Insomnia-related rodent models in drug discovery.

Despite the widespread prevalence and important medical impact of insomnia, effective agents with few side effects are lacking in clinics. This is most likely due to relatively poor understanding of the etiology and pathophysiology of insomnia, and the lack of appropriate animal models for screening new compounds. As the main homeostatic, circadian, and neurochemical modulations of sleep remain essentially similar between humans and rodents, rodent models are often used to elucidate the mechanisms of insomnia and to develop novel therapeutic targets. In this article, we focus on several rodent models of insomnia induced by stress, diseases, drugs, disruption of the circadian clock, and other means such as genetic manipulation of specific neuronal activity, respectively, which could be used to screen for novel hypnotics. Moreover, important advantages and constraints of some animal models are discussed. Finally, this review highlights that the rodent models of insomnia may play a crucial role in novel drug development to optimize the management of insomnia.

Sex-specific expression of distinct serotonin receptors mediates stress vulnerability of adult hippocampal neural stem cells in mice.

Women are more vulnerable to stress and have a higher likelihood of developing mood disorders. The serotonin (5HT) system has been highly implicated in stress response and mood regulation. However, sex-dependent mechanisms underlying serotonergic regulation of stress vulnerability remain poorly understood. Here, we report that adult hippocampal neural stem cells (NSCs) of the Ascl1 lineage (Ascl1-NSCs) in female mice express functional 5HT1A receptors (5HT1ARs), and selective deletion of 5HT1ARs in Ascl1-NSCs decreases the Ascl1-NSC pool only in females. Mechanistically, 5HT1AR deletion in Ascl1-NSCs of females leads to 5HT-induced depolarization mediated by upregulation of 5HT7Rs. Furthermore, repeated restraint stress (RRS) impairs Ascl1-NSC maintenance through a 5HT1AR-mediated mechanism. By contrast, Ascl1-NSCs in males express 5HT7R receptors (5HT7Rs) that are downregulated by RRS, thus maintaining the Ascl1-NSC pool. These findings suggest that sex-specific expression of distinct 5HTRs and their differential interactions with stress may underlie sex differences in stress vulnerability.

Anterior cingulate cortex projections to the dorsal medial striatum underlie insomnia associated with chronic pain.

Chronic pain often leads to the development of sleep disturbances. However, the precise neural circuit mechanisms responsible for sleep disorders in chronic pain have remained largely unknown. Here, we present compelling evidence that hyperactivity of pyramidal neurons (PNs) in the anterior cingulate cortex (ACC) drives insomnia in a mouse model of nerve-injury-induced chronic pain. After nerve injury, ACC PNs displayed spontaneous hyperactivity selectively in periods of insomnia. We then show that ACC PNs were both necessary for developing chronic-pain-induced insomnia and sufficient to mimic sleep loss in naive mice. Importantly, combining optogenetics and electrophysiological recordings, we found that the ACC projection to the dorsal medial striatum (DMS) underlies chronic-pain-induced insomnia through enhanced activity and plasticity of ACC-DMS dopamine D1R neuron synapses. Our findings shed light on the pivotal role of ACC PNs in developing chronic-pain-induced sleep disorders.

Ventral pallidal glutamatergic neurons regulate wakefulness and emotion through separated projections.

Insomnia is often comorbid with depression, but the underlying neuronal circuit mechanism remains elusive. Recently, we reported that GABAergic ventral pallidum (VP) neurons control wakefulness associated with motivation. However, whether and how other subtypes of VP neurons regulate arousal and emotion are largely unknown. Here, we report glutamatergic VP (VPVglut2) neurons control wakefulness and depressive-like behaviors. Physiologically, the calcium activity of VPVglut2 neurons was increased during both NREM sleep-to-wake transitions and depressive/anxiety-like behaviors in mice. Functionally, activation of VPVglut2 neurons was sufficient to increase wakefulness and induce anxiety/depressive-like behaviors, whereas inhibition attenuated both. Dissection of the circuit revealed that separated projections of VPVglut2 neurons to the lateral hypothalamus and lateral habenula promote arousal and depressive-like behaviors, respectively. Our results demonstrate a subtype of VP neurons is responsible for wakefulness and emotion through separated projections, and may provide new lines for the intervention of insomnia and depression in patients.

