The current study showed immense impacts of 72 h SD on the neurons and microglia in adolescent brains. SD produced a hyperdopaminergic status, with escalated locomotor activity, increased neuronal activity in the striatum and midbrain DA neurons under Amph stimulation, as well as altered expression of striatal DA receptors. We also discovered distinct features of microglial activation in the striatum of SD mice. However, whereas treatment with an anti-neuroinflammation agent MINO normalized the increment of the striatal microglial density in SD mice, it did not rescue the behavioral changes in the Amph challenge test, implying that microglia reactivity might not be the major stem of the SD-induced hyperdopaminergia. In contrast, SD mice showed increased activity and sensitivity of the CRF signaling, which potently primes both DA system and microglia activity. Collectively, these findings support a fueling role of stress responses evoked by SD to the DA circuitry and the striatal microglia in adolescents. Our results also unraveled a potential mechanism of how sleep deprivation navigates adolescent individuals to the susceptibility to psychiatric illnesses.
4.1. 72 h SD induces a CRF signaling-mediated hyperdopaminergic status in adolescent mice
In good agreement with previous studies [14, 20], we found that SD elicited exaggerated responses to a novel environment and acute Amph stimulation in adolescent mice. Whereas most studies focused on changes in DA afferent regions such as the striatum and prefrontal cortex [14, 31], we herein demonstrated the significance of functional alternations in the midbrain DA neurons in the SD-induced hyperdopaminergic status. Using c-fos labeling as the indicator of neuronal activity, we found that aside from increased striatal c-fos density, SD also evoked robust c-fos expression in the VTA. It is to note that acute psychostimulant administration generally exerts inhibition effects through the somatodendritic-released DA [56] or forebrain innervation to the VTA DA neurons [57]. The increased VTA DA neuronal activity following the Amph challenge in the SD animals was akin to the pattern of repetitive drug administration [28, 29] as well as repeated stress exposure [58]. Notably, appreciable evidence demonstrated that drugs of abuse and stressful experience trigger common pathways to generate behavioral and neuronal sensitization by evoking neuroplastic changes in the VTA DA neurons [13]. Taken together, our findings postulate that the hyperdopaminergic status might attribute to the SD-induced sensitization in the mesolimbic DA system.
One of the prominent findings in the current study was that 72 h SD enhanced hypothalamic CRF signaling and increased midbrain CRF-R1 level. These results suggested that SD arouses CRF-mediated stress response and sensitivity. It is well known that the activation of the CRF system is a crucial aspect of the stress-induced sensitization of DA circuitries [52, 53, 13]. The CRF-containing neurons in the PVN, central amygdala, and bed nucleus of the stria terminalis innervate the VTA DA neurons and form excitatory synapses [59]. In the VTA, locally released CRF acts in concert with glutamate to modulate the activity of DA neurons mainly through CRF-R1 [53]. The regulatory effects of CRF on the VTA DA neurons are predominantly excitatory, which encompasses increasing neuronal firing [60], potentiating N-methyl-D-aspartate (NMDA) receptors [61], and evoking DA release in the target brain areas [62]. Systemic or intra-VTA administration of CRF receptor antagonists has demonstrated a vital role of CRF signaling in stress-induced drug-seeking behaviors [63, 64]. Besides, CRF may influence the DA circuitries indirectly by activating the HPA axis and the subsequent release of glucocorticoids. In our previous study, we found that 72 h SD elevated plasma corticosterone [24], which is highly lipophilic and able to diffuse into the CNS. Glucocorticoid receptors are widely expressed in neurons of the DA system [13]. Animal studies showed that corticosterone augments DA release into the NAc [65], and increases Amph or cocaine self-administration, and locomotor sensitization [66, 67]. Together, 72 h SD could trigger CRF-mediated stress responses, probably potentiates by both intra- and extra-hypothalamic CRF signaling pathways, leading to neuronal and behavioral sensitization of the DA system.
CRF signaling also mediates microglial functions directly under stressful conditions since microglia express both CRF-1R and CRF-2R [68]. CRF stimulation was found to enhance microglial production of proinflammatory cytokines TNF-α or IL-18 [69, 70], which is proposed to modulate the stress responses [71]. Taken together, enhanced CRF signaling upon SD might act as a potent trigger to both neuronal and microglial reactivity of the DA system.
