in circadian rhythms where an increase in serotonin induces phase shifts (Prosser, 2003). The VPLO is associated with sleep promotion and neurons housed in this brain area are primarily galanin and GABAergic. These inhibitory neurons help promote sleep by inhibiting the ascending arousal system (Kim, 2020). Together the SCN and VPLO are important to sleep as they independently mediate the two interacting sleep processes: sleep homeostasis and circadian rhythms.

Another important aspect of sleep is waste clearance. During sleep, the brain clears waste via the glymphatic pathway. This pathway is reliant on glial cells to clear proteins, toxins, and metabolic wastes by cerebrospinal fluid (CSF) flow through perivascular spaces (Hablitz, 2020). While multiple cell types comprise the glymphatic system, astrocytes are a primary mediator of waste processing. Astrocytes tile the entire brain and mediate extracellular homeostasis. These cells create a syncytium throughout the brain connected by gap junctions. Increasing research has supported that astrocytes participate in chemical transmission by releasing gliotransmitters and expressing neurochemical receptors. While most of our knowledge of sleep at the cellular level revolves around neurons, recent research has focused on how astrocytes contribute to sleep processes (Haydon, 2017). Knowing that these glial cells can participate in neurochemical transmission and are expressed in brain areas associated with sleep, a relationship between astrocytes and sleep can be hypothesized. In this review, I explore the relationship between astrocytes and sleep and their contribution to these processes.

SCN and VPLO astrocytes aid in circadian rhythms and sleep homeostasis

As mentioned, the SCN and VPLO are important brain areas associated with mediating sleep homeostasis and circadian rhythms. One study used optogenetics in mice to assess if VPLO astrocytes contribute to sleep promotion where upon activation, VPLO astrocytes promoted sleep and increased sleep duration (Kim, 2020). Researchers observed that astrocyte stimulation during the mice’s wake state induced sleep with a percent increase in NREM slow wave sleep and REM sleep as well as a decrease in percent time awake. Along with sleep promotion, optogenetic stimulation of VPLO astrocytes increased extracellular ATP and c-Fos expression in VPLO neurons. To further support these astrocyte mechanisms, researchers metabolically inhibited VPLO astrocytes which reduced overall sleep duration, extracellular ATP, and neuronal c-Fos expression and increased percent time spent awake in the mice (Kim, 2020). Other studies have noted that VPLO astrocyte gliotransmission, specifically adenosine, aids in homeostatic sleep drive also denoted as sleep pressure (Nadjar, 2013). One study used DnSNARE expression in mouse VPLO astrocytes to see how inhibiting astrocyte adenosine release affected sleep pressure. When DnSNARE was expressed in astrocytes, NREM sleep pressure was significantly reduced compared to controls supporting that astrocyte adenosine release is important for sleep promotion (Halassa, 2009).

Another study investigated how SCN astrocytes contribute to circadian rhythm mediation. In the SCN, neurons are more active during circadian day while astrocytes are more active during circadian night (Brancaccio, 2017).  During circadian night the activity of SCN neurons is suppressed and this inhibition can be attributed to increased extracellular glutamate concentration (Brancaccio, 2017). GABAergic neurons can respond to glutamate by expressing glutamate receptors. When glutamate binds it increases the release of GABA which subsequently increases inhibition. One study genetically ablated SCN astrocytes in mice to investigate the relationship between astrocytes and increased nighttime extracellular glutamate concentrations. Extracellular glutamate levels in the SCNs of mice were fluoresced and compared between astrocytic or neuronal ablation (Brancaccio, 2017). Florescent level analysis showed a significant decrease with astrocytic ablation but not neuronal ablation suggesting that extracellular glutamate levels in the SCN rely heavily on astrocytes (Brancaccio, 2017). Another study found that genetically ablating SCN mouse astrocytes significantly lengthened the period of circadian rhythms supporting that astrocytes act as circadian oscillators. To further support this finding, this study genetically ablated the clock genes expressed in SCN astrocytes which resulted in the same increase in circadian period length (Tso, 2017). Combined, astrocytes in the VPLO and SCN perform different tasks but ultimately affect and directly contribute to the essential sleep processes that these brain areas are associated with.

