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Abstract Background Depressive symptoms often occur in patients with Alzheimer's disease (AD) and exacerbate the pathogenesis of AD. However, the neural circuit mechanisms underlying the AD-associated depression remain unclear. The serotonergic system plays crucial roles in both AD and depression. Methods We used a combination of in vivo trans-synaptic circuit-dissecting anatomical approaches, chemogenetic manipulations, optogenetic manipulations, pharmacological methods, behavioral testing, and electrophysiological recording to investigate dorsal raphe nucleus serotonergic circuit in AD-associated depression in AD mouse model. Results We found that the activity of dorsal raphe nucleus serotonin neurons (DRN5-HT) and their projections to the dorsal hippocampal CA1 (dCA1) terminals (DRN5-HT-dCA1CaMKII) both decreased in brains of early 5×FAD mice. Chemogenetic or optogenetic activation of the DRN5-HT-dCA1CaMKII neural circuit attenuated the depressive symptoms and cognitive impairments in 5×FAD mice through serotonin receptor 1B (5-HT1BR) and 4 (5-HT4R). Pharmacological activation of 5-HT1BR or 5-HT4R attenuated the depressive symptoms and cognitive impairments in 5×FAD mice by regulating the DRN5-HT-dCA1CaMKII neural circuit to improve synaptic plasticity. Conclusions These findings provide a new mechanistic connection between depression and AD and provide potential pharmaceutical prevention targets for AD.

Summary: Self-grooming is an innate stereotyped behavior influenced by sense and emotion. It is considered an important characteristic in various disease models. However, the neural circuit mechanism underlying sensory-induced and emotion-driven self-grooming remains unclear. We found that the ventral zona incerta (Ziv) was activated during spontaneous self-grooming (SG), corn oil-induced sensory self-grooming (OG), and tail suspension-induced stress self-grooming (TG). Optogenetic excitation of Ziv parvalbumin (PV) neurons increased the duration of SG. Conversely, optogenetic inhibition of ZivPV neurons significantly reduced self-grooming in all three models. Furthermore, glutamatergic inputs from the primary sensory cortex activated the Ziv and contributed to OG. Activation of GABAergic inputs from the central amygdala to the Ziv increased SG, OG, and TG, potentially through local negative regulation of the Ziv. These findings suggest that the Ziv may play a crucial role in processing sensory and emotional information related to self-grooming, making it a potential target for regulating stereotyped behavior.

Abstract Ca2+ signaling is essential for oligodendrocyte (OL) development and myelin formation. Inositol 1,4,5‐trisphosphate receptor type 2 (ITPR2) is an endoplasmic reticulum calcium channel and shows stage‐dependent high levels in postmitotic oligodendrocyte precursor cells (OPCs). The role and potential mechanism of ITPR2 in OLs remain unclear. In this study, it is revealed that loss of Itpr2 in OLs disturbs Ca2+ homeostasis and inhibits myelination in adolescent mice. Animals with OL‐specific deletion of Itpr2 exhibit anxiety/depressive‐like behaviors and manifest with interrupted OPC proliferation, leading to fewer mature OLs in the brain. Detailed transcriptome profiling and signal pathway analysis suggest that MAPK/ERK‐CDK6/cyclin D1 axis underlies the interfered cell cycle progression in Itpr2 ablated OPCs. Besides, blocking MAPK/ERK pathway significantly improves the delayed OPC differentiation and myelination in Itpr2 mutant. Notably, the resting [Ca2+]i is increased in Itpr2 ablated OPCs, with the elevation of several plasma calcium channels. Antagonists against these plasma calcium channels can normalize the resting [Ca2+]i level and enhance lineage progression in Itpr2‐ablated OPCs. Together, the findings reveal novel insights for calcium homeostasis in manipulating developmental transition from OPCs to pre‐OLs; additionally, the involvement of OLs‐originated ITPR2 in depressive behaviors provides new therapeutic strategies to alleviate myelin‐associated psychiatric disorders.

IntroductionAutism spectrum disorder (ASD) is a group of diseases often characterized by poor sociability and challenges in social communication. The anterior cingulate cortex (ACC) is a core brain region for social function. Whether it contributes to the defects of social communication in ASD and whether it could be physiologically modulated to improve social communication have been poorly investigated. This study is aimed at addressing these questions.MethodsFragile X mental retardation 1 (FMR1) mutant and valproic acid (VPA)-induced ASD mice were used. Male–female social interaction was adopted to elicit ultrasonic vocalization (USV). Immunohistochemistry was used to evaluate USV-activated neurons. Optogenetic and precise target transcranial magnetic stimulation (TMS) were utilized to modulate anterior cingulate cortex (ACC) neuronal activity.ResultsIn wild-type (WT) mice, USV elicited rapid expression of c-Fos in the excitatory neurons of the left but not the right ACC. Optogenetic inhibition of the left ACC neurons in WT mice effectively suppressed social-induced USV. In FMR1−/−- and VPA-induced ASD mice, significantly fewer c-Fos/CaMKII-positive neurons were observed in the left ACC following USV compared to the control. Optogenetic activation of the left ACC neurons in FMR1−/− or VPA-pretreated mice significantly increased social activity elicited by USV. Furthermore, precisely stimulating neuronal activity in the left ACC, but not the right ACC, by repeated TMS effectively rescued the USV emission in these ASD mice.DiscussionThe excitatory neurons in the left ACC are responsive to socially elicited USV. Their silence mediates the deficiency of social communication in FMR1−/− and VPA-induced ASD mice. Precisely modulating the left ACC neuronal activity by repeated TMS can promote the social communication in FMR1−/− and VPA-pretreated mice.