Mounting evidence suggested a reciprocal relationship between sleep loss and stress responses [54, 55]. On one hand, the facilitated HPA activity is essential for maintaining wakefulness during the SD regimen, as a positive correlation was found between plasma cortisol level and arousal state [72]. On the other hand, exogenous CRF and glucocorticoid treatments could increase wakefulness, suppress sleep, and alter sleep architecture through interacting with sleep-wake regulatory neural circuitries, including the wake-promoting locus coeruleus and sleep-activating nuclei in the preoptic hypothalamus [73, 74]. Notably, the activity of VTA DA neurons and their projections to the NAc is necessary for arousal [75]. Correspondingly, the physiological arousing effects of the DA system were supported by pharmacological or genetic manipulations targeting DA receptors [76, 77]. The above pieces of literature, accompanied by our observations on elevated CRF and DA signaling after SD, have raised that the SD-dependent heightened CRF signaling might increase the excitability of the DA system, to fulfill the demand for inducing or sustaining arousal.
4.2. 72 h SD alters striatal DA receptor expression in adolescent mice
Besides exacerbated behavioral and neuronal responses to the Amph challenge, the current study also identified an SD-induced downregulation of striatal D2R at both mRNA and protein levels. Similarly, downregulated D2R had been recognized in the ventral striatum after chronic treatments of psychostimulants or agonists [78, 79]. One potential mechanism for SD-induced D2R reduction is the prolonged DA stimulation, which may lead to the surface internalization, endocytosis, and degradation of D2R [80, 81]. Previous studies demonstrated that SD elevates DA concentration or its metabolites in the striatum [82, 20]. Although D2R plays a crucial role in the maintenance of wakefulness [75, 76], sustained DA stimulation might result in the degradation of striatal D2R. Another tenable cause might be the activation of the adenosine receptor [83], which is co-expressed with D2R on striatal MSNs [84]. As a derivative of energy metabolism, extracellular adenosine accumulates during the waking state and employs modulatory effects through the activation of A1 and A2 receptors [85], which might decrease D2R binding affinity [86] or drive D2R surface internalization [87]. In addition, chronic stress exposure has been shown to reduce striatal D2R mRNA expression, emphasizing that the DA system is the affected pathway between sleep loss and stress responses [88, 89]. Collectively, the heightened DA signaling or energy expenditure evoked by SD or SD-related stress might be responsible for the reduction of D2R biosynthesis and expression.
In contrast to the downregulated striatal D2R expression, an increase in striatal D1R protein was observed in the SD mice. Specifically, the significance has occurred in the protein but not the mRNA level, raising the assumption of decreased D1R elimination rather than upregulated biosynthesis in the SD paradigm. In rodents, functional availability of D1R and D2R in the CPu and NAc peaks approximately between P28 to P40 and declines thereafter to adult levels [33, 34]. A similar developmental pattern of DA receptors was also reported in the human postmortem striatum, implicating a highly-conserved pruning process of DA receptors during adolescence [90]. Remarkably, it has been shown that microglia and complement-mediated immune signaling might contribute to the elimination of D1R in the adolescent NAc [38]. This developmental D1R pruning is associated with the successful transition of social behaviors from adolescent to adult, emphasizing the importance of proper neuron-microglia interactions in the maturating DA system [38].
Hypersensitivity of D1R and reduction of D2R, which result in the imbalance between the signaling of these two receptors, have been proposed to predispose the individual to addiction [91, 92]. It is supported by a correlation between sleep disturbance and susceptibility to cocaine addiction [93]. Therefore, alternations of DA receptors, as maladaptive responses engaging neuronal and microglial changes, elicited by sleep loss are likely to bring detrimental consequences such as vulnerability to substance-use disorders or extended neuropathology in the DA system. The intricate mechanisms underpin should be carefully elucidated in the future.
