Electroacupuncture alleviates chemotherapy-induced peripheral neuropathy and anxiety by reducing TRPC6/PKC-dependent activation of glutamatergic neurons in the paraventricular thalamic nucleus.

Noradrenergic projections from the locus coeruleus (LC) to the thalamus and anterior cingulate cortex (ACC) contribute to pain‒like behaviors, yet their hierarchical organization remains unclear. Here, we examined how LC‒derived norepinephrine (NE) inputs to the paraventricular thalamic nucleus (PVA) and ACC differentially regulate nociceptive sensitization. In adult male and female mice, complete Freund's adjuvant (CFA) was used to induce pain‒like behaviors. To examine functional connectivity among LC, PVA, and ACC, we combined targeted recombination in active populations (Fos‒TRAP), in vivo recordings, and viral tracing. We used optogenetic and chemogenetic tools to selectively manipulate LC projections and assess their impact on neural activity and pain behaviors. CFA led to enhanced c‒Fos expression in LC, PVA, and ACC (Cells per microscopic field; LC: 13.60 ± 2.24 vs. 44.50 ± 7.72; PVA: 8.00 ± 1.58 vs. 66.40 ± 9.45; ACC: 12.80 ± 2.28 vs. 36.70 ± 2.59; p < 0.001), alongside increased gamma‒band activity and single‒unit firing rates. Monosynaptic LC-ACC and polysynaptic LC-PVA-ACC circuits were identified. Notably, nociception‒related LC neurons preferentially projected to PVA, which subsequently targeted hyperactive ACC neurons. Under inflammatory pain conditions, activation of the LC-PVA-ACC circuits evoked greater ACC firing (Hz; LC-PVA-ACC vs. LC-ACC: 15.75 ± 2.88 vs. 9.72 ± 2.06; P < 0.001) and tactile‒evoked responses (Hz; 22.98 ± 2.60 vs. 15.34 ± 1.86; P < 0.001) than direct LC-ACC activation. Consistently, optogenetic or chemogenetic manipulation of the LC-PVA-ACC circuit produced stronger modulation of mechanical and thermal pain sensitivity than direct LC-ACC stimulation. We identify the LC-PVA-ACC pathway as a hierarchical noradrenergic circuit that modulates nociceptive sensitization via a thalamocortical relay, thereby revealing a circuit‒specific mechanism by which the LC-NE system regulates pain processing.

Using in vivo MRI to reveal circuits that are sensitive to traumatic stress in male and female rats.

Environmental threats are typically encountered when animals are searching for food and other necessities. Adaptive behavior must balance competition between fear behavior and reward seeking. We gave rats local neuronal deletions of the ventral pallidum (VP) or specifically deleted paraventricular thalamic nucleus (PVT) neurons projecting directly to the VP. Rats were then assessed in a conditioned suppression procedure in which cues predicting unique foot shock probabilities were presented during, but independent from, reward seeking. Foot shock introduction generally suppressed reward seeking in rats, and recovery from shock introduction was facilitated in rats with VP or PVT → VP pathway deletions. Discriminative fear was observed in controls, and this fear responding reduced over a single extinction session. VP deletion enhanced extinction fear responding, and PVT → VP pathway deletion abolished within-session fear reductions. The results demonstrate the VP and its inputs from the PVT shape reward seeking in threat settings and govern fear extinction responding. (PsycInfo Database Record (c) 2025 APA, all rights reserved).

The substantial contribution of glial cells, particularly astrocytes, in the progression of chronic pain is increasingly acknowledged in the spinal dorsal horn. However, the precise alterations and roles of astrocytes in the midline nuclei of the thalamus during the development of chronic pain remain unclear. In our investigation, spared nerve injury (SNI) induced mechanical pain hypersensitivity in mice, which was associated with the activation and proliferation of astrocytes in the posterior region of the paraventricular thalamic nucleus (pPVT). The chemogenetic activation of astrocyte activity was observed to induce pain hypersensitivity, whereas the inhibition of astrocyte activity was found to alleviate pain. To elucidate the phenotypic regulatory mechanisms of astrocytes, a variety of techniques were employed, including immunofluorescence staining, Western blot analysis, and RT-qPCR. It was confirmed that the activated and proliferating astrocytes within the pPVT in the SNI model were predominantly A1-reactive astrocytes, as evidenced by the expression of complement C3. It is noteworthy that the blockade of the C3a receptor (C3aR) resulted in a significant reduction in pain perception in the SNI mouse model, accompanied by a decrease in the release of pro-inflammatory factors. In conclusion, our results demonstrate that the activation and proliferation of reactive A1 astrocytes in the pPVT are involved in the pathogenesis of SNI. Consequently, targeting type A1 astrocytes may offer a potential strategy to alleviate chronic pain.