Autism spectrum disorder (ASD) is a group of developmental diseases characterized by social dysfunction and repetitive stereotype behaviors. Besides genetic mutations, environmental factors play important roles in the development of ASD. Valproic acid (VPA) is widely used for modeling environmental factor induced ASD in rodents. However, traditional VPA modeling is low-in-efficiency and the phenotypes often vary among different batches of experiments. To optimize this ASD-modeling method, we tested “two-hit” hypothesis by single or double exposure of VPA and poly:IC at the critical time points of embryonic and postnatal stage. The autistic-like behaviors of mice treated with two-hit schemes (embryonic VPA plus postnatal poly:IC, embryonic poly:IC plus postnatal VPA, embryonic VPA plus poly: IC, or postnatal VPA plus poly:IC) were compared with mice treated with traditional VPA protocol. The results showed that all single-hit and two-hit schemes produced core ASD phenotypes as VPA single treatment did. Only one group, namely, mice double-hit by VPA and poly:IC simultaneously at E12.5 showed severe impairment of social preference, social interaction and ultrasonic communication, as well as significant increase of grooming activity and anxiety-like behaviors, in comparation with mice treated with the traditional VPA protocol. These data demonstrated that embryonic two-hit of VPA and poly:IC is more efficient in producing ASD phenotypes in mice than the single-hit of VPA, indicating this two-hit scheme could be utilized for modeling environmental factors induced ASD.

Abstract Impaired extinction of fear memory is one of the most common symptoms in post‐traumatic stress disorder (PTSD), with limited therapeutic strategies due to the poor understanding of its underlying neural substrates. In this study, functional screening is performed and identified hyperactivity in the mediodorsal thalamic nucleus (MD) during fear extinction. Furthermore, the encoding patterns of the hyperactivated MD is investigated during persistent fear responses using multiple machine learning algorithms. The anterior cingulate cortex (ACC) is also identified as a functional downstream region of the MD that mediates the extinction of fear memory. The thalamocortical circuit is comprehensively analyzed and found that the MD‐ACC parvalbumin interneurons circuit is preferentially enhanced in PTSD mice, disrupting the local excitatory and inhibitory balance. It is found that decreased phosphorylation of the Kv3.2 channel contributed to the hyperactivated MD, primarily to the malfunctioning thalamocortical circuit. Using a lipid nanoparticle‐based RNA therapy strategy, channelopathy is corrected via a methoxylated siRNA targeting the protein phosphatase 6 catalytic subunit and restored fear memory extinction in PTSD mice. These findings highlight the function of the thalamocortical circuit in PTSD‐related impaired extinction of fear memory and provide therapeutic insights into Kv3.2‐targeted RNA therapy for PTSD.

Low back pain (LBP) is a leading cause of global disabilities. Numerous molecular, cellular, and anatomical factors are implicated in LBP. Current issues regarding neurologic alterations in LBP have focused on the reorganization of peripheral nerve and spinal cord, but neural mechanisms of exactly what LBP impacts on the brain required further researches. Based on existing clinical studies that chronic pain problems were accompanying alterations in brain structures and functions, researchers proposed logical conjectures that similar alterations occur in LBP patients as well. With recent extensive studies carried out using noninvasive neuroimaging technique, increasing number of abnormalities and alterations has been identified. Here, we reviewed brain alterations including white matters, grey matters, and neural circuits between brain areas, which are involved in chronic LBP. Moreover, brain structural and functional connectivity abnormalities are correlated to the happening and transition of LBP. The negative emotions related to back pain indicate possible alterations in emotional brain regions. Thus, the aim of this review is to summarize current findings on the alterations corresponding to LBP in the brain. It will not only further our understanding of etiology of LBP and understanding of negative emotions accompanying with back pain but also provide ideas and basis for new accesses to the diagnosis, treatment, and rehabilitation afterward based on integral medicine.

Abstract Ischemic stroke is a neurological disorder caused by vascular stenosis or occlusion, accounting for approximately 87% of strokes. Clinically, the most effective therapy for ischemic stroke is vascular recanalization, which aims to rescue neurons undergoing ischemic insults. Although reperfusion therapy is the most effective treatment for ischemic stroke, it still has limited benefits for many patients, and ischemia-reperfusion (I/R) injury is a widely recognized cause of poor prognosis. Here, we aim to investigate the mechanism of protein phosphatase Mg2+/Mn2+ dependent 1 K (PPM1K) mediates metabolic disorder of branched-chain amino acids (BCAA) by promoting fatty acid oxidation led to ferroptosis after cerebral I/R injury. We established the I/R model in mice and used BT2, a highly specific BCAA dehydrogenase (BCKD) kinase inhibitor to promote BCAA metabolism. It was further verified by lentivirus knocking down PPM1K in neurons. We found that BCAA levels were elevated after I/R injury due to dysfunctional oxidative degradation caused by phosphorylated BCKD E1α subunit (BCKDHA). Additionally, the level of phosphorylated BCKDHA was determined by decreased PPM1K in neurons. We next demonstrated that BCAA could induce oxidative stress, lipid peroxidation, and ferroptosis in primary cultured cortical neurons in vitro. Our results further showed that BT2 could reduce neuronal ferroptosis by enhancing BCAA oxidation through inhibition of BCKDHA phosphorylation. We further found that defective BCAA catabolism could induce neuronal ferroptosis by PPM1K knockdown. Furthermore, BT2 was found to alleviate neurological behavior disorders after I/R injury in mice, and the effect was similar to ferroptosis inhibitor ferrostatin-1. Our findings reveal a novel role of BCAA in neuronal ferroptosis after cerebral ischemia and provide a new potential target for the treatment of ischemic stroke.

Abstract Refractory status epilepticus (RSE) is a neurological emergency where sustaining seizure causes severe neuronal death. Currently, there is no available neuroprotectant effective in RSE. Aminoprocalcitonin (NPCT) is a conserved peptide cleaved from procalcitonin, but its distribution and function in the brain remain enigmatic. Survival of neurons relies on sufficient energy supply. Recently, we found that NPCT was extensively distributed in the brain and had potent modulations on neuronal oxidative phosphorylation (OXPHOS), suggesting that NPCT might be involved in neuronal death by regulating energy status. In the present study, combining biochemical and histological methods, high-throughput RNA-sequence, Seahorse XFe analyser, an array of mitochondria function assays, and behavior-electroencephalogram (EEG) monitoring, we investigated the roles and translational values of NPCT in neuronal death after RSE. We found that NPCT was extensively distributed throughout gray matters in rat brain while RSE triggered NPCT overexpression in hippocampal CA3 pyramidal neurons. High-throughput RNA-sequence demonstrated that the influences of NPCT on primary hippocampal neurons were enriched in OXPHOS. Further function assays verified that NPCT facilitated ATP production, enhanced the activities of mitochondrial respiratory chain complexes I, IV, V, and increased neuronal maximal respiration capacity. NPCT exerted multiple neurotrophic effects including facilitating synaptogenesis, neuritogenesis, spinogenesis, and suppression of caspase-3. A polyclonal NPCT immunoneutralization antibody was developed to antagonize NPCT. In the in vitro 0-Mg2+ seizure model, immunoneutralization of NPCT caused more neuronal death, while exogenous NPCT supplementation, though did not reverse death outcomes, preserved mitochondrial membrane potential. In rat RSE model, both peripheral and intracerebroventricular immunoneutralization of NPCT exacerbated hippocampal neuronal death and peripheral immunoneutralization increased mortality. Intracerebroventricular immunoneutralization of NPCT further led to more serious hippocampal ATP depletion, and significant EEG power exhaustion. We conclude that NPCT is a neuropeptide regulating neuronal OXPHOS. During RSE, NPCT was overexpressed to protect hippocampal neuronal survival via facilitating energy supply.

