PV network plasticity mediated by neuregulin1-ErbB4 signalling controls fear extinction.

Neuroplasticity in the medial prefrontal cortex (mPFC) is essential for fear extinction, the process of which forms the basis of the general therapeutic process used to treat human fear disorders. However, the underlying molecules and local circuit elements controlling neuronal activity and concomitant induction of plasticity remain unclear. Here we show that sustained plasticity of the parvalbumin (PV) neuronal network in the infralimbic (IL) mPFC is required for fear extinction in adult male mice and identify the involvement of neuregulin 1-ErbB4 signalling in PV network plasticity-mediated fear extinction. Moreover, regulation of fear extinction by basal medial amygdala (BMA)-projecting IL neurons is dependent on PV network configuration. Together, these results uncover the local molecular circuit mechanisms underlying mPFC-mediated top-down control of fear extinction, suggesting alterative therapeutic approaches to treat fear disorders.

Integrating traditional apprenticeship and modern educational approaches in traditional Chinese medicine education.

BACKGROUND: The traditional apprenticeship system and modern educational models both contribute to traditional Chinese medicine (TCM) education in unique ways. This study aims to evaluate the advantages and disadvantages of these approaches and investigates their potential integration for optimal TCM teaching. METHODS: The study employs a comprehensive literature search strategy with specific keywords through Boolean Operators, focusing on articles discussing TCM education, sourced from the databases PubMed, Scopus, and CNKI. For comparative analysis, the study analyzes the advantages and disadvantages of three distinct approaches-traditional, modern, and hybrid-in TCM education. Finally, we use a spider plot methodology to visually evaluate across 11 critical educational aspects for these approaches. RESULTS: A comprehensive review of 70 articles on TCM education highlights the value of both conventional and modern approaches. Conventional methods often emphasize teacher-centered clinical training that aligns well with the apprenticeship model, serving to bridge formal academic learning with hands-on experience. Modern methods incorporate elements like technology and formal accreditation but caution that the absence of traditional apprenticeship could dilute core TCM principles. One recurring theme across multiple studies is the enduring importance of apprenticeship-a pedagogical cornerstone rooted in TCM's historical and cultural context-as well as its tension with modern methodologies that incorporate online resources and scientific frameworks. An integrated approach attempts to harmonize these strengths but reveals a gap in interactivity, suggesting that incorporating apprenticeship could offer a practical, hands-on method to improve student engagement. CONCLUSIONS: Conventional methods in TCM education emphasize teacher-centered clinical training akin to apprenticeship, modern methods incorporate technological advances and formal accreditation; however, the absence of traditional apprenticeship could compromise core TCM principles, and an integrated model, though striving to harmonize these elements, still falls short in the area of student interactivity.

Three New Steriodal Saponins from the Rhizomes of Tupistra chinensis Baker.

The phytochemical constituent investigation on the 70 % ethanol extract of the rhizomes of Tupistra chinensis Baker resulted in the isolation of three new steroidal saponins which were named tuchinosides A-C (1-3). Their structures were determined by extensive spectrum analysis and chemical evidence, especially 2D NMR and HR-ESI-MS techniques. In addition, the cytotoxicity of compounds 1-3 against several human cancer cell lines was evaluated.

Cinnamaldehyde ameliorates LPS-induced cardiac dysfunction via TLR4-NOX4 pathway: The regulation of autophagy and ROS production.

Cinnamaldehyde (CA), a major bioactive compound extracted from the essential oil of Cortex Cinnamomi, exhibits anti-inflammatory activity on endotoxemia. Accumulating evidence indicates reactive oxygen species (ROS) and autophagy play a vital role in the cardiac dysfunction during endotoxemia. The aim of this study was to unveil the mechanism of CA on ROS production and autophagy during endotoxemia. Male Sprague-Dawley rats were stimulated by LPS (20mg/kg i.v.) with or without treatment of CA. Cardiac function and histopathological staining were preformed 4h after LPS stimulation. The levels of TNF-α, IL-1ß and IL-6 were detected by ELISA. The expression of p-JNK, p-ERK, p-p38, TLR4, NOX4, NOX2, ATG5 and LC3 proteins were determined by Western blot. The results showed that CA inhibited cardiac dysfunction, inflammatory infiltration and the levels of TNF-α, IL-1ß and IL-6 in LPS stimulated rats by blocking the TLR4, NOX4, MAPK and autophagy signalings. In order to obtain further confirmation of the mechanism of CA on endotoxemia in vitro, a limited time-course study was firstly performed by Western blot. TLR4, NOX4 and LC3 were significantly increased after 4h LPS stimulation. CA reversed the intracellular ROS production and MAPK signaling activation induced by LPS. Electron microscopy, mRFP-GFP-LC3 transfection and western blot results revealed autophagic flux were attenuated after CA treatment. The siRNA and molecular docking results suggest that CA can suppress both TLR4 and NOX4 during endotoxemia. Our data revealed that CA ameliorated LPS-induced cardiac dysfunction by inhibiting ROS production and autophagy through TLR4-NOX4 pathway.