The purpose of this study was to investigate the anxiety-like behaviors, circadian rhythms and sleep, and to elucidate the possible underlying mechanisms of the abnormal sleep behavior in Shank3 gene knockout (Shank3-KO) mice. The anxiety-like behaviors were detected by elevated plus-maze (EPM) test, open field test (OFT) and tail suspension test (TST). The circadian rhythms were detected by running wheel test. The electroencephalogram (EEG)/electromyogram (EMG) recordings were performed synchronically by polysomnograph. The distribution of SHANK3 in anterior cingulate cortex (ACC), paraventricular thalamus (PVT), nucleus accumbens (NAc), basolateral amygdala (BLA) and hippocampal CA2 region in wild type (WT) mice was detected by immunofluorescence assay. The protein expression of c-Fos in PVT, ACC and NAc was also detected by immunofluorescence assay during light cycle. The colocalization of c-Fos and vesicular glutamate transporter 2 (Vglut2, a marker for glutamatergic neurons) in the PVT was detected by immunofluorescence double labeling experiment. The results of EPM test showed that, compared with the WT mice, the Shank3-KO mice showed less time in open arms and less number of open arm entries. The results of OFT showed that the Shank3-KO mice showed less time in central area and less number of central area entries. The immobility time of Shank3-KO mice was increased in the TST. The results of running wheel rhythm test showed that the phase shift time of Shank3-KO mice in the continuous dark period was increased. The results of EEG/EMG recording showed that, compared with the WT mice, the duration of wakefulness in Shank3-KO mice was increased and the duration of non-rapid eye movement (NREM) sleep was decreased during light phase; The bout number of wakefulness was increased, the bout number of NREM sleep was decreased, NREM-wake transitions were increased, and wake-NREM transitions were decreased during light phase. SHANK3 was expressed in ACC, PVT, NAc and BLA in the WT mice. The expression of c-Fos in the PVT of Shank3-KO mice was up-regulated 2 h after entering the light phase, and majority of c-Fos was co-localized with Vglut2. These results suggest that the anxiety level of Shank3-KO mice is increased, the regulation of the internal rhythms is decreased, and the bout number of wakefulness is increased during light phase. The glutamatergic neurons in PVT may be involved in the regulation of abnormal sleep behavior in Shank3-KO mice during the light phase.

The paraventricular thalamic nucleus (PVT) integrates subcortical signals related to arousal, stress, addiction, and anxiety with top-down cortical influences. Increases or decreases in PVT activity exert profound, long-lasting effects on behavior related to motivation, addiction, and homeostasis. Yet the sources of its subcortical excitatory and inhibitory afferents, their distribution within the PVT, and their integration with layer-specific cortical inputs remain unclear. Using transgenic male and female mice selective for GABAergic and glutamatergic neurons, or for different cortical layers, we found that the input organization of PVT is unique among thalamic nuclei. PVT received subcortical GABAergic and glutamatergic inputs from multiple, distinct hypothalamic and brainstem regions. Most regions provided either excitatory or inhibitory afferents; however, subcortical inputs with dual components have also been found. Most of these subcortical inputs selectively targeted the core region of the PVT that contained large number of densely packed calretinin-positive (CR+) neurons. Cortical afferents to PVT displayed layer-specific segregation. Layer 5 neurons of the medial prefrontal cortex preferentially innervated the CR+ core, whereas layer 6 input was more abundant in the transition zone between PVT and the mediodorsal nucleus. These findings demonstrate extensive convergence of excitatory and inhibitory inputs from diverse subcortical sources, selectively, in a sharply delineated CR+ core region of PVT which is also under strong top-down control from layer 5. This unique organization may explain why the CR+ PVT core serves as a critical bottleneck in the subcortex–cortex communication involved in affective behavior.

The paraventricular thalamic nucleus is involved in sleep-fragmentation-associated cardiac dysfunction after acute myocardial infarction.