The C-C motif chemokine ligand 2 (CCL2) has been implicated in chronic pain, but its exact mechanism of peripheral sensitization is unknown. In this study, we aimed to clarify the mechanism of CCL2 regulation of ion channels. Our behavioral experiments revealed that ZD7288, a blocker of Ih current, can inhibit CFA and CCL2-mediated mechanical and thermal nociceptive sensitization. Furthermore, patch clamp studies demonstrated that CFA-induced peripheral sensitization primarily affects the excitability of small-diameter DRG neurons. Further studies revealed that inflammatory pain caused by CFA or incubation of DRG with CCL2 mainly affected Ih currents in small-diameter DRG neurons, which were blocked by co-incubation CCR2 antagonist INCB3344 or adenylate cyclase inhibitor SQ22536. Immunohistochemical staining showed that both intraplantar injection of CFA as well as DRG injection of CCL2 resulted in significant upregulation of CCR2+/HCN2+ expression. In conclusion, we suggest in the inflammatory pain state, CCL2 can act on small-diameter DRG neurons, leading to upregulation of HCN2 expression and consequently Ih, which in turn leads to neuronal hyperexcitability.

During development, apoptosis removes redundant cells and ensures proper organ morphogenesis. Necrosis is long known as an adult-bound inflammatory and pathologic cell death. Whether there exists physiological necrosis during early development has been speculated but yet clearly demonstrated. Here, we report evidence of necroptosis, a type of programmed necrosis, specifically in perivascular cells of cerebral cortex and skin at the early stage of development. Phosphorylated Mixed Lineage Kinase Domain-Like protein (MLKL), a key molecule in executing necroptosis, co-expressed with blood endothelial marker CD31 and venous-lymphatic progenitor marker Sox18. Depletion of Mlkl did not affect the formation of blood vessel network but increased the differentiation of venous-lymphatic lineage cells in postnatal cerebral cortex and skin. Consistently, significant enhancement of cerebrospinal fluid diffusion and lymphatic drainage was found in brain and skin of Mlkl-deficient mice. Under hypobaric hypoxia induced cerebral edema and inflammation induced skin edema, Mlkl mutation significantly attenuated brain-blood-barrier damage and edema formation. Our data, for the first time, demonstrated the presence of physiological vascular-associated necroptosis and its potential involvement in the development of venous-lymphatic vessels.

Abstract Reactive astrogliosis usually bears some properties of neural progenitors. How injury triggers astrocyte dedifferentiation remains largely unclear. Here, we report that ischemia induces rapid up-regulation of Wnt2 protein in apoptotic neurons and activation of canonical Wnt signaling in reactive astrocytes in mice, primates and human. Local delivery of Wnt2 shRNA abolished the dedifferentiation of astrocytes while over-expressing Wnt2 promoted progenitor marker expression and neurogenesis. Both the activation of Wnt signaling and dedifferentiation of astrocytes was compromised in ischemic caspase-3−/− cortex. Over-expressing stabilized β-catenin not only facilitated neurogenesis but also promoted functional recovery in ischemic caspase-3−/− mice. Further analysis showed that apoptotic neurons up-regulated Wnt2 protein via internal ribosome entry site (IRES)-mediated translation. Knocking down death associated protein 5 (DAP5), a key protein in IRES-mediated protein translation, significantly diminished Wnt activation and astrocyte dedifferentiation. Our data demonstrated an apoptosis-initiated Wnt-activating mechanism which triggers astrocytic dedifferentiation and facilitates neuronal regeneration.

IntroductionThe pre-Bötzinger complex (pre-BötC), a kernel of inspiratory rhythmogenesis, is a heterogeneous network with excitatory glutamatergic and inhibitory GABAergic and glycinergic neurons. Inspiratory rhythm generation relies on synchronous activation of glutamatergic neuron, whilst inhibitory neurons play a critical role in shaping the breathing pattern, endowing the rhythm with flexibility in adapting to environmental, metabolic, and behavioral needs. Here we report ultrastructural alterations in excitatory, asymmetric synapses (AS) and inhibitory, symmetric synapses (SS), especially perforated synapses with discontinuous postsynaptic densities (PSDs) in the pre-BötC in rats exposed to daily acute intermittent hypoxia (dAIH) or chronic (C) IH.MethodsWe utilized for the first time a combination of somatostatin (SST) and neurokinin 1 receptor (NK1R) double immunocytochemistry with cytochrome oxidase histochemistry, to reveal synaptic characteristics and mitochondrial dynamic in the pre-BötC.ResultsWe found perforated synapses with synaptic vesicles accumulated in distinct pools in apposition to each discrete PSD segments. dAIH induced significant increases in the PSD size of macular AS, and the proportion of perforated synapses. AS were predominant in the dAIH group, whereas SS were in a high proportion in the CIH group. dAIH significantly increased SST and NK1R expressions, whereas CIH led to a decrease. Desmosome-like contacts (DLC) were characterized for the first time in the pre-BötC. They were distributed alongside of synapses, especially SS. Mitochondria appeared in more proximity to DLC than synapses, suggestive of a higher energy demand of the DLC. Findings of single spines with dual AS and SS innervation provide morphological evidence of excitation-inhibition interplay within a single spine in the pre-BötC. In particular, we characterized spine-shaft microdomains of concentrated synapses coupled with mitochondrial positioning that could serve as a structural basis for synchrony of spine-shaft communication. Mitochondria were found within spines and ultrastructural features of mitochondrial fusion and fission were depicted for the first time in the pre-BötC.ConclusionWe provide ultrastructural evidence of excitation-inhibition synapses in shafts and spines, and DLC in association with synapses that coincide with mitochondrial dynamic in their contribution to respiratory plasticity in the pre-BötC.