A Novel Mechanism of Spine Damages in Stroke via DAPK1 and Tau.

Synaptic spine loss is one of the major preceding consequences of stroke damages, but its underlying molecular mechanisms remain unknown. Here, we report that a direct interaction of DAPK1 with Tau causes spine loss and subsequently neuronal death in a mouse model with stroke. We found that DAPK1 phosphorylates Tau protein at Ser262 (pS(262)) in cortical neurons of stroke mice. Either genetic deletion of DAPK1 kinase domain (KD) in mice (DAPK1-KD(-/-)) or blocking DAPK1-Tau interaction by systematic application of a membrane permeable peptide protects spine damages and improves neurological functions against stroke insults. Thus, disruption of DAPK1-Tau interaction is a promising strategy in clinical management of stroke.

Enrichment of cardiac differentiation by a large starting number of embryonic stem cells in embryoid bodies is mediated by the Wnt11-JNK pathway.

Embryoid bodies (EBs) with large starting numbers of embryonic stem cells (ESCs) have a greater degree of cardiac differentiation than from low numbers of EBs. However, the biological roles of signaling molecules in these effects are not well understood. Here, we show that groups of EBs with different starting numbers of ESCs had differential gene expression patterns for Wnt5a and Wnt11. Wnt11 significantly increased the percentage of beating EBs by up-regulating the expression of the cardiac-specific genes. Wnt5a did not show these effects. Moreover, Wnt11 significantly increased the level of phosphorylated Jun N-terminal kinase. The inhibition of the JNK pathway by SP600125 blocked the effects of Wnt11. Thus, enrichment of cardiac differentiation in groups of EBs with a larger starting number of ESCs is mediated by the Wnt11-JNK pathway.

Ventral subiculum promotes wakefulness through several pathways in male mice.

The ventral subiculum (vSUB), the major output structure of the hippocampal formation, regulates motivation, stress integration, and anxiety-like behaviors that rely on heightened arousal. However, the roles and underlying neural circuits of the vSUB in wakefulness are poorly known. Using in vivo fiber photometry and multichannel electrophysiological recordings in mice, we found that the vSUB glutamatergic neurons exhibited high activities during wakefulness. Moreover, activation of vSUB glutamatergic neurons caused an increase in wakefulness and anxiety-like behaviors and induced a rapid transition from sleep to wakefulness. In addition, optogenetic stimulation of vSUB glutamatergic terminals and retrograde-targeted chemogenetic activation of vSUB glutamatergic neurons revealed that vSUB promoted arousal by innervating the lateral hypothalamus (LH), nucleus accumbens (NAc) shell, and prefrontal cortex (PFC). Nevertheless, local microinjection of dopamine D1 or D2/D3 receptor antagonist blocked the wake-promoting effect induced by chemogenetic activation of vSUB pathways. Finally, chemogenetic inhibition of vSUB glutamatergic neurons decreased arousal. Altogether, our findings reveal a prominent contribution of vSUB glutamatergic neurons to the control of wakefulness through several pathways.

Two previously undescribed cholestanol saponins from the rhizomes of Paris fargesii var. petiolata.

Two previously undescribed cholestanol saponins, parpetiosides F - G (1-2), and six known analogs (3-8) were isolated from the rhizomes of Paris fargesii var. petiolata. Their structures were elucidated by extensive spectroscopic data analysis and chemical methods. Compound 1 was a rare 6/6/6/5/5 fused-rings cholestanol saponin with disaccharide moiety linked at C-26 of aglycone which was hardly seen in genus Paris. All of these compounds were discovered in this plant for the first time. In addition, the cytotoxicities of saponins (1-8) against three human cancer cell lines (U87, HepG2 and SGC-7901) were evaluated by CCK-8 method, and saponins 5-8 displayed certain cytotoxicities. The strong interactions between saponins 5-8 and SCUBE3, an oncogene for glioma cells, were displayed by molecular docking.