AimRecent neuropathological studies suggest that the accumulation of neurodegenerative disease–associated proteins in subcortical structures may contribute to mood symptoms. Animal models have highlighted the role of the paraventricular thalamic nucleus (PVT) in bipolar disorder (BD) pathophysiology. However, neuropathological investigations in the thalamus in BD remain limited. This study aimed to examine neurodegenerative pathology in the thalamus and medial temporal region including the hippocampus in patients with BD.MethodsPostmortem brain tissues of the thalamus and medial temporal region of nine patients with BD and nine age‐matched controls were obtained from Matsuzawa Hospital, with additional medial temporal samples of 14 BD cases acquired from the Stanley Foundation Brain Bank. Immunohistochemical analyses were performed using antibodies against phosphorylated tau, amyloid‐β, α‐synuclein, TDP‐43, and granulovacuolar degeneration (GVD) markers including CHMP2B and CK‐1δ.ResultsThe 23 BD cases exhibited a significantly greater burden of tau pathologies, including higher neurofibrillary tangle Braak stages (P = 0.015) and more severe argyrophilic grain Saito stage (P = 0.029), compared with the nine controls. Notably, CHMP2B‐positive GVD was significantly more frequently observed in the PVT of BD cases than in the controls (five of nine vs. zero of nine, P = 0.029).ConclusionsThese findings suggest that neurodegenerative processes, particularly tau pathology and CHMP2B‐positive GVD in the PVT may play a role in BD pathophysiology.

nNOS-expressing neurons in the mPFC mediate depression-related behaviors in mice through pPVT-mPFC projections.

BACKGROUNDHypobaric hypoxia exposure (HHE) often causes neuropsychiatric disorders. Due to its complex mechanism, efficient strategies for alleviating HHE-induced anxiety- and depression-like behaviors remain limited.AIMTo characterize alterations in the oral and gut microbiota following HHE and to explore a potential microbiota-based intervention to mitigate associated psychiatric symptoms.METHODSC57BL/6J mice were exposed to simulated high-altitude hypoxia (5000 m) for 1, 3, 5, or 7 days. Behavioral assessments, including the open field test, elevated plus maze, and forced swim test, were conducted to evaluate anxiety- and depression-like behaviors. Oral and fecal microbiota were analyzed using 16S rRNA sequencing to assess changes in microbial composition and diversity. Immunofluorescence staining was performed to examine c-Fos expression in brain nuclei. A probiotic formulation containing Lactobacillus rhamnosus (L. rhamnosus) DSM17648, Lactobacillus acidophilus DDS-1, and L. rhamnosus UALR-06 was administered to mice subjected to one day of HHE (HH1) to evaluate its therapeutic efficacy.RESULTSBehavioral tests revealed that HHE caused anxiety- and depression-like behaviors, which were most pronounced after 1 day of exposure. The IF data revealed significantly increased expression of c-Fos in various brain nuclei after HHE, including the anterior cingulate cortex, paraventricular thalamic nucleus, lateral habenula nucleus, paraventricular hypothalamic nucleus, lateral hypothalamus, and periaqueductal gray. The 16S rRNA sequencing results demonstrated a sharp decline in the abundance of Lactobacillus in the oral microbiota of mice exposed to HH1 and a marked decrease in the abundance of Lactobacillus and Bifidobacterium in the fecal microbiota of mice exposed to three days of HHE. Finally, oral administration and gavage of Lactobacillus significantly alleviated anxiety- and depression-like behaviors in HH1 mice.CONCLUSIONHHE caused significant variations in the oral and fecal microbiota of mice. Lactobacillus supplementation alleviated anxiety- and depression-like behaviors in mice. Improving oral flora may relieve HHE-induced psychiatric disorders.

Neurons in the paraventricular nucleus of the thalamus (PVT) integrate visceral and limbic inputs and project to multiple brain regions to bias behavior toward aversive or defensive states. This study examines MOR signaling in anterior PVT neurons in brain slices from untreated and morphine-treated animals. Imaging in a MOR-Cre reporter rat revealed extensive expression in aPVT cells, and the application of [Met]5− enkephalin (ME) induced outward currents which were abolished by the MOR-selective antagonist CTAP. A saturating concentration of ME resulted in desensitization that was blocked by compound 101, indicating a phosphorylation-dependent process. The opioid sensitivity of amygdala-, nucleus accumbens-, and prefrontal cortex-projecting neurons was then examined. Neurons that projected to the amygdala were more sensitive to ME than cortical- and accumbal-projecting cells. Following chronic treatment, tolerance to morphine was found in neurons projecting to the amygdala and nucleus accumbens with a trend toward tolerance observed in neurons projecting to the prefrontal cortex. The results reveal that adaptations to chronic opioid exposure are in the aPVT circuits contribute to affective pain processing and may provide specific insights into the etiology of withdrawal following the cessation of opioid use.