Compensatory remodeling of a septo-hippocampal GABAergic network in the triple transgenic Alzheimer's mouse model.

BACKGROUND: Alzheimer's disease (AD) is characterized by a progressive loss of memory that cannot be efficiently managed by currently available AD therapeutics. So far, most treatments for AD that have the potential to improve memory target neural circuits to protect their integrity. However, the vulnerable neural circuits and their dynamic remodeling during AD progression remain largely undefined. METHODS: Circuit-based approaches, including anterograde and retrograde tracing, slice electrophysiology, and fiber photometry, were used to investigate the dynamic structural and functional remodeling of a GABAergic circuit projected from the medial septum (MS) to the dentate gyrus (DG) in 3xTg-AD mice during AD progression. RESULTS: We identified a long-distance GABAergic circuit that couples highly connected MS and DG GABAergic neurons during spatial memory encoding. Furthermore, we found hyperactivity of DG interneurons during early AD, which persisted into late AD stages. Interestingly, MS GABAergic projections developed a series of adaptive strategies to combat DG interneuron hyperactivity. During early-stage AD, MS-DG GABAergic projections exhibit increased inhibitory synaptic strength onto DG interneurons to inhibit their activities. During late-stage AD, MS-DG GABAergic projections form higher anatomical connectivity with DG interneurons and exhibit aberrant outgrowth to increase the inhibition onto DG interneurons. CONCLUSION: We report the structural and functional remodeling of the MS-DG GABAergic circuit during disease progression in 3xTg-AD mice. Dynamic MS-DG GABAergic circuit remodeling represents a compensatory mechanism to combat DG interneuron hyperactivity induced by reduced GABA transmission.

GABAergic neurons in the rostromedial tegmental nucleus are essential for rapid eye movement sleep suppression.

Rapid eye movement (REM) sleep disturbances are prevalent in various psychiatric disorders. However, the neural circuits that regulate REM sleep remain poorly understood. Here, we found that in male mice, optogenetic activation of rostromedial tegmental nucleus (RMTg) GABAergic neurons immediately converted REM sleep to arousal and then initiated non-REM (NREM) sleep. Conversely, laser-mediated inactivation completely converted NREM to REM sleep and prolonged REM sleep duration. The activity of RMTg GABAergic neurons increased to a high discharge level at the termination of REM sleep. RMTg GABAergic neurons directly converted REM sleep to wakefulness and NREM sleep via inhibitory projections to the laterodorsal tegmentum (LDT) and lateral hypothalamus (LH), respectively. Furthermore, LDT glutamatergic neurons were responsible for the REM sleep-wake transitions following photostimulation of the RMTgGABA-LDT circuit. Thus, RMTg GABAergic neurons are essential for suppressing the induction and maintenance of REM sleep.

A cluster of mesopontine GABAergic neurons suppresses REM sleep and curbs cataplexy.

Physiological rapid eye movement (REM) sleep termination is vital for initiating non-REM (NREM) sleep or arousal, whereas the suppression of excessive REM sleep is promising in treating narcolepsy. However, the neuronal mechanisms controlling REM sleep termination and keeping sleep continuation remain largely unknown. Here, we reveal a key brainstem region of GABAergic neurons in the control of both physiological REM sleep and cataplexy. Using fiber photometry and optic tetrode recording, we characterized the dorsal part of the deep mesencephalic nucleus (dDpMe) GABAergic neurons as REM relatively inactive and two different firing patterns under spontaneous sleep-wake cycles. Next, we investigated the roles of dDpMe GABAergic neuronal circuits in brain state regulation using optogenetics, RNA interference technology, and celltype-specific lesion. Physiologically, dDpMe GABAergic neurons causally suppressed REM sleep and promoted NREM sleep through the sublaterodorsal nucleus and lateral hypothalamus. In-depth studies of neural circuits revealed that sublaterodorsal nucleus glutamatergic neurons were essential for REM sleep termination by dDpMe GABAergic neurons. In addition, dDpMe GABAergic neurons efficiently suppressed cataplexy in a rodent model. Our results demonstrated that dDpMe GABAergic neurons controlled REM sleep termination along with REM/NREM transitions and represented a novel potential target to treat narcolepsy.