Distinct glutamatergic projections of the posteroventral medial amygdala play different roles in arousal and anxiety.

Sleep disturbance usually accompanies anxiety disorders and exacerbates their incidence rates. The precise circuit mechanisms remain poorly understood. Here, we found that glutamatergic neurons in the posteroventral medial amygdala (MePVGlu) are involved in arousal and anxiety-like behaviors. Excitation of MePVGlu neurons not only promoted wakefulness but also increased anxiety-like behaviors. Different projections of MePVGlu neurons played various roles in regulating anxiety-like behaviors and sleep-wakefulness. MePVGlu neurons promoted wakefulness through the MePVGlu-posteromedial cortical amygdaloid area (PMCo) pathway and the MePVGlu-bed nucleus of the stria terminals (BNST) pathway. In contrast, MePVGlu neurons increased anxiety-like behaviors through the MePVGlu-ventromedial hypothalamus (VMH) pathway. Chronic sleep disturbance increased anxiety levels and reduced reparative sleep, accompanied by the enhanced excitability of MePVGlu-PMCo and MePVGlu-VMH circuits but suppressed responses of glutamatergic neurons in the BNST. Inhibition of the MePVGlu neurons could rescue chronic sleep deprivation-induced phenotypes. Our findings provide important circuit mechanisms for chronic sleep disturbance-induced hyperarousal response and obsessive anxiety-like behavior, and are expected to provide a promising strategy for treating sleep-related psychiatric disorders and insomnia.

Time-dependent regulation of neuregulin-1ß/ErbB/ERK pathways in cardiac differentiation of mouse embryonic stem cells.

Neuregulin-1ß (NRG-1ß)/ErbB signaling plays crucial roles in the cardiac differentiation of mouse embryonic stem cells (ESCs), but its roles and the underlying mechanisms in cardiac differentiation are incompletely understood. This study showed that NRG-1ß significantly increased the percentage of beating embryoid bodies (EBs) and up-regulated the gene expressions of Nkx2.5, GATA4, α-actin, MLC-2v, and ANF in a time-dependent manner, with no effect on the gene expressions of HCN4 and Tbx3. Inhibition of ErbB receptors with AG1478 significantly decreased the percentage of beating EBs; down-regulated the gene expressions of Nkx2.5, GATA4, MLC-2v, ANF, and α-actin; and concomitantly up-regulated the gene expressions of HCN4 and Tbx3 in a time-dependent manner. Moreover, the up-regulation of transcripts for Nkx2.5 and GATA4 by NRG-1ß was blocked by the extracellular signal-related kinases (ERK) 1/2 inhibitor, U0126. However, U0126 could not inhibit the transcript up-regulations of MLC-2v and ANF by NRG-1ß. The protein quantitation results were consistent with those of gene quantitation. Our results suggest that NRG-1ß/ErbB signaling plays critical roles in the cardiac differentiation of mouse ESCs and in the subtype specification of cardiomyocytes in a time-dependent manner. The ERK1/2 pathway may be involved in the early cardiogenesis, but not in the subtype specification of cardiomyocytes.

Anticancer effects of bufalin on human hepatocellular carcinoma HepG2 cells: roles of apoptosis and autophagy.

The traditional Chinese medicine bufalin, extracted from toad's skin, has been demonstrated to exert anticancer activities in various kinds of human cancers. The mechanisms of action lie in its capacity to induce apoptosis, or termed type I programmed cell death (PCD). However, type II PCD, or autophagy, participates in cancer proliferation, progression, and relapse, as well. Recent studies on autophagy seem to be controversial because of the dual roles of autophagy in cancer survival and death. In good agreement with previous studies, we found that 100 nM bufalin induced extensive HepG2 cell apoptosis. However, we also noticed bufalin triggered autophagy and enhanced Beclin-1 expression, LC3-I to LC3-II conversion, as well as decreased p62 expression and mTOR signaling activation in HepG2 cells. Blockage of autophagy by selective inhibitor 3-MA decreased apoptotic ratio in bufalin-treated HepG2 cells, suggesting a proapoptotic role of bufalin-induced autophagy. Furthermore, we investigated the underlying mechanisms of bufalin-induced autophagy. Bufalin treatment dose-dependently promoted AMPK phosphorylation while AMPK inhibition by compound C significantly attenuated bufalin-induced autophagy. Taken together, we report for the first time that bufalin induces HepG2 cells PCD, especially for autophagy, and the mechanism of action is, at least in part, AMPK-mTOR dependent.