Novelty has significant effects on feeding behavior. New foods and unfamiliar environments suppress consumption, and adaptation to novelty is fundamental to survival. Yet, little is known about habituation to eating in a novel environment. The aim of the current study was to determine if context familiarity impacts habituation to novel food and to identify underlying neural substrates. Adult male and female rats were tested for consumption of a novel, palatable food in a novel or familiar environment across four habituation sessions and a final test session. Test-induced Fos expression was measured in amygdalar, thalamic, prefrontal, and hippocampal regions known to be recruited during the first exposure to novelty. Rats in the novel context ate less compared to rats in the familiar context during each habituation session and test, and females ate less than males during the first session. Habituation to eating in the novel context robustly induced Fos in the majority of regions analyzed, including the central, basolateral, and basomedial nuclei of the amygdala, thalamic paraventricular and reuniens nuclei, and the hippocampal field CA1. Females had overall higher Fos induction in most regions analyzed and higher in the novel condition in the reuniens nucleus. Bivariate correlation analyses of Fos induction between regions found a large number of correlations in the novel context condition. Females tested in the novel context had uniquely large number of correlations between all regions analyzed, except for one thalamic subregion. These results suggest that novelty from context remains relevant late in habituation and recruits a distinct and more interactive network in females than in males.

Repeated ethanol exposure during adolescence increases the risk for displaying anxiogenic phenotype in adulthood, but the underlying mechanisms are not fully understood. The paraventricular nucleus of thalamus (PVT) has been considered a hub brain area for controlling the anxiety network in the brain. Recent structural and functional investigations indicate that the PVT exhibits diverse neural signals aligned with early-life events, which are highly linked with anxiety-like behaviors. However, it remains unknown if repeated ethanol exposure during adolescence will affect the coordinated brain activities of the PVT in adulthood, and consequent behavioral adaptation. Here we show that adolescent repeated intermittent ethanol exposure (AIE) triggers anxiety-like behaviors and parallelly induces the glutamatergic adaptation in the PVT after four weeks withdrawal from the last ethanol exposure. The firing rates, along with the spatiotemporal calcium transients in the PVT neurons during behavior were increased in the AIE mice compared to those in the counterpart control mice. Importantly, with the chemogenetic inhibition of the PVT neurons, we found alleviated the anxiety-like behavior in the AIE mice.The increased neuronal activities in the PVT of AIE mice was, at least partly, via the reduction of GLT1 (an astrocyte dominant glutamate transporter, known as EAAT2, slc1a2). Our non-invasive magnetic resonance spectroscopy (MRS) measures also showed an increase in glutamate/GABA ratio in the thalamic area including the PVT of the GLT1 conditional knock-down mice, which exhibited the heightened anxiety-like behavior. In addition, while the selective knock-out of GLT1 in the astrocytes of PVT in the alcohol naïve mice induces anxiogenic phenotypes, the selective upregulation of GLT1 in the PVT astrocytes of the mice that were treated with AIE paradigm alleviated the anxiety-like behaviors as well. These findings highlight the significant role of PVT astrocytic GLT1 in the anxiogenic phenotype in adulthood induced by withdrawal from adolescent repeated ethanol exposure, suggesting that GLT1 in the PVT could serve as a therapeutic target for alcohol use disorder and comorbid emotional disorders.

Preclinical research highlights the paraventricular thalamic nucleus as important in various stages of substance use disorder. However, there is limited research on it in relation to methamphetamine, especially regarding its functional changes after long-term abstinence. This study aims to understand the alterations in functional connectivity of the paraventricular thalamic nucleus in methamphetamine abstainers and its correlation with drug craving at two different withdrawal periods. A total of 49 subjects were allocated to the short-term withdrawal group, 44 to the long-term withdrawal group, and 42 to the healthy control group, all of whom are male and adult. Craving scores were assessed using a visual analogue scale. Functional connectivity was evaluated through resting-state functional MRI, which reflects the correlation between connectivity in different brain regions. Significant differences in functional connectivity between the paraventricular thalamic nucleus and the left caudate nucleus were observed across the three groups. The healthy control group exhibited the strongest connectivity, followed by the long-term withdrawal group, while the short-term withdrawal group demonstrated the weakest connectivity. Within the short-term withdrawal group, functional connectivity of the paraventricular thalamic nucleus with both the left parahippocampal gyrus (r = -0.45, p = 0.001) and the left inferior temporal gyrus (r = -0.43, p = 0.002) was significantly correlated with craving scores. This study confirms abnormalities in the paraventricular thalamic nucleus among male methamphetamine abstainers, emphasizes its potential role in regulating methamphetamine use disorder and craving mechanisms, and offers insights into long-term changes in brain function after abstinence.