Astrocytes control NREM sleep depth and duration independently through different GPCR pathways

While there are many types of G-protein coupled receptors (GPCRs), there are only four families of the G-proteins coupled with these receptors. Two of which astrocytes can express in conjunction with many possible GPCRs are inhibitory Gi and stimulatory or inhibitory Gq proteins (Nagai, 2021). Analysis of mouse neocortical astrocytic calcium movement by G-protein activation has been shown to regulate slow wave sleep. One study monitored this calcium movement in mice during natural sleep where a relationship between astrocytic calcium movement and transitions from high to low slow wave activity was observed (Bojarskaite, 2020). Compounding on this, another study used mouse cortical astrocytes to investigate if these calcium events were mediated by the astrocytic Gi-coupled IP3 receptor 2 (IP3R2) pathway. Slow wave activity and IP3R2 calcium events for sleep and stationary wake were monitored for wild type mice and IP3R2 knockout mice (Vaidyanathan, 2021). The IP3R2 knockout mice had less calcium movement and subsequently less slow wave activity. Thus, indicating that slow wave activity (sleep depth) is mediated by calcium events that follow astrocytic IP3R2 pathway activation.

To further assess the contribution of astrocytic G-protein pathways in sleep related processes, mouse cortical astrocyte Gq protein pathways were activated through Gq-DREADD (Vaidyanathan, 2021). It was found that astrocytic Gq protein pathway activation effects sleep duration where a significant percent increase in time spent in NREM sleep and a significant decrease in time spent awake was shown (Vaidyanathan, 2021). This supports that astrocytes, specifically Gq proteins coupled to GPCRs expressed on astrocytes, play a role in NREM sleep duration. Furthermore, these results support independent cortical astrocyte mediation of NREM sleep depth and duration by Gi and Gq protein pathway activation.

Astrocytes support glymphatic flow

During the nighttime cleansing processes of the glymphatic pathway, glymphatic fluid flows through perivascular spaces in the brain. This flow is mediated by aquaporin channel four (AQP4) that is specifically localized on the end feet of astrocytes that surround brain vasculature (Hablitz, 2020).  One study assessed the importance of AQP4 for glymphatic flow by comparing fluorescence levels between wild type and AQP4 knockout mice. The amount of fluorescence was correlated with glymphatic fluid penetration throughout the brain. A red fluorescent tracer was injected intracisternally into wild type and AQP4 knockout mice where brain slices were assessed for percent area of fluorescence.  The AQP4 knockout mice exhibited less fluorescence compared wild type mice indicating less penetration of glymphatic fluid (Mestre, 2018). Although the glymphatic system is reliant on glial cells, astrocytes are specifically important as they allow for full penetration of glymphatic fluid within the brain via the astrocyte specific AQP4.

Conclusion

As mentioned, sleep is an essential regulatory process that is important for our overall well-being. Gaining an understanding of sleep is paramount as it provides insight to sleep related disorders and a greater understanding of the human body. The new research outlined in this review highlighted the importance of astrocytes in sleep and sleep related processes. The results of these studies indicated that astrocytes of the VPLO induced sleep, SCN astrocytes mediated extracellular glutamate levels, astrocytic Gi and Gq coupled GPCRs regulated sleep depth and duration, and astrocyte localized APQ4 is essential for glymphatic fluid flow. This research also provides a new avenue for further studies as there is still much unknown about astrocytes and their contribution to sleep.

Understanding sleep at the cellular level and knowing astrocytes contribute to this opens new doors for potential drug targets for pharmaceutical companies. Future research based on these studies could focus on how astrocytes in brain areas associated with sleep are affected by common sleep medications. Many pharmaceuticals for insomnia on the market are benzodiazepines which act as GABA agonists. Astrocytes can express GABA receptors and release GABA and research has supported the relationship between benzodiazepines action and astrocytes (Hertz, 2006). However, none to date has investigated how this relationship effects sleep. Another avenue of potential research stemming from these studies could investigate how astrocytes of the reticular formation contribute to transitions from sleep to wakefulness. Previous research has investigated how astrocytes of the reticular formation contribute to endogenous benzodiazepine inhibition by releasing GABA (Christian, 2013). Although, little is known about how astrocytes contribute to the ascending arousal actions that the reticular formation is known for. Future research investigating the relationship between astrocytes and sleep processes will hopefully provide greater insight into the complex and essential process that is sleep.

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[+] Other Work By Alexandra Pederson