4.3. 72 h SD activates striatal microglia in adolescent mice
Microglia activation is well recognized in various SD regimens [21–23]. Likewise, our data of increased microglia density, altered microglial morphology, and upregulated transcription of a plethora of pro-inflammatory components suggest that 72 h SD primes microglia activation and neuroinflammation in the adolescent striatum. In our current model, SD effectively evokes stress responses and activates the neuroendocrine system which has a direct impact on microglia. Stress-coping hormones such as glucocorticoids, CRF, and norepinephrine could elicit microglia activation and subsequent cytokine productions [68]. Besides, the increase of danger-associated molecular patterns (DAMPs) is also a potent activator of microglia and neuroinflammation in the context of SD [94]. Endogenous DAMPs bind to their associated pattern recognition receptors on microglia and activate the downstream inflammatory pathways, such as increasing the transcription of NLRP3 or pro-inflammatory cytokines [95]. We, therefore, proposed that DAMPs generated by increased synaptic activity or accumulated cellular stress during SD contribute to the priming of inflammatory pathways in the striatum.
In addition to the profiles of neuroinflammation-related cytokines, we observed an increase in the complexity of microglial processes in the CPu of the SD mice. Hyper-ramified microglia have been identified in various chronic stress models [96–98]. Microglia hyper-ramification might be a transition phase from the resting to the activated state in response to injury [99]. Emerging evidence further suggests that microglia, equipped with a wild array of receptors, are activated by neurotransmitters and purinergic signaling [68], and elaborate their processes to intensify the surveillance, contacts, and modulation of the surrounding neuronal structures [100]. In the SD paradigm, aberrant neuronal activity might stimulate microglial morphological changes and interactions with the surrounding microenvironment.
Microglial-mediated neuronal activity and structural remodeling might take place during the sleep state [39–41]. In particular, our previous studies demonstrated that the lack of sleep prevents microglial engulfment and phagocytosis of synaptic materials in adolescent mice [22, 24]. Here, we provided evidence of downregulated CX3CR1 and CD11b, in the striatal homogenate of the SD mice. These molecules are microglia-specific components that participate in the developmental synaptic refinement [101] and synaptic remodeling of the DA system [38, 48]. Resonating with the mRNA assessment, the lysosomal marker CD68 was also found to decrease in the NAcC microglia of SD mice, implying a reduction in the microglial phagocytic capacity. Since microglia and complement-mediated immune signaling play role in the elimination of D1R in the adolescent NAc [38], our results suggested that the deficiency in microglia phagocytic capacity is a potential culprit for the increased remnant of striatal D1R after SD.
Microglia activation and neuroinflammation have been acknowledged by converging clinical and animal studies as risk factors for developing substance use disorders or other psychiatric illnesses associated with abnormal DA functions [25]. MINO administration has been shown to inhibit the microglial activation and neuroinflammatory events associated with the DA system, including drug-seeking behaviors, behavioral sensitization to psychostimulants, and reduced PPI [48–50]. In the present study, six days of MINO treatment reduced the SD-induced microglia increase in the striatum; however, it did not prevent the hyperactivity after Amph stimulation. These results implicated that microglia activation is not the only source of SD-induced hyperdopaminergic status. On a cautionary note, the abnormal DA signaling might be a convergence of different etiologies or multiple factors. Besides the activation of stress response systems, attenuated pruning processes, and priming of pro-inflammatory cascades occurred in our SD paradigm, which might not be prevented by a single MINO treatment. The consequences of various harmful factors brought by SD are highly intertwined with each other; we might develop therapeutic strategies using combined pathways.
4.4. Conclusion
Our current findings present an interplay between neuroendocrine, DA, and immune systems in sleep-deprived adolescent mice. We proposed that prolonged sleep loss stimulates the DA system to achieve the demand of arousal maintenance by activating the stress response systems. Furthermore, the elevated stress responses result in various neuronal and microglial adaptions in the DA systems, including altered striatal DA receptors, decreased microglial pruning capacity, and primed neuroinflammatory signaling, which contributes to enhanced sensitivity to psychostimulants in sleep-deprived adolescents. While a wealth of studies showed that SD models have prominent face validity to neuropsychiatric disorders such as schizophrenia, herein we provide evidence indicating similar underlying mechanisms covering the three major aspects, namely the aberrant neuroendocrine, DA, and immune signaling, between SD and a wild range of neuropsychiatric illnesses. The detrimental effects of sleep insufficiency on the DA system are potent risk factors for the development of mental disorders and should be considered deliberately in the prevention or therapeutic strategy for neuropsychiatric disorders.