As the core area of emotion regulation, the amygdala is involved in and regulates many related behaviors, such as fear, anxiety, depression, as well as reward, learning, and memory. Most previous connectional studies have focused on the anterior and middle parts of the basolateral nucleus (BL) and basomedial nucleus (BM) of the amygdala. Little is known about the brain-wide connections of the posterior part of the BL and BM (termed parvicellular subdivision of the BL and BM, i.e., BLpc and BMpc). In this study, brain-wide afferent and efferent projections of the BLpc and BMpc in the rats are investigated using both retrograde and anterograde tracing methods. Both common and differential connections of the BLpc and BMpc are revealed. Major common inputs of both regions originate from the ventral hippocampal CA1 and prosubiculum, sublenticular extended amygdala, anterior basomedial nucleus, midline thalamic nuclei, endopiriform nucleus, dorsal raphe, piriform cortex and lateral entorhinal cortex. The BLpc receives preferential inputs from agranular insular cortex, amygdalopiriform transition area, periaqueductal gray, parataenial nucleus and anterior cortical nucleus of the amygdala. The BMpc preferentially receives its inputs from the peripeduncular nucleus, paraventricular nucleus of thalamus, ventromedial hypothalamic nucleus (VMH), caudal bed nucleus of stria terminalis (BST), medial amygdaloid nucleus and posterior cortical nucleus of the amygdala. Major differential outputs of the BLpc and BMpc are also obvious. The BLpc projects mainly to nucleus accumbens, rostral BST, lateral central amygdaloid nucleus (Ce), intermediate BL and BM. The BMpc sends its main outputs to VMH, medial Ce, caudal BST, prosubiculum, and perirhinal-ectorhinal cortices. These major findings are further confirmed with anterograde viral tracing in mice. Compared with previous findings in monkeys, our findings in rodents suggest that the BLpc and BMpc have overall similar connectional patterns across species. In addition, some gene markers for BM subdivisions are identified. All these findings would provide an important anatomical basis for the understanding of emotion-related neuronal circuits and diseases and for cross-species comparison of the subcircuits in amygdaloid complex.

Efficient treatment of temporal lobe epilepsy (TLE) remains challenging due to limited understanding of cellular and network changes and the interference of novel antiepileptic drugs (AEDs) with tissue reorganisation. This study compared the effects of brivaracetam and levetiracetam on histological alterations in key brain regions of the epileptic circuitry, namely, the hippocampus, amygdala, piriform cortex (PC), endopiriform nucleus (EPN) and paraventricular thalamic nucleus (PVT), using the kainic acid (KA) rat model of TLE. Male Wistar rats were assigned to sham-operated (SHAM), epileptic (EPI), brivaracetam- (BRV-EPI) and levetiracetam-treated (LEV-EPI) epileptic groups. Epileptic groups received KA in the right lateral ventricle, which induced status epilepticus followed by a 3-week recovery and latent period. Rats then underwent 3weeks of oral brivaracetam, levetiracetam or placebo treatment with continuous video monitoring for seizure analysis. Subsequently, triple fluorescent immunolabeling assessed microglial, astrocytic, and neuronal changes. The results showed a drastic increase in microglia density in the EPI and BRV-EPI groups compared to control and LEV-EPI. The BRV-EPI group displayed a significantly higher microglia density than SHAM and EPI groups in the right CA1, CA3 and left CA1 regions, bilateral amygdalae, EPN, PVT and left PC. Astrocyte density was significantly elevated in hippocampal regions of the BRV-EPI group, while neuronal density decreased. Furthermore, brivaracetam did not reduce seizure activity in this disease phase. Significance: Brivaracetam treatment increased microglial activation under epileptic conditions in vivo in all examined brain-regions participating in the epileptic circuitry, in contrast to the effects of levetiracetam, highlighting differences in AED-induced histological alterations.