Hypothalamic modulation of adult hippocampal neurogenesis in mice confers activity-dependent regulation of memory and anxiety-like behavior.

Adult hippocampal neurogenesis plays a critical role in memory and emotion processing, and this process is dynamically regulated by neural circuit activity. However, it remains unknown whether manipulation of neural circuit activity can achieve sufficient neurogenic effects to modulate behavior. Here we report that chronic patterned optogenetic stimulation of supramammillary nucleus (SuM) neurons in the mouse hypothalamus robustly promotes neurogenesis at multiple stages, leading to increased production of neural stem cells and behaviorally relevant adult-born neurons (ABNs) with enhanced maturity. Functionally, selective manipulation of the activity of these SuM-promoted ABNs modulates memory retrieval and anxiety-like behaviors. Furthermore, we show that SuM neurons are highly responsive to environmental novelty (EN) and are required for EN-induced enhancement of neurogenesis. Moreover, SuM is required for ABN activity-dependent behavioral modulation under a novel environment. Our study identifies a key hypothalamic circuit that couples novelty signals to the production and maturation of ABNs, and highlights the activity-dependent contribution of circuit-modified ABNs in behavioral regulation.

Striatal neurons expressing dopamine D1 receptor promote wakefulness in mice.

Patients with Parkinson's disease (PD) suffer from severe sleep disorders. Pathophysiology of the basal ganglia (BG) underlies PD, and the dorsal striatum represents the major input pathway of the BG. However, the roles and mechanisms of the dorsal striatum in controlling sleep-wake cycles remain unknown. To demonstrate the contribution of dopamine D1 receptor (D1R)-positive neurons within the dorsal striatum in promoting wakefulness, we combined optogenetic manipulations and fiber photometry with electroencephalography/electromyography recording in D1R-Cre mice. As a result, optogenetic activation of striatal D1R neurons induced immediate transitions from non-rapid eye movement (NREM) sleep to wakefulness, whereas inhibition of striatal D1R neurons attenuated wakefulness by chemogenetics. Multi-channel fiber photometry recordings revealed that the activity of striatal D1R neurons synchronized with that of BG upstreams, namely the prefrontal cortex and mediodorsal thalamus, in terms of immediate increase in activity during NREM-to-wake transitions and rapid decease during wake-to-NREM transitions. Further optogenetic manipulations revealed a prominent contribution of striatal D1R neurons in control of wakefulness by upstream, corticostriatal, thalamostriatal, and nigrostriatal projections and via downstream, striato-entopeduncular, or striatonigral pathways. Taken together, our findings revealed a circuit regulating wakefulness through striatal D1R neurons. Striatal D1R neurons play an important role in controlling wakefulness by integrating the corticostriatal, thalamostriatal, and nigrostriatal projections and innervation of striato-entopeduncular or striatonigral pathways.

Presynaptic inputs to vasopressin neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus in mice.

Arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) are involved in important physiological behaviors, such as controling osmotic stability and thermoregulation. However, the presynaptic input patterns governing AVP neurons have remained poorly understood due to their heterogeneity, as well as intermingling of AVP neurons with other neurons both in the SON and PVN. In the present study, we employed a retrograde modified rabies-virus system to reveal the brain areas that provide specific inputs to AVP neurons in the SON and PVN. We found that AVP neurons of the SON and PVN received similar input patterns from multiple areas of the brain, particularly massive afferent inputs from the diencephalon and other brain regions of the limbic system; however, PVNAVP neurons received relatively broader and denser inputs compared to SONAVP neurons. Additionally, SONAVP neurons received more projections from the median preoptic nucleus and organum vasculosum of the lamina terminalis (a circumventricular organ), compared to PVNAVP neurons, while PVNAVP neurons received more afferent inputs from the bed nucleus of stria terminalis and dorsomedial nucleus of the hypothalamus, both of which are thermoregulatory nuclei, compared to those of SONAVP neurons. In addition, both SONAVP and PVNAVP neurons received direct afferent projections from the bilateral suprachiasmatic nucleus, which is the master regulator of circadian rhythms and is concomitantly responsible for fluctuations in AVP levels. Taken together, our present results provide a comprehensive understanding of the specific afferent framework of AVP neurons both in the SON and PVN, and lay the foundation for further dissecting the diverse roles of SONAVP and PVNAVP neurons.

