This protocol details the procedure of Correlative Light Microscopy and Electron Microscopy (CLEM) with 3D Focus Ion Beam Scanning Electron Microscopy (FIBSEM) technique for mouse brain tissue in Zeiss Crossbeam 550 FIBSEM system.

Mental stress serves as a major contributor to a spectrum of esophageal motility disorders, including distal esophageal spasm (DES), a condition marked by premature contractile activity of the lower esophageal sphincter (LES). These spasms are often exacerbated by psychiatric conditions such as distress, anxiety, and depression. This study investigates the impact of mental stress on LES contractile function in mice. Chronic unpredictable mild stress (CUMS) was observed to reduce food intake, prolong esophageal emptying time, and increase LESP, along with elevated levels of inflammatory factors, IL-1β and TNFα in mice. Retrograde viral tracing identified a neuronal projection from the hypothalamus paraventricular nucleus (PVN) to the LES via the dorsal motor nucleus of the vagus (DMV) and the vagus nerve. Optogenetic activation of the PVN-DMV-vagus pathway increased the frequency and amplitude of LES electromyogram signals in mice, an effect negated by vagotomy or acetylcholine receptor antagonists. Conversely, chemogenetic inhibition of the PVN-DMV-vagus pathway alleviated CUMS-induced LES contraction abnormalities. High-throughput RNA sequencing revealed an upregulation of myosin binding protein C2 (MYBPC2) in the gastroesophageal junction (GEJ) following sustained activation of the PVN-DMV-vagus pathway. Similarly, cultured GEJ cells exposed to acetylcholine exhibited increased MYBPC2 levels. Notably, downregulation of MYBPC2 expression in the GEJ restored normal function in mice with PVN-DMV-vagus-acetylcholine pathway activation. In summary, this study demonstrates that mental stress induces LES contraction disorders through a PVN-DMV-vagus dependent pathway, which drives an increase in MYBPC2 expression in the GEJ.

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

This protocol describes the post-staining procedure for ultrathin Epon sections on EM grids.

Affective empathy describes the capacity to share others' emotions, yet the molecular mechanisms governing individual differences in empathic sensitivity remain poorly understood. Using a male mouse model of social transmission of depression (STD), we demonstrated that STD‑sensitive observer mice exhibit heightened neuronal excitability and upregulated major urinary protein 1 (MUP1) expression in the medial prefrontal cortex (mPFC) compared with STD‑insensitive observer mice. Mechanistically, MUP1 drives the STD‑sensitive phenotype by modulating astrocytic glutamate transporter 1 (GLT1) to augment mPFC neuronal excitability. Via virtual screening, we identified two small‑molecule compounds (C25 and C11) that bidirectionally modulate STD‑related behaviors. In summary, our work establishes MUP1 as a master modulator of STD vulnerability, deciphers mechanisms driving heterogeneous affective empathy across individuals, and unveils actionable therapeutic candidates for empathy-related psychiatric illnesses.

BACKGROUND:Insomnia represents a major global health challenge, with profound detrimental effects on physical and mental well-being. Paeoniflorin (PF), a bioactive component derived from Paeonia lactiflora Pall. (known as "Shao Yao" in traditional Chinese medicine, TCM), has garnered growing interest for its potential in the management of mental disorders. PURPOSE:The present study was designed to: 1) evaluate the effects of PF on anxiety-like behaviors, body weight changes, and the levels of inflammatory factors in sleep-deprived (SD) mice; 2) determine its optimal therapeutic dose; and 3) explore the underlying mechanisms of action. METHODS:Male C57BL/6 mice (n=8 per group) were randomly divided into six experimental groups: Control, SD + Saline, SD + 15 mg/kg PF, SD + 30 mg/kg PF, SD + 60 mg/kg PF, and SD + 0.5 mg/kg Diazepam (DZP). Mice in the PF-treated groups received daily intraperitoneal injections of PF for the entire duration of the SD protocol. To identify the brain regions targeted by PF, c-Fos immunofluorescence staining and chemogenetic approaches were employed. Additionally, network pharmacology, molecular docking, and molecular dynamics simulations were utilized to predict and validate the potential molecular targets of PF. Finally, the western blot experiment was used for verification. RESULTS:This study demonstrated that PF effectively attenuated anxiety-like behaviors in SD mice. Mechanistically, this beneficial effect was associated with the inhibition of three key pathological processes in SD mice: excessive activation of the paraventricular nucleus of the hypothalamus (PVN), aberrant phosphorylation of the epidermal growth factor receptor/phosphatidylinositol 3-kinase/protein kinase B (EGFR/PI3K/AKT) signaling pathway, and abnormally elevated inflammatory responses. CONCLUSION:PF alleviates SD-induced anxiety by curbing PVN hyperactivity, EGFR/PI3K/AKT hyper-phosphorylation and neuroinflammation, offering a multi-target therapeutic strategy for SD-related disorders.

Mounting evidence has validated the social transmissibility of depression-like behaviors. This study identifies the essential roles of dopamine signaling and the olfactory system in mediating socially transmitted depression (STD) in male mice. Breath odors from defeated conspecifics induce approach behaviors through the main olfactory bulb-piriform cortex (MOB-PiC) pathway, whereas urine odors trigger avoidance via the accessory olfactory bulb-medial amygdala (AOB-MeA) pathway. Both chemosensory inputs ultimately converge in the medial prefrontal cortex (mPFC). Social interactions with defeated conspecifics markedly enhance dopamine release from the ventral tegmental area (VTA) to the mPFC. In STD-sensitive mice, upregulated dopamine transporter (DAT) expression in the mPFC reduces basal dopamine levels and facilitates depression-like phenotypes. The MOB-PiC-mPFC-VTA axis mediates social interaction-evoked dopamine elevation, and DAT modulates dopamine reduction. Elevated dopamine variability within the mPFC is sufficient to drive STD. Notably, DAT-targeted intervention confers superior therapeutic efficacy against STD compared with serotonin system modulation.

This protocol details the procedure of our mouse brain immunofluorescence for detecting DAergic axonal dystrophies in Synj^^RQ KI mice.

Background: Depression, a prevalent mental health disorder, benefits from traditional Chinese medicine (TCM). Echinacoside (ECH), a natural phenolic compound extracted from Cistanche tubulosa and Echinacea angustifolia, exhibits neuroprotective and antioxidant properties. However, research on the potential mechanism of ECH's antidepressant activity is limited. This study explored the antidepressant potential of ECH in mice subjected to chronic unpredictable mild stress (CUMS) and its underlying molecular mechanisms. Methods: Mice received ECH (10, 20, 40 mg/kg/d, i.p.) during the last 14 days of a 28-day CUMS protocol. The therapeutic effect was assessed via the sucrose preference test (SPT), tail suspension test (TST) and forced swim test (FST). Hematoxylin-eosin (H&E) and Nissl staining were employed to evaluate the changes of neuronal injury in hippocampus. Network pharmacology was used to explore the potential targets and pathway enrichment in ECH-mediated antidepression. The expression changes of PI3K/AKT/Nrf2/HO-1 were evaluated by Western blotting. Furthermore, the neuroprotection effects of ECH were assessed on cultured primary neurons injured by corticosterone (CORT) using CCK-8 assay. Results: The results indicated that ECH significantly alleviated depression-like behaviors in CUMS mice characterized as the improved sucrose intake in SPT, reduced immobility duration in TST and FST, reversed weight loss and hippocampal neuronal injury induced after CUMS. PI3K/AKT was selected as core targets by network pharmacology and supported by molecular docking and dynamics simulations. WB results indicated that ECH administration offered neuroprotection by recovering the expression levels of p-PI3K, p-AKT, Nrf2, and HO-1 in CUMS mice hippocampus. Moreover, ECH rescued CORT-induced neuron death in vitro by activating PI3K/AKT/Nrf2/HO-1 pathway, which was abolished by PI3K inhibitor LY294002. Conclusion: These findings demonstrated that ECH alleviated depressive-like behaviors via PI3K/AKT/Nrf2/HO-1 activation, highlighting its potential as a novel antidepressant.

Anxiety disorders—including generalized anxiety disorder, posttraumatic stress disorder, and social anxiety disorder—are highly prevalent psychiatric conditions that impose substantial clinical and social burdens. Preclinical and clinical studies have shown that electroacupuncture (EA) can effectively alleviate anxiety-like behaviors; however, the specific neural circuits and molecular mechanisms underlying EA’s therapeutic effects remain incompletely elucidated. We first assessed the impacts of EA at four classical acupoints—Zusanli (ST36), Neiguan (PC6), Tianshu (ST25), and Baihui (GV20)—delivered with distinct stimulation waveforms on anxiety-like behaviors in conventionally housed mice, using the elevated plus maze and open field test paradigms. To identify the neural circuit underlying the behavioral effects of Baihui (GV20) EA, we employed pseudorabies virus expressing enhanced green fluorescent protein (PRV-EGFP) for retrograde tracing from Baihui (GV20) and quantified c-Fos expression across the whole brain as a marker of neuronal activation. ELISA was utilized to measure plasma oxytocin (OXT) levels following EA at Baihui (GV20). Furthermore, a selective pharmacological antagonist of the oxytocin receptor (OXT-R) was administered to verify the critical role of OXT signaling in mediating the anxiolytic benefits of Baihui (GV20) EA. EA at GV20 using intermittent electrical wave stimulation exhibited the most robust anxiolytic effects compared to EA at other acupoints or alternative stimulation parameters. Retrograde virus tracing from GV20 revealed a direct neuronal connection between the PVN and the GV20 acupoint region. Further experiments showed that GV20 EA significantly increased the activation of OXT-synthesizing neurons in the PVN and elevated peripheral OXT concentrations in mouse plasma. Critically, intraperitoneal injection of an OXTR antagonist completely abrogated the anxiolytic effects of GV20 EA, confirming that OXT signaling is indispensable for this therapeutic action. Intermittent 1.5 mA EA at Baihui (GV20) mitigates anxiety-like behavior in mice via a PVN-derived, OXT-dependent pathway. This work clarifies the anatomical and molecular mechanisms underlying EA-mediated anxiety relief and provides a basis for further exploring functional connections between specific acupoints and brain regions.

Proper fuelling of the brain is critical to sustain cognitive function, but the role of fatty acid (FA) combustion in this process has been elusive. Here we show that acute block of a neuron-specific triglyceride lipase, DDHD2 (a genetic driver of complex hereditary spastic paraplegia), or of the mitochondrial lipid transporter CPT1 leads to rapid onset of torpor in adult male mice. These data indicate that in vivo neurons are probably constantly fluxing FAs derived from lipid droplets (LDs) through β-oxidation to support neuronal bioenergetics. We show that in dissociated neurons, electrical silencing or blocking of DDHD2 leads to accumulation of neuronal LDs, including at nerve terminals, and that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion to drive local mitochondrial ATP production. These data demonstrate that nerve terminals can make use of LDs during electrical activity to provide metabolic support and probably have a critical role in supporting neuron function in vivo.

The underlying mechanisms of post-traumatic stress disorder (PTSD) are still not fully understood, creating significant obstacles for developing effective therapeutic strategies. Recently, ferroptosis, an iron-dependent form of regulated cell death, has been shown to play a role in several psychiatric disorders, such as major depressive disorder (MDD), stress-induced anxiety, Alzheimer’s disease (AD), and Parkinson’s disease (PD). While direct evidence for the role of ferroptosis in PTSD is still limited, an increasing number of studies suggest that the pathological features of PTSD may trigger the ferroptosis cascade. Additionally, the typical hallmarks of ferroptosis, such as iron dysregulation, lipid peroxidation, and failure of antioxidant defense systems, may intersect with the pathogenesis of PTSD. Importantly, some treatments for PTSD, such as antioxidants and free radical scavengers, have been proven to inhibit ferroptosis, which further supports the case for ferroptosis as a potential pathogenic mechanism in PTSD. To thoroughly investigate the mechanistic links between ferroptosis and PTSD, we analyze the relevant literature on ferroptosis and PTSD in this review. Our aim is to elucidate the potential relationships between ferroptosis and PTSD, thereby providing novel insights for future research directions. Furthermore, we call for more experimental and clinical studies to explore this relationship further, with the ultimate goal of developing more effective therapeutic strategies for PTSD.

IntroductionFerroptosis, an iron-dependent regulated cell death pathway, shares several features of Parkinson’s disease (PD) physiopathology, and efficient neuroprotective therapies are required to prevent DAergic neuron death initiated by ferroptosis. Electroacupuncture (EA), a treasure of Traditional Chinese Medicine, exerted therapeutic effects against PD to avoid the side effects of dopamine (DA)-based therapies. However, its underlying mechanisms still need to be fully understood.MethodsMPTP-induced PD mice were treated with EA to evaluate its neuroprotective effects. Behavioral assessments, histopathological analysis of DAergic neurons, and quantification of ferroptosis biomarkers-including malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, glutathione (GSH), and mitochondrial integrity-were performed. Protein expression levels of SLC7A11, GPX4, ferritin heavy chain 1 (FTH1), and nuclear factor erythroid 2-related factor 2 (Nrf2) were analyzed via immunoblotting. To validate pathway specificity, the Nrf2 inhibitor trigonelline (AT) was co-administered with EA.ResultsEA treatment significantly mitigated MPTP-induced DAergic neuron loss and motor deficits. Mechanistically, EA suppressed ferroptosis by reducing lipid peroxidation and iron accumulation while restoring GSH levels. It upregulated ferroptosis-suppressive proteins SLC7A11, GPX4, FTH1, and Nrf2, alongside ameliorating mitochondrial dysfunction. Crucially, AT administration abolished EA’s protective effects, confirming Nrf2 pathway dependency.DiscussionThese findings demonstrate that EA exerts neuroprotection in PD by inhibiting ferroptosis through activation of the Nrf2/SLC7A11/FTH1/GPX4 signaling axis. This study not only elucidates a novel mechanism underlying EA’s efficacy in PD but also highlights ferroptosis modulation as a therapeutic strategy, bridging traditional medicine with molecular pathophysiology. This study has provided new ideas for exploring the mechanism of EA in PD treatment.

Recent studies have identified a family of rod-shaped proteins thought to mediate lipid transfer at intracellular membrane contacts by a bridge-like mechanism. We show that one such protein, bridge-like lipid transfer protein 3A (BLTP3A)/UHRF1BP1 binds VAMP7 vesicles via its C-terminal region, and anchors them to lysosomes via its chorein domain-containing N-terminal region binding to Rab7. Upon lysosome damage, BLTP3A-positive vesicles rapidly (within minutes) dissociate from lysosomes. Lysosome damage is known to activate the CASM (Conjugation of ATG8 to Single Membranes) pathway, leading to lipidation and lysosomal recruitment of mammalian ATG8 (mATG8) proteins. We find that this process drives the reassociation of BLTP3A with damaged lysosomes via an interaction of its LIR motif with mATG8 which coincides with a dissociation from the vesicles. Our findings reveal that BLTP3A is an effector of CASM, potentially as part of a mechanism to help repair or minimize lysosome damage.

BLTP2/KIAA0100, a bridge-like lipid transfer protein, was reported to localize at contacts of the endoplasmic reticulum (ER) with either the plasma membrane (PM) or recycling tubular endosomes depending on the cell type. Our findings suggest that mediating bulk lipid transport between the ER and the PM is a key function of this protein as BLTP2 tethers the ER to tubular endosomes only after they become continuous with the PM and that it also tethers the ER to macropinosomes in the process of fusing with the PM. We further identify interactions underlying binding of BLTP2 to the PM, including phosphoinositides, the adaptor proteins FAM102A and FAM102B, and also N-BAR domain proteins at membrane-connected tubules. The absence of BLTP2 results in the accumulation of intracellular vacuoles, many of which are connected to the plasma membrane, pointing to a role of the lipid transport function of BLTP2 in the control of PM dynamics.

Current pharmacotherapies for post-traumatic stress disorder (PTSD) are limited by delayed onset and side effects. Despite ketamine exhibiting rapid relief of the core symptoms of PTSD, its clinical efficacy varies considerably depending on the timing of drug delivery. However, the underlying mechanism remains unclear. In this study, the therapeutic effects of early (day 1) and late (day 7) administration of S-Ketamine on behavioral phenotypes in rodent's models of PTSD are compared. It is observed that early rather than late administration of S-Ketamine significantly ameliorates PTSD symptoms, especially impaired fear extinction. The firing and burst rates of VTADA neurons consecutively decrease following PTSD modeling and are restored by early S-Ketamine intervention. In particular, VTADA neurons respond to the conditioned stimuli, mediating the replacement of aversive memory encoding during fear extinction. The inhibition of VTADA-OFC interrupts the PTSD treatment induced by S-Ketamine. A non-invasive temporally interfering brain stimulation targeting the OFC is further developed, sensitizing cortical dopaminergic transmission and extending the effective time window of S-Ketamine for anti-PTSD. Overall, a neural mechanism for the heterogeneous VTADA-OFC neurocircuit-mediated time-dependent therapeutic effect of S-Ketamine is illustrated. In addition, a novel technique is developed to optimize the strategy of ketamine-assisted psychotherapy for PTSD treatment.

Ultraviolet radiation B (UVB), the most biologically active ultraviolet ray in sunlight, exert broad effects on physiological and behavioral functions, including circadian rhythm, mood, and cognition. However, its underlying mechanisms are still unknown. In this study, in order to verify effects of UVB on anxiety and social behaviors, C57BL/6 mice receiving 2 h UVB exposure after chronic restraint stress were used. UVB exposure improved anxiety-like behaviors and social activities in normal and restraint stressed mice. Meanwhile, UVB exposure increased the neural excitability in mPFC according to cFos staining and electrophysiology results. And benefits of UVB exposure could be blocked by chemogenetical inhibition of mPFC or inhibiting mPFC to basal lateral amygdala (BLA) pathway. In conclusion, we identify UVB exposure ameliorate chronic stress-induced anxiety and social impairment by activating mPFC to BLA pathway. The series of research may lead to the development of UVB as a novel therapeutic approach for treating anxiety and social avoidance in the future.

Central nervous system (CNS) disorders, which include both neurodegenerative and neuroinflammatory conditions, present significant therapeutic challenges due to their complex pathological mechanisms, limited treatment options, and difficulties in drug delivery across the blood-brain barrier (BBB). Therefore, exploring therapeutic targets capable of overcoming these obstacles is of paramount importance. The liver X receptor (LXR) plays a crucial role in lipid homeostasis, inhibition of neuroinflammation, and promotion of neurogenesis, making it a promising therapeutic target for CNS diseases. However, the potential adverse effects associated with LXR agonists, such as hepatic steatosis and hypertriglyceridemia, may hinder their future clinical application. In this review, we examine the dual roles of LXR agonists in CNS diseases, highlighting their therapeutic benefits alongside their adverse effects. To address these challenges, we propose several optimization strategies, including the development of LXRβ selective agonists, advanced nano-delivery systems, and combination therapies, with the ultimate goal of providing a theoretical foundation for future therapies centered on LXR.