ABSTRACTThe zona incerta (ZI) supports diverse behaviors including binge feeding, sleep–wake cycles, nociception, and hunting. Diverse ZI functions can be attributed to its heterogeneous neurochemical characterization, cytoarchitecture, and efferent connections. The ZI is predominantly GABAergic, but we recently identified a subset of medial ZI GABA cells that are marked by the enzyme tyrosine hydroxylase (TH) and produce dopamine (DA). While the role of GABA within the ZI is well studied, less is known about the functions of ZI DA cells. To identify potential roles of ZI DA cells, we further phenotyped them and mapped their efferent fiber projections. We showed that wild‐type TH‐immunoreactive (‐ir) ZI cells did not express somatostatin or calretinin immunoreactivity. We next validated a Th‐cre;L10‐Egfp mouse line and found that medial Egfp ZI cells were more likely to be TH‐ir. We therefore delivered a Cre‐dependent virus into the medial ZI of Th‐cre or Th‐cre;L10‐Egfp mice and selected two injection cases for full brain mapping, namely, cases with the lowest and highest colocalization between TH‐ir and virally transduced, DsRed‐labeled cells, to identify common target sites. Overall, DsRed‐labeled fibers were distributed brainwide and were most prominent within the motor‐related midbrain (MBmot), notably the periaqueductal gray area and superior colliculus. We also observed numerous DsRed‐labeled fibers within the polymodal association cortex‐related thalamus (DORpm), like paraventricular thalamic nucleus and nucleus of reunions, that processes external and internal sensory input. Overall, ZI DA cells displayed a similar fiber profile to ZI GABA cells and may integrate sensory input to coordinate motor output at their target sites.

Ntrk1 (also known as TrkA) is a nerve growth factor receptor with essential roles in the development and function of the cholinergic nervous system. Ntrk1 is expressed in a few specific and defined brain areas. Specific antibodies are necessary to identify the expression and localization of Ntrk1 in the brain, and validating signal authenticity is critical. These issues have not been investigated sufficiently. We evaluated the utility of commercial antibodies for Ntrk1 using western blotting in brain lysates from Ntrk1 knockout mice and tested the utility of the antibody that showed specificity in western blotting for immunohistochemistry applications in the adult mouse brain. We confirmed specificity for one of the seven commercial antibodies in western blots, in which the specific bands were absent in the knockout samples. Using this antibody, we performed immunohistochemical staining of the brain tissues of adult mice to examine Ntrk1 localization. Distinct signals were observed in regions with known Ntrk1 expression, such as the striatum and basal forebrain. The characteristic expression pattern of Ntrk1 in the paraventricular thalamic nucleus (PVT) was verified at the protein level, with high and low expression levels in the anterior and posterior PVT, respectively.

Spinosin, a key flavonoids component found in Semen Zizhiphi spinosae, is known to enhance pentobarbital-induced sleep, which is primarily assessed with the loss-of-righting reflex (LORR). This research focused on investigating the impact of spinosin on sleep regulation in typical murine models. We used electroencephalogram (EEG) and electromyogram (EMG) recordings to evaluate the effects of spinosin (10, 20, 40 mg/kg, i.p.) on sleep-wake state. Immunohistochemical techniques were employed to investigate the c-Fos expression in various sleep-wake brain regions following the injection of spinosin. In the initial three-hour period following administration, spinosin administered at a dose of 40 mg/kg exhibited a notable augmentation in the duration of non-rapid eye movement (NREM) sleep, with a 2.04-fold increase (P < 0.0001), accompanied by a reduction in wakefulness by approximately 42.84% (P < 0.0001) compared to the vehicle group. Immunohistochemical analysis revealed an enhancement in c-Fos expression within the accumbens nucleus (Acb) when treated with spinosin at 40 mg/kg. In contrast, a notable reduction in c-Fos expression was detected across various brain regions, including the paraventricular thalamic nucleus (PV), lateral hypothalamic area (LHA), ventrolateral periaqueductal gray (VLPAG), dorsal raphe nucleus (DR), and lateral parabrachial nucleus (LPB) (P < 0.05). In addition, the treatment resulted in an increase in c-Fos expression within gamma-aminobutyric acid (GABAergic) neurons in the Acb, while simultaneously decreasing c-Fos expression in orexin neurons within the LHA. The results indicate that spinosin (40 mg/kg, i.p.) enhances NREM sleep in mice. Moreover, heightened activity of GABAergic neurons in the Acb and reduced activity of orexin neurons in the LHA may be the pathway through which spinosin promotes sleep.