Hypnotic activities of Zao Ren An Shen capsule, a traditional Chinese medicine, in an anxiety-like mouse model.

PURPOSE: Zao Ren An Shen capsule (ZRASC) which is composed of three kinds of traditional Chinese herbs is a popular Chinese medicine for the treatment of insomnia. This study investigated the hypnotic effect of ZRASC in an anxiety-like mouse model. METHODS: We determined the role of ZRASC in anxiety and co-morbid insomnia using electroencephalogram and electromyogram recordings. Anxiety-like behaviors were tested by using the open-field, light/dark box, or elevated plus-maze in mice. Immunohistochemical techniques were employed to reveal the mechanism by which ZRASC regulated anxiety and insomnia. RESULTS: ZRASC at 680 mg/kg prolonged the time spent in the central area, open arms area, and light box by 1.9, 2.3, and 1.7-fold respectively, compared with the vehicle control group in immobilization stress (IMS) mice. ZRASC at 680 mg/kg given at 08:00 h increased the amount of non-rapid eye movement sleep by 1.4-fold in a 2-h period after dosing in IMS mice. However, it did not alter the sleep-wake behaviors in normal mice. Immunohistochemistry showed that IMS increased c-Fos expression in the neurons of the stria terminalis and tuberomammillary nucleus by 1.8 and 1.6-fold, respectively. In addition, ZRASC (680 mg/kg) reversed the IMS-induced c-Fos expression. CONCLUSIONS: Our results suggest that ZRASC is an effective therapeutic strategy for both anxiety disorder and sleep disturbances in an anxiety-like mouse model.

Ablation of olfactory bulb glutamatergic neurons induces depressive-like behaviors and sleep disturbances in mice.

RATIONALE: Major depression is a serious, but common, psychological disorder, which consists of a long-lasting depressive mood, feelings of helplessness, anhedonia, and sleep disturbances. It has been reported that rats with bilateral olfactory bulbectomies (OBXs) exhibit depressive-like behaviors which indicates that the olfactory bulb (OB) plays an important role in the formation of depression. However, which type of OB neurons plays an important role in the formation of depression remains unclear. OBJECTIVE: To determine the role of OB neuronal types in depression and related sleep-wake dysfunction. METHODS: Firstly, we established and evaluated a conventional physical bilateral OBX depression model. Secondly, we used chemical methods to ablate OB neurons, while maintaining the original shape, and evaluated depressive-like behaviors. Thirdly, we utilized AAV-flex-taCasp3-TEVp and transgenetic mice to specifically ablate the OB GABAergic or glutamatergic neurons, then evaluated depressive-like behaviors. RESULTS: Compared with measured parameters in sham mice, mice with OBXs or ibotenic acid-induced OB lesions exhibited depressive-like behaviors and sleep disturbances, as demonstrated by results of depressive-like behavior tests and sleep recordings. Selective lesioning of OB glutamatergic neurons, but not GABAergic neurons induced depressive-like behaviors and increased rapid eye movement sleep during the light phase of the circadian cycle. CONCLUSIONS: These results indicate that OB glutamatergic neurons play a key role in olfactory-related depression and sleep disturbance.

Whole-Brain Neural Connectivity to Lateral Pontine Tegmentum GABAergic Neurons in Mice.