Cytotoxic steroidal glycosides from the rhizomes of Paris polyphylla var. yunnanensis.

Paris polyphylla var. yunnanensis (Franch.) Hand.-Mazz. (Melanthiaceae), an important specie of the genus Paris, has long been in a traditional Chinese medicine (TCM) for a long time. This study aimed to isolate and identify the structures of bioactive saponins from the rhizomes of P. polyphylla var. yunnanensis and evaluate their cytotoxicity against BxPC-3, HepG2, U373 and SGC-7901 carcinoma cell lines. Seven previously undescribed and seven known saponins were identified, and Paris saponins VII (PSVII) showed significant cytotoxicity against the BxPC-3 cell line with IC50 values of 3.59 µM. Furthermore, flow cytometry, transmission electron microscopy and western-bolt analysis revealed that PSVII inhibited the proliferation of BxPC-3 cells and might be involved in inducing apoptosis and pyroptosis by activating caspase-3, -7 and caspase-1, respectively.

Rational Design and Acoustic Assembly of Human Cerebral Cortex-Like Microtissues from hiPSC-Derived Neural Progenitors and Neurons.

Development of biologically relevant and clinically relevant human cerebral cortex models is demanded by mechanistic studies of human cerebral cortex-associated neurological diseases and discovery of preclinical neurological drug candidates. Here, rational design of human-sourced brain-like cortical tissue models is demonstrated by reverse engineering and bionic design. To implement this design, the acoustic assembly technique is employed to assemble hiPSC-derived neural progenitors and neurons separately in a label-free and contact-free manner followed by subsequent neural differentiation and culture. The generated microtissues encapsulate the neuronal microanatomy of human cerebral-cortex tissue that contains six-layered neuronal architecture, a 400-µm interlayer distance, synaptic connections between interlayers, and neuroelectrophysiological transmission. Furthermore, these microtissues are infected with herpes simplex virus type I (HSV-1) virus, and the HSV-induced pathogenesis associated with Alzheimer's disease is determined, including neuron loss and the expression of Aß. Overall, a high-fidelity human-relevant in vitro histotypic model is provided for the cerebral cortex, which will facilitate wide applications in probing the mechanisms of neurodegenerative diseases and screening the candidates for neuroprotective agents.

Distinct Hypothalamic Paraventricular Nucleus Inputs to the Cingulate Cortex and Paraventricular Thalamic Nucleus Modulate Anxiety and Arousal.

Insomnia and anxiety are two common clinical diseases that threaten people's physical and mental health. Insomnia and anxiety may share some similar underlying neural circuit mechanisms in the brain. In this study, we combine techniques including chemo-fMRI, optogenetics, and chemogenetics to reveal that the glutamatergic neurons of the paraventricular hypothalamic nucleus (PVN) regulate both anxiety and arousal through two different downstream neural circuits. Optogenetic activation of the PVN-cingulate cortex (Cg) neural circuit triggers anxiety-like behaviors in mice without affecting the wakefulness, while optogenetic activation of the PVN-paraventricular thalamic nucleus (PVT) neural circuit promotes wakefulness in mice without affecting anxiety-like behaviors. Our research reveals that PVN is a key brain area for controlling anxiety and arousal behaviors. We also provide a neurological explanation for anxiety disorder and insomnia which may offer guidance for treatments including drugs or transcranial magnetic stimulation for the patients.

Neuregulin-1-dependent control of amygdala microcircuits is critical for fear extinction.

The posttraumatic stress disorder is marked by an impaired ability to extinct fear memory acquired in trauma. Although previous studies suggest that fear extinction depends on the function of the amygdala, the underlying mechanisms are unclear. We found that NRG1 receptors (ErbB4) were abundantly expressed in the intercalated cells mass of amygdala (ITC). The NRG1-ErbB4 pathway in the ITC promotes fear extinction. The NRG1-ErbB4 pathway in the ITC did not affect excitatory input to ITC neurons from BLA neurons but increased feed-forward inhibition of (the central medial nucleus of the amygdala) CeM neurons through increased GABAergic neurotransmission of ITC neurons. We also found that the NRG1-ErbB4 signaling pathway in ITC might regulate fear extinction through P/Q-type voltage-activated Ca2+ channels (VACCs) but not through L- or N-type VACCs. Overall, our results suggest that the NRG1-ErbB4 signaling pathway in the ITC might represent a potential target for the treatment of anxiety disorders.

Paris saponin H inhibits the proliferation of glioma cells through the A1 and A3 adenosine receptor­mediated pathway.

Paris saponin H (PSH) is a type of steroid saponin derived from Rhizoma Paridis (RP; the rhizome of Paris). In our previous studies, saponins from RP exerted antiglioma activity in vitro. However, the effects of PSH on glioma have not been elucidated. The aim of the present study was to evaluate the effects of PSH on U251 glioblastoma cells and elucidate the possible underlying mechanism. The cells were treated with PSH at various concentrations for 48 h, and the cell viability, invasion, apoptosis and cycle progression were assessed using specific assay kits. The activation of Akt, 44/42­mitogen­activated protein kinase (MAPK) and the expression levels of A1 adenosine receptor (ARA1) and ARA3 were assessed by western blotting. The results demonstrated that PSH inhibited cell viability, migration and invasion, and induced apoptosis. Treatment of U251 cells with PSH induced the upregulation of p21 and p27, and the downregulation cyclin D1 and S­phase kinase associated protein 2 protein expression levels, which induced cell cycle arrest at the G1 phase. The results also demonstrated that PSH inhibited the expression of ARA1, and the agonist of ARA1, 2­chloro­N6­cyclopentyladenosine, reversed the effects of PSH. Hypoxia induced increases in the ARA3, hypoxia­inducible factor­1α (HIF­1α) and vascular endothelial growth factor (VEGF) protein expression levels, which were associated with the activation of the Akt and P44/42 MAPK pathways. Compared with the hypoxia group, PSH inhibited the expression levels of ARA3, HIF­1α and VEGF, as well as the phosphorylation levels of Akt and 44/42 MAPK, and repressed HIF­1α transcriptional activity. Furthermore, the results demonstrated that PSH inhibited the expression of HIF­1α by inhibiting the phosphorylation of Akt and 44/42 MAPK mediated by ARA3. Taken together, these results suggested that PSH reduced U251 cell viability via the inhibition of ARA1 and ARA3 expression, and further inhibited Akt and 44/42 MAPK phosphorylation, induced apoptosis and cell cycle arrest.

An updated model-ready emission inventory for Guangdong Province by incorporating big data and mapping onto multiple chemical mechanisms.

An accurate characterization of spatial-temporal emission patterns and speciation of volatile organic compounds (VOCs) for multiple chemical mechanisms is important to improving the air quality ensemble modeling. In this study, we developed a 2017-based high-resolution (3 km × 3 km) model-ready emission inventory for Guangdong Province (GD) by updating estimation methods, emission factors, activity data, and allocation profiles. In particular, a full-localized speciation profile dataset mapped to five chemical mechanisms was developed to promote the determination of VOC speciation, and two dynamic approaches based on big data were used to improve the estimation of ship emissions and open fire biomass burning (OFBB). Compared with previous emissions, more VOC emissions were classified as oxygenated volatile organic compound (OVOC) species, and their contributions to the total ozone formation potential (OFP) in the Pearl River Delta (PRD) region increased by 17%. Formaldehyde became the largest OFP species in GD, accounting for 11.6% of the total OFP, indicating that the model-ready emission inventory developed in this study is more reactive. The high spatial-temporal variability of ship sources and OFBB, which were previously underestimated, was also captured by using big data. Ship emissions during typhoon days and holidays decreased by 23-55%. 95% of OFBB emissions were concentrated in 9% of the GD area and 31% of the days in 2017, demonstrating their strong spatial-temporal variability. In addition, this study revealed that GD emissions have changed rapidly in recent years due to the leap-forward control measures implemented, and thus, they needed to be updated regularly. All of these updates led to a 5-17% decrease in the emission uncertainty for most pollutants. The results of this study provide a reference for how to reduce uncertainties in developing model-ready emission inventories.

Glutamatergic Neurons of the Paraventricular Nucleus are Critical for the Control of Wakefulness.

Normal sleep-wake behavior is extremely important for humans to maintain basic physiological and cognitive activities. However, the neural mechanisms underlying sleep-wake regulation are not fully understood. The paraventricular nucleus (PVN) of the hypothalamus has been classically defined as a region for the regulation of the hypothalamoneurohypophysial system and autonomic nervous system. Here, we identify the glutamatergic neurons in the PVN that play a unique role in sleep-wake regulation. Firstly using in vivo fiber photometry, we found altered calcium activities of PVN glutamatergic neurons during three sleep state transitions in freely behaving mice. The calcium activities of PVN glutamatergic neurons began to increase before non-rapid-eye movement (NREM) sleep to wake transitions and NREM sleep to rapid-eye-movement (REM) sleep transitions and began to decrease before wake to NREM sleep transitions. Then we used chemogenetic manipulations together with polysomnographic recordings, activation of PVN neurons increased wakefulness and decreased NREM sleep, while inhibition of PVN neurons caused a reduction in wakefulness and an increase in NREM sleep. Altogether, our findings revealed an important role for PVN glutamatergic neurons in the regulation of wake state.

Tetrahydroxystilbene Glucoside Ameliorates Infrasound-Induced Central Nervous System (CNS) Injury by Improving Antioxidant and Anti-Inflammatory Capacity.

BACKGROUND: Infrasound is a major threat to global health by causing injuries of the central nervous system (CNS). However, there remains no effective therapeutic agent for preventing infrasound-caused CNS injury. 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glycoside (THSG) exerts protective function against CNS injuries and may have beneficial effects on infrasound-induced CNS impairment. METHODS: A mouse model with CNS (oxidative stress-induced inflammation and neuronal apoptosis) injuries was established when the mouse was exposed to the infrasound of 16 Hz at 130 dB for 2 h each day and the duration of treatment was 8 d. The mice were divided into the control (CG, healthy mice), the model (MG, model mice), and the THSG (EG, experimental group, model mice treated with THSG) groups. The learning and memory impairments caused by infrasound were examined using a Morris water maze test. Lipid profiles, antioxidant biomarkers, and inflammatory cytokines in hippocampus tissue were measured by using corresponding ELISA kits. Meanwhile, BCL-2/BAX/caspase-3 signaling pathway was measured in the hippocampi and prefrontal cortex of the mouse brain using real-time qPCR and Western blot. Nissl's stain was used to measure neuronal necrosis in the hippocampi and prefrontal cortex of the mouse brain. RESULTS: THSG significantly ameliorated the learning and memory impairments caused by infrasound. On the other hand, THSG improved lipid profiles, increased antioxidant properties by affecting the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA), and displayed anti-inflammatory action via the downregulation of IL- (interleukin-) 6, IL-8, IL-10, TNF- (tumor necrosis factor-) α, and hs-CRP (high-sensitivity C-reactive protein) in the hippocampal tissues of the mouse model (P < 0.05). Additionally, Nissl's stain showed that THSG inhibited infrasound-induced neuronal necrosis in the hippocampi and prefrontal cortex. Besides, THSG exerted antiapoptosis function by upregulating the level of Bcl-2 and downregulating the levels of BAX and caspase-3 in the hippocampi. CONCLUSION: THSG may be an effective anti-infrasound drug against CNS injury by improving antioxidant, anti-inflammatory, antiapoptosis, and antinecrosis capacities. Further research is still needed to confirm the exact molecular mechanism.

Orchestrating Opiate-Associated Memories in Thalamic Circuits.

Disrupting memories that associate environmental cues with drug experiences holds promise for treating addiction, yet accessing the distributed neural network that stores such memories is challenging. Here, we show that the paraventricular nucleus of the thalamus (PVT) orchestrates the acquisition and maintenance of opiate-associated memories via projections to the central nucleus of the amygdala (CeA) and nucleus accumbens (NAc). PVT→CeA activity associates morphine reward to the environment, whereas transient inhibition of the PVT→NAc pathway during retrieval causes enduring protection against opiate-primed relapse. Using brain-wide activity mapping, we revealed distributed network activities that are altered in non-relapsing mice, which enabled us to find that activating the downstream NAc→lateral hypothalamus (LH) pathway also prevents relapse. These findings establish the PVT as a key node in the opiate-associated memory network and demonstrate the potential of targeting the PVT→NAc→LH pathway for treating opioid addiction.