Abstract Social dysfunction is the core syndrome of autism spectrum disorder (ASD) and lacks effective medicine. Although numerous risk genes and relevant environmental factors have been identified, the convergent molecular mechanism underlying ASD‐associated social dysfunction remains largely elusive. Here, we report aberrant activation of canonical Wnt signaling and increased glycolysis in the anterior cingulate cortex (ACC, a key brain region of social function) of two ASD mouse models (Shank3−/− and valproic acid‐treated mice) and their corresponding human neurons. Overexpressing β‐catenin in the ACC of wild‐type mice induces both glycolysis and social deficits. Suppressing glycolysis in ASD mice partially rescued synaptic and social phenotype. Axin2, a key inhibitory molecule in Wnt signaling, interacts with the glycolytic enzyme enolase 1 (ENO1) in ASD neurons. Surprisingly, an Axin2 stabilizer, XAV939, effectively blocked Axin2/ENO1 interaction, switched glycolysis/oxidative phosphorylation balance, promoted synaptic maturation, and rescued social function. These data revealed excessive neuronal Wnt‐glycolysis signaling as an important underlying mechanism for ASD synaptic deficiency, indicating Axin2 as a potential therapeutic target for social dysfunction.

Pregnancy exposure of valproic acid (VPA) is widely adopted as a model of environmental factor induced autism spectrum disorder (ASD). Increase of excitatory/inhibitory synaptic transmission ratio has been proposed as the mechanism of VPA induced ASD. How this happened, particularly at the level of excitatory neuron differentiation in human neural progenitor cells (NPCs) remains largely unclear. Here, we report that VPA exposure remarkably inhibited human NPC proliferation and induced excitatory neuronal differentiation without affecting inhibitory neurons. Following VPA treatment, mitochondrial dysfunction was observed before neuronal differentiation, as showed by ultrastructural changes, respiratory complex activity, mitochondrial membrane potential and oxidation levels. Meanwhile, extracellular acidification assay revealed an elevation of glycolysis by VPA stimulation. Interestingly, inhibiting glycolysis by 2-deoxy-d-glucose-6-phosphate (2-DG) efficiently blocked the excitatory neuronal differentiation of human NPCs induced by VPA. Furthermore, 2-DG treatment significantly compromised the VPA-induced expression of H3ac and H3K9ac, and the VPA-induced binding of H3K9ac on the promoter of Ngn2 and Mash1, two key transcription factors of excitatory neuron fate determination. These data, for the first time, demonstrated that VPA biased excitatory neuron differentiation by glycolysis-mediated histone acetylation of neuron specific transcription factors.

IntroductionSchizophrenia is a severe psychiatric disorder with a high prevalence worldwide, however, its pathogenesis remains poorly understood.Methods and resultsIn this study, we used the non-competitive NMDA receptor antagonist MK-801 to induce schizophrenia-like behaviors and confirmed that mice exhibited stereotypic rotational behavior and hyperlocomotion, social interaction defects and cognitive dysfunction, similar to the clinical symptoms in patients. Here, the anterior cingulate cortex (ACC) and basolateral amygdala (BLA) were involved in the schizophrenia-like behaviors induced by MK-801. Furthermore, we confirmed BLA sent glutamatergic projection to the ACC. Chemogenetic and optogenetic regulation of BLA-ACC projecting neurons affected social and cognitive deficits but not stereotypic rotational behavior in MK-801-treated mice.DiscussionOverall, our study revealed that the BLA-ACC circuit plays a major role and may be a potential target for treating schizophrenia-related symptoms.

Myelin formation is regulated by epigenetic mechanisms and ensures proper neuronal function during development and after demyelination. Here, the authors show that TET1, a DNA hydroxymethylase, regulates myelination during development and remyelination in mice.

Schizophrenia is a severe mental disorder characterized by positive, negative, and cognitive symptoms. Cognitive symptoms are a kind of symptoms with high incidence and great impact on patients. There is no effective treatment in clinical practice. N-methyl-d-aspartic acid (NMDA) receptor hypofunction may be an important cause of cognitive symptoms. MK-801 (also named Dizocilpine), a noncompetitive antagonist of NMDA receptor, is often used to construct a model of NMDA receptor dysfunction. In terms of treatment, environmental enrichment (EE) as an environmental intervention can effectively improve the symptoms of cognitive impairment in rodents. In this paper, we first briefly introduce the background of cognitive symptoms and EE in schizophrenia, and then investigate the manifestations of MK-801 induced cognitive impairment, the improvement of EE on these cognitive impairments based on the MK-801 induced schizophrenia rodent models, and the possible mechanism of EE in improving cognitive symptoms. This article reviews the literature in recent years, which provides an important reference for MK-801 to construct a cognitive symptom model of schizophrenia and the mechanism of EE in improving cognitive symptoms of schizophrenia.

Chronic sleep deprivation (SD) is a common problem for humans and can lead to many deleterious effects, including depression, anxiety, stroke, permanent cognitive deficits, stress, and other physiological diseases. It is vital to acquire information about the relevant neural activities at the whole-brain level to systematically explore the mechanisms of brain dysfunction related to SD. Expression of the immediate-early gene (IEG) Fos in the mouse brain has been widely used as a functional marker of brain activity in the field of neuroscience. However, most previous studies only analyzed the change of c-Fos in several specific brain regions using traditional research methods or in short-term SD model. Here, we applied c-Fos mapping through the fluorescence micro-optical sectioning tomography (fMOST) technique and AAV-PHP.eB to comprehensive analysis the state of cumulative activation across the whole brain in a mouse model of chronic SD. The chronic rapid eyes movement (REM) SD model was induced by moving mice to a separate holding area filled with water. The experimental period lasted for 6 h per day. The results showed that after 14 days of SD, the mice displayed anxiety-like behaviors in open field test and elevated plus maze test, and displayed depression-like behaviors in tail suspension test and the sucrose preference test. The c-Fos + cells were detected in a maximum of 230 brain regions. SD-induced stress model evoked c-Fos expression in several brain regions compared to the control group. In particular, the isocortex-cerebral cortex plate area, including the retrosplenial, anterior cingulate, agranular insular, gustatory, and parasubiculum, appear to be the most sensitive regions after chronic REM SD.

Toxoplasma gondii (T. gondii) is a zoonotic parasite that is widely distributed and seriously endangers public health and human health. Therefore, accurate and effective detection of T. gondii is crucial. This study proposes a microfluidic biosensor using a thin-core microfiber (TCMF) coated with molybdenum disulfide (MoS2) for immune detection of T. gondii. The single-mode fiber was fused with the thin-core fiber, and the TCMF was obtained by arc discharging and flame heating. In order to avoid interference and protect the sensing structure, the TCMF was encapsulated in the microfluidic chip. MoS2 and T. gondii antigen were modified on the surface of TCMF for the immune detection of T. gondii. Experimental results showed that the detection range of the proposed biosensor for T. gondii monoclonal antibody solutions was 1 pg/mL to 10 ng/mL with sensitivity of 3.358 nm/log(mg/mL); the detection of limit was calculated to be 87 fg/mL through the Langmuir model; the dissociation constant and the affinity constant were calculated to be about 5.79 × 10−13 M and 1.727 × 1014 M−1, respectively. The specificity and clinical characteristics of the biosensor was explored. The rabies virus, pseudorabies virus, and T. gondii serum were used to confirm the excellent specificity and clinical characteristics of the biosensor, indicating that the proposed biosensor has great application potential in the biomedical field.

Paclitaxel is widely used as a first-line chemotherapy agent to treat malignant tumors. However, paclitaxel causes peripheral nerve fiber damage and neuropathic pain in some patients. In addition, patients received paclitaxel chemotherapy are often accompanied by negative emotions such as anxiety. The amygdala is critically involved in regulating pain signals, as well as anxiety. The purpose of this study is to clarify the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII)-positive glutamatergic neurons in the amygdala in paclitaxel-induced pain and negative affective symptoms. Intraperitoneal injection of paclitaxel into mice caused mechanical and thermal allodynia, as measured by Von Frey test and Hargreaves test, and anxiety, as measured by open field test and elevated plus maze test. Immunofluorescence staining revealed that c-fos-positive neurons were significantly more in the basolateral amygdala (BLA) and central amygdala (CeA) in paclitaxel-treated mice than untreated mice. Furthermore, part of c-fos-positive neurons in the BLA were immunoreactive of CaMKII. Engineered Designer receptors exclusively activated by designer drugs (DREADD) receptor hM4Di or hM3Dq was selectively expressed on CaMKII neurons by injection of adeno-associated virus (AAV) vectors containing CaMKII and hM4Di or hM3Dq. Administration of DREADD agonist CNO to selectively inhibit the CaMKII neurons in the BLA significantly increased the paw withdrawal thresholds and paw withdrawal latencies. In addition, selectively inhibition of CaMKII neurons in the BLA alleviated anxiety behavior without affecting the motor activity. In summary, our findings suggest that CaMKII neurons in the amygdala are critical for neuropathic pain and anxiety behaviors induced by paclitaxel chemotherapy.

Environmental factors, such as medication during pregnancy, are one of the major causes of autism spectrum disorder (ASD). Valproic acid (VPA) intake during pregnancy has been reported to dramatically elevate autism risk in offspring. Recently, researchers have proposed that VPA exposure could induce excitatory or inhibitory synaptic dysfunction. However, it remains to be determined whether and how alterations in the excitatory/inhibitory (E/I) balance contribute to VPA-induced ASD in a mouse model. In the present study, we explored changes in the E/I balance during different developmental periods in a VPA mouse model. We found that typical markers of pre- and postsynaptic excitatory and inhibitory function involved in E/I balance markedly decreased during development, reflecting difficulties in the development of synaptic plasticity in VPA-exposed mice. The expression of brain-derived neurotrophic factor (BDNF), a neurotrophin that promotes the formation and maturation of glutamatergic and GABAergic synapses during postnatal development, was severely reduced in the VPA-exposed group. Treatment with exogenous BDNF during the critical E/I imbalance period rescued synaptic functions and autism-like behaviors, such as social defects. With these results, we experimentally showed that social dysfunction in the VPA mouse model of autism might be caused by E/I imbalance stemming from BDNF deficits during the developmental stage.

Pain and itch are intricately entangled at both circuitry and behavioral levels. Emerging evidence indicates that parvalbumin (PV)-expressing neurons in zona incerta (ZI) are critical for promoting nocifensive behaviors. However, the role of these neurons in itch modulation remains elusive. Herein, by combining FOS immunostaining, fiber photometry, and chemogenetic manipulation, we reveal that ZI PV neurons act as an endogenous negative diencephalic modulator for itch processing. Morphological data showed that both histamine and chloroquine stimuli induced FOS expression in ZI PV neurons. The activation of these neurons was further supported by the increased calcium signal upon scratching behavior evoked by acute itch. Behavioral data further indicated that chemogenetic activation of these neurons reduced scratching behaviors related to histaminergic and non-histaminergic acute itch. Similar neural activity and modulatory role of ZI PV neurons were seen in mice with chronic itch induced by atopic dermatitis. Together, our study provides direct evidence for the role of ZI PV neurons in regulating itch, and identifies a potential target for the remedy of chronic itch.

Astrocytic glycogen plays an important role in brain energy metabolism. However, the contribution of glycogen metabolism to stress-induced depression remains unclear. Chronic social defeat stress was used to induce depression-like behaviors in mice, assessed with behavioral tests. Glycogen concentration in the medial prefrontal cortex (mPFC) and the expression of key enzymes of the glycogen metabolism were investigated using Western blots, immunofluorescent staining, electron microscopy, and biochemical assays. Stereotaxic surgery and viral-mediated gene transfer were applied to knockdown or overexpress brain-type glycogen phosphorylase (PYGB) in the mPFC. The glycogen content increased in the mPFC after stress. Glycogenolytic dysfunction due to inactivation of PYGB was responsible for glycogen accumulation. Behavioral tests on astrocyte-specific PYGB overexpression mice showed that augmenting astrocytic PYGB reduces susceptibility to depression when compared with stress-susceptible mice. Conversely, PYGB genetic down-regulation in the mPFC was sufficient to induce glycogen accumulation and depression-like behaviors. Furthermore, PYGB overexpression in the mPFC decreases susceptibility to depression, at least partially by rescuing glycogen phosphorylase activity to maintain glycogen metabolism homeostasis during stress. These findings indicate that (1) glycogen accumulation occurs in mice following stress and (2) glycogenolysis reprogramming leads to glycogen accumulation in astrocytes and PYGB contributes to stress-induced depression-like behaviors. Pharmacological tools acting on glycogenolysis might constitute a promising therapy for depression.

Abstract Ten-eleven translocation (TET) proteins, encoding dioxygenase for DNA hydroxymethylation, are important players in nervous system development and disease. In addition to their proverbial enzymatic role, TET proteins also possess non-enzymatic activity and function in multiple protein–protein interaction networks, which remains largely unknown during oligodendrocyte differentiation. To identify partners of TET1 in the myelinating cells, we performed proteome-wide analysis using co-immunoprecipitation coupled to mass spectrometry (IP-MS) in purified oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (mOLs), respectively. Following a stringent selection of MS data based on identification reliability and protein enrichment, we identified a core set of 1211 partners that specifically interact with TET1 within OPCs and OLs. Analysis of the biological process and pathways associated with TET1-interacting proteins indicates a significant enrichment of proteins involved in regulation of cellular protein localization, cofactor metabolic process and regulation of catabolic process, et al. We further validated TET1 interactions with selected partners. Overall, this comprehensive analysis of the endogenous TET1 interactome during oligodendrocyte differentiation suggest its novel mechanism in regulating oligodendrocyte homeostasis and provide comprehensive insight into the molecular pathways associated with TET1.

Abstract The anterior cingulate cortex (ACC) serves as a critical hub for the anxiety and pain perception. The large-conductance Ca2+-activated potassium channels, or BKCa channels, are ubiquitously expressed throughout the central nervous system including the cingulate cortex. However, what changes of cortical BKCa channels undergo in the ACC remains unknown in pain-related anxiety. In the present study, a significant upregulation of synaptic and non-synaptic BKCa channel accessory β4 subunits in the ACC was accompanied with pain-associated anxiety-like behaviors in the chronic compression of multiple dorsal root ganglia (mCCD) of the rat. NS1619, an opener of BKCa channels, significantly rescued the alteration of fAHP and AP duration of ACC pyramidal neurons in mCCD rats. The mRNA expression of BKCa β4 subunits was extremely upregulated in the ACC after mCCD with the increased amount of both synaptic and non-synaptic BKCa β4 subunit protein. Meanwhile, NS1619 reversed the enhanced AMPA receptor-mediated spontaneous excitatory postsynaptic current (sEPSC) frequency and the attenuated PPR of ACC neurons in mCCD rats. Local activation of BKCa channels in the ACC reversed mechanical allodynia and anxiety-like behaviors. These results suggest that the upregulation of postsynaptic and presynaptic BKCa β4 subunit may contribute to neuronal hyperexcitability and the enhanced synaptic transmission in the ACC in neuropathic pain state, and then may result in anxiety-like behavior induced by neuropathic pain.

Abstract Purpose The aim of this work was to evaluate the prognostic value of tumor length and diameter for patients with esophageal squamous cell cancer (ESCC) treated with definitive (chemo)radiotherapy to identify potential indicators for separate nonsurgical T staging, which are needed in clinical practice. Materials and Methods A total of 682 patients with ESCC who underwent definitive (chemo)radiotherapy between 2009 and 2015 were reviewed. Esophageal tumor length and diameter were determined by barium esophagography and computed tomography before treatment. Univariate and multivariate analyses were used to assess the impact of tumor length and diameter on long‐term overall survival (OS) and progression‐free survival (PFS). Propensity score matching (PSM) analysis was also used to control intergroup heterogeneity. Results The median OS and PFS were 22.2 months and 15.4 months, respectively, in the tumor length ≤ 6 cm group, which were significantly longer than those in the tumor length > 6 cm group (13.4 and 8.5 months, respectively). The median OS and PFS were 23.3 months and 15.9 months, respectively, in the tumor diameter ≤ 3.5 cm group, which were also significantly longer than those in the tumor diameter > 3.5 cm group (13.3 and 8.8 months, respectively). Similar results were found after PSM. Univariate and multivariate analyses showed that tumor length and diameter were both independent predictors of long‐term survival. Conclusion Tumor length and diameter are both independent prognostic factors for ESCC patients treated with definitive (chemo)radiotherapy. These two imaging parameters have the potential for development and use in nonsurgical T staging.

Summary: Terahertz (THz) frequency occupies a large portion of the electromagnetic spectrum that is between the infrared and microwave regions. Recent advances in THz application have stimulated interests regarding the biological effects within this frequency range. In the current study, we report that irradiation with a single-frequency THz laser on mice cortical neuron cultures increases excitatory synaptic transmission and neuronal firing activities. Microarray assay reveals gene expression dynamics after THz exposure, which is consistent with morphology and electrophysiology results. Besides, certain schedule of THz irradiation inhibits the proliferation of oligodendrocyte precursor cells (OPCs) and promotes OPC differentiation. Of note, the myelination process is enhanced after THz exposure. In summary, our observations suggest that THz irradiation can modulate the functions of different neuronal cells, with different sensitivity to THz. These results provide important understanding of the mechanisms that govern THz interactions with nervous systems and suggest THz wave as a new strategy for neuromodulation.

Post-traumatic stress disorder (PTSD) is a severe, long-term psychological disorder triggered by distressing events. The neural basis and underlying mechanisms of PTSD are not completely understood. Therefore, it is important to determine the pathology of PTSD using reliable animal models that mimic the symptoms of patients. However, the lack of evidence on the clinical relevance of PTSD animal models makes it difficult to interpret preclinical studies from a translational perspective. In this study, we performed a comprehensive screening of the behavioral, neuronal, glial, and electroencephalographic (EEG) profiles in the single prolonged stress and electric foot shock (SPS&S) mouse model. Based on the clinical features of PTSD, we observed fearful and excessive responses to trauma-related environments in the SPS&S mouse model that lasted longer than 14 days. The mice exhibited a defective and strong resistance to the extinction of fear memories caused by auditory cues and also showed enhanced innate fear induced by visual stimuli with concomitant phobias and anxiety. Furthermore, neurons, astrocytes, and microglia in PTSD-related brain regions were activated, supporting abnormal brain activation and neuroimmune changes. EEG assessment also revealed decreased power and impaired coupling strength between cortical regions. These results demonstrated that the SPS&S mouse model recapitulates the behavioral symptoms as well as neural and EEG profiles of PTSD patients, justifying the preclinical use of this mouse model.

Self-grooming is an innate, cephalo-caudal progression of body cleaning behaviors that are found in normal rodents but exhibit repetitive and stereotyped patterns in several mouse models, such as autism spectrum disorders (ASDs). It is also recognized as a marker of stress and anxiety. Mice with Shank3B gene knockout (KO) exhibit typical ASD-like behavioral abnormalities, including stereotyped self-grooming and increased levels of anxiety. However, the exact relationship between anxiety and stereotyped self-grooming in certain types of animal models is not clear. We selected three animal models with high anxiety to compare their self-grooming parameters. First, we confirmed that Shank3B KO mice (ASD model), acute restraint stress mouse model (stress model), and chronic inflammatory pain mouse model (pain model) all showed increased anxiety levels in the open field test (OFT) and elevated plus maze (EPM). We found that only the ASD model and the stress model produced increased total grooming duration. The pain model only exhibited an increasing trend of mean self-grooming duration. We used the grooming analysis algorithm to examine the self-grooming microstructure and assess the cephalo-caudal progression of grooming behavior. The results showed distinct self-grooming microstructures in these three models. The anxiolytic drug diazepam relieved the anxiety level and the total time of grooming in the ASD and stress models. The grooming microstructure was not restored in Shank3B KO mice but was partially relieved in the stress model, which suggested that anxiety aggravated stereotyped self-grooming duration but not the grooming microstructure in the ASD mouse model. Our results indicated that stereotyped behavior and anxiety may be shared by separate, but interacting, neural circuits in distinct disease models, which may be useful to understand the mechanisms and develop potential treatments for stereotyped behaviors and anxiety.

Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric oxide synthase (NOS) pathway. We firstly explored the effects of NO/NOS signaling on oxidative stress and apoptosis in in vivo and in vitro models of cerebral ischemia. Further, we evaluated the protective effects of Leo against oxygen and glucose deprivation (OGD)-induced oxidative stress and apoptosis in PC12 cells. We found that the rats showed anxiety-like behavior, and the morphology and number of neurons were changed in a model of photochemically induced cerebral ischemia. Both in vivo and in vitro results show that the activity of superoxide dismutase (SOD) and glutathione (GSH) contents were decreased after ischemia, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were increased, indicating that cerebral ischemia induced oxidative stress and neuronal damage. Moreover, the contents of NO, total NOS, constitutive NOS (cNOS) and inducible NOS (iNOS) were increased after ischemia in rat and PC12 cells. Treatment with L-nitroarginine methyl ester (L-NAME), a nonselective NOS inhibitor, could reverse the change in NO/NOS expression and abolish these detrimental effects of ischemia. Leo treatment decreased ROS and MDA levels and increased the activity of SOD and GSH contents in PC12 cells exposed to OGD. Furthermore, Leo reduced NO/NOS production and cell apoptosis, decreased Bax expression and increased Bcl-2 levels in OGD-treated PC12 cells. All the data suggest that Leo protected against oxidative stress and neuronal apoptosis in cerebral ischemia by inhibiting the NO/NOS system. Our findings indicate that Leo could be a potential agent for the intervention of ischemic stroke and highlighted the NO/NOS-mediated oxidative stress signaling.

Psychiatric disorders are complex and heterogeneous disorders arising from the interaction of multiple factors based on neurobiology, genetics, culture, and life experience. Increasing evidence indicates that sustained abnormalities are maintained by epigenetic modifications in specific brain regions. Over the past decade, the critical, non-redundant roles of the ten-eleven translocation (TET) family of dioxygenase enzymes have been identified in the brain during developmental and postnatal stages. Specifically, TET-mediated active demethylation, involving the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and subsequent oxidative derivatives, is dynamically regulated in response to environmental stimuli such as neuronal activity, learning and memory processes, and stressor exposure. Here, we review the progress of studies designed to provide a better understanding of how profiles of TET proteins and 5hmC are powerful mechanisms by which to explain neuronal plasticity and long-term behaviors, and impact transcriptional programs operative in the brain that contribute to psychiatric disorders.

PurposeComorbid anxiety and depression in people with epilepsy (PWE) are highly prevalent and contribute to low quality of life (QOL) and may even lead to poor outcomes of epilepsy. Among the various factors that affect these negative emotional comorbidities, possible gender differences remain poorly understood and are often neglected. This research aimed to determine whether there are discrepancies in the incidence and influence factors of anxiety and depression between men and women with epilepsy in a hospital in northwest China.MethodsA total of 158 adult PWE (female: N = 65; 41.1%) completed self-report questionnaires, including the Self-rating Anxiety Scale (SAS), the Self-rating Depression Scale (SDS), the Chinese version of the Quality of Life in Epilepsy-31 (QOLIE-31) inventory and the Pittsburgh Sleep Quality Inventory (PSQI). The comparison between male and female PWE was made by regression analysis.ResultsFor the prevalence of anxiety and depression in PWE, no gender difference was found in this study. However, the moderating factors of psychiatric comorbidities were significantly different between men and women: male PWE with comorbid anxiety were more likely to be affected by sleep quality, while anxiety symptoms in female PWE were closely associated with the frequency of seizures. Education years and QOL social function were significant indicators of depression in male PWE but not in female PWE. The important and common predictor for anxiety and depressive symptoms in PWE was QOL energy/fatigue, with male patients being more affected.ConclusionFor the PWE included in this study, the incidence of comorbid anxiety and depression in PWE was similar for men and women, but the moderating factors affecting comorbid anxiety and depressive disorders differed between genders: male PWE were more likely to be affected by psychosocial factors, while female PWE were more influenced by epilepsy itself. This exploration suggests that gender-specific health care should be considered in epilepsy therapy to improve the psychiatric condition and QOL of PWE, and different treatments should be conducted for male and female PWE to prevent negative emotional comorbidities.

Neuropathic pain (NP) is a complex, chronic pain condition caused by injury or dysfunction affecting the somatosensory nervous system. This study aimed to identify crucial mRNAs and microRNAs (miRNAs) in the prelimbic cortex (PL) of NP rats. mRNA and miRNA microarrays were applied in the present study. The miRNA-mRNA regulatory network was constructed by using ingenuity pathway analysis (IPA). A total of 35 differentially expressed (DE) RNAs (24 miRNAs and 10 mRNAs) were identified in the spared nerve injury (SNI) group compared with the control group. The DE miRNA-mRNA network showed that IL-6 and tumor necrosis factor (TNF) were core components. Mir-30c-5p and mir-16-5p were the most connected miRNAs in the network. Interestingly, four mRNAs (Rnase 4, Egr2, Rexo4, and Klf2) with significantly increased expression were abundantly expressed in microglia, which was verified by the real-time quantitative polymerase chain reaction (qPCR). Furthermore, the expression of Rnase4 and Egr2 decreased in M1-polarized macrophages and increased in M2-polarized macrophages. In conclusion, we screened dozens of DE mRNAs and miRNAs in the PL of SNI rats. The core of the DE mRNA and miRNA network pointed to molecules associated with inflammation. Four mRNAs (Rnase4, Egr2, Rexo4, and Klf2) might be the potential markers of M2 polarization.

IntroductionAccumulating evidence indicates that the glutamatergic system plays an important role in the development of depression. Notably, the antidepressant effect of metabotropic glutamate receptor 5 (mGluR5) modulation is inconsistent across studies. Here, we attempted to identify the involvement of the gut microbiota and inflammation in mGluR5−/− mice.MethodsmGluR5−/− mice and their wild-type littermates were used in our study. We used the open field (OF) and elevated plus maze (EPM) tests to assess anxiety-like behaviors, and we used the two-day forced swim test (FST) and tail suspension test (TST) to test despair-like behaviors. 16S rDNA was used to analyze the gut microbiota. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of inflammatory factors. Western blotting was used to detect the levels of various proteins.ResultsmGluR5−/− mice had no significant increase or decrease of despair-like behavior in the absence of stress exposure. However, mGluR5−/− mice exhibited despair-like behaviors following stress exposure. No significant changes in other glutamate receptors or representative synaptic proteins were detected in the prefrontal cortex (PFC) or hippocampus of mGluR5−/− mice. Very similar bacterial groups were observed in mGluR5−/− mice and wild-type controls. In addition, there was no significant difference in the α-diversity of the microbiota between mGluR5−/− mice and wild-type controls. The levels of all measured cytokines (IL-1β, IL-2, IL-4, IL-6, IL-10, and TNF-α) did not change significantly in the PFCs or colons of mGluR5−/− mice.ConclusionIn conclusion, we deduced that mGluR5−/− mice are susceptible to despair-like behavior. The systemic knockout of mGluR5 did not affect the gut microbiota or inflammatory factors in mice.

Social deficiency is one of the core syndromes of autism spectrum disorders (ASD), for which the underlying developmental mechanism still remains elusive. Anterior cingulate cortex (ACC) plays a key role in integrating social information and regulating social behavior. Recent studies have indicated that synaptic dysfunction in ACC is essential for ASD social defects. In the present study, we investigated the development of synapses and the roles of glycogen synthase kinase 3β (GSK-3β), which mediates multiple synaptic signaling pathways in ACC by using Shank3b−/− mice (a widely used ASD mouse model). Our data revealed that Shank3b mutation abolished the social induced c-Fos expression in ACC. From 4 weeks post-birth, neurons in Shank3b−/− ACC exhibited an obvious decrease in spine density and stubby spines. The length and thickness of post-synaptic density (PSD), the expression of vesicular glutamate transporter 2 (vGlut2) and glutamate receptor 2 (GluR2), and the frequency of miniature excitatory post-synaptic currents (mEPSCs) were significantly reduced in Shank3b−/−ACC. Interestingly, the levels of phosphorylated GSK-3β (Ser9), which inhibits the activity of GSK-3β, decreased along the same time course as the levels of GluR2 increased in ACC during development. Shank3b mutation leads to a dramatic increase of pGSK-3β (Ser9), and decrease of pPSD95 (a substrate of GSK-3β) and GluR2. Local delivery of AAV expressing constitutively active GSK-3β restored the expression of GluR2, increased the spine density and the number of mature spines. More importantly, active GSK-3β significantly promoted the social activity of Shank3b−/− mice. These data, in together, indicate that decrease of GSK-3β activity in ACC may contribute to the synaptic and social defects of Shank3b−/− mice. Enhancing GSK-3β activity may be utilized to treat ASD in the future.

Summary: BDNF/TrkB neurotrophic signaling regulates neuronal development, differentiation, and survival, and deficient BDNF/TrkB activity underlies neurodegeneration in Alzheimer’s disease (AD). However, exactly how BDNF/TrkB participates in AD pathology remains unclear. Here, we show that deprivation of BDNF/TrkB increases inflammatory cytokines and activates the JAK2/STAT3 pathway, resulting in the upregulation of transcription factor C/EBPβ. This, in turn, results in increased expression of δ-secretase, leading to both APP and Tau fragmentation by δ-secretase and neuronal loss, which can be blocked by expression of STAT3 Y705F, knockdown of C/EBPβ, or the δ-secretase enzymatic-dead C189S mutant. Inhibition of this pathological cascade can also rescue impaired synaptic plasticity and cognitive dysfunctions. Importantly, reduction in BDNF/TrkB neurotrophic signaling is inversely coupled with an increase in JAK2/STAT3, C/EBPβ, and δ-secretase escalation in human AD brains. Therefore, our findings provide a mechanistic link between BDNF/TrkB reduction, C/EBPβ upregulation, δ-secretase activity, and Aβ and Tau alterations in murine brains. : Deficient BDNF/TrkB activity underlies AD pathogenesis. Wang et al. report that deprivation of BDNF/TrkB increases inflammatory cytokines and activates the JAK2/STAT3 pathway, resulting in the upregulation of C/EBPβ/AEP signaling. Reduction of BDNF is inversely coupled with the aforementioned pathway in AD brains. Inhibition of JAK2/STAT3/C/EBPβ/AEP prevents BDNF-depletion-mediated pathology. Keywords: BDNF, JAK2/STAT3, neuroinflammation, C/EBPβ, δ-secretase, Alzheimer’s diesase

Although several studies have found that metabotropic glutamate 5 receptor (mGluR5) may play an important role in