The GABAergic neurons in the lateral pontine tegmentum (LPT) play key roles in the regulation of sleep and locomotion. The dysfunction of the LPT is related to neurological disorders such as rapid eye movement sleep behavior disorder and ocular flutter. However, the whole-brain neural connectivity to LPT GABAergic neurons remains poorly understood. Using virus-based, cell-type-specific, retrograde and anterograde tracing systems, we mapped the monosynaptic inputs and axonal projections of LPT GABAergic neurons in mice. We found that LPT GABAergic neurons received inputs mainly from the superior colliculus, substantia nigra pars reticulata, dorsal raphe nucleus (DR), lateral hypothalamic area (LHA), parasubthalamic nucleus, and periaqueductal gray (PAG), as well as the limbic system (e.g., central nucleus of the amygdala). Further immunofluorescence assays revealed that the inputs to LPT GABAergic neurons were colocalized with several markers associated with important neural functions, especially the sleep-wake cycle. Moreover, numerous LPT GABAergic neuronal varicosities were observed in the medial and midline part of the thalamus, the LHA, PAG, DR, and parabrachial nuclei. Interestingly, LPT GABAergic neurons formed reciprocal connections with areas related to sleep-wake and motor control, including the LHA, PAG, DR, parabrachial nuclei, and superior colliculus, only the LPT-DR connections were in an equally bidirectional manner. These results provide a structural framework to understand the underlying neural mechanisms of rapid eye movement sleep behavior disorder and disorders of saccades.

Adenosine A2A receptor mediates hypnotic effects of ethanol in mice.

Ethanol has extensive effects on sleep and daytime alertness, causing premature disability and death. Adenosine, as a potent sleep-promoting substance, is involved in many cellular and behavioral responses to ethanol. However, the mechanisms of hypnotic effects of ethanol remain unclear. In this study, we investigated the role of adenosine in ethanol-induced sleep using C57BL/6Slac mice, adenosine A2A receptor (A2AR) knockout mice, and their wild-type littermates. The results showed that intraperitoneal injection of ethanol (3.0 g/kg) at 21:00 decreased the latency to non-rapid eye movement (NREM) sleep and increased the duration of NREM sleep for 5 h. Ethanol dose-dependently increased NREM sleep, which was consistent with decreases in wakefulness in C57BL/6Slac mice compared with their own control. Caffeine (5, 10, or 15 mg/kg), a nonspecific adenosine receptor antagonist, dose-dependently and at high doses completely blocked ethanol-induced NREM sleep when administered 30 min prior to (but not after) ethanol injection. Moreover, ethanol-induced NREM sleep was completely abolished in A2AR knockout mice compared with wild-type mice. These findings strongly indicate that A2AR is a key receptor for the hypnotic effects of ethanol, and pretreatment of caffeine might be a strategy to counter the hypnotic effects of ethanol.

[Senescence-associated Beta Galactosidase Expression in Rat Schwann Cells after Chronic Denervation].

OBJECTIVE: To investigate the effect of prolonged axon depletion on senescence-associated beta galactosidase (SA-ß-gal) expression in Schwann cells (SCs) of adult rats. METHODS: Male adult Sprague-Dawley (SD) rats were randomize grouped into sham-operated group and denervation groups for 1 week, 2 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks. Rats were subjected to right sciatic nerve transection. After particular denervation duration for the distal stumps, animals were anesthetized and perfused. Proximal stumps of 5 mm and distal stumps of 10 mm from injured nerves, and the corresponding segments from the sham groups and contralateral nerves were harvested and prepared for SA-ß-gal staining to detect SA-ß-gal expression. Then, additional injured distal stumps denervated for 8 weeks were employed for determining cellular distribution of SA-ß-gal expression by co-labeling of SA-ß-gal and SC-specific protein (S100ß). RESULTS: SA-ß-gal expression transiently increased in distal tips of proximal stumps 2 weeks after adult rat sciatic nerve transection without suture. In contrast, in the distal stumps of transected adult rat sciatic nerves, axon depletion for 2 weeks increased SA-ß-gal expression, and the increased expression of SA-ß-gal remained constant after prolonged denervation durations. Furthermore, combination of SA-ß-gal staining with S100ß immunofluorescence staining showed that SA-ß-gal expression. was exclusively present in denervated SCs. CONCLUSION: Prolonged axon depletion increased SA-ß-gal expression in adult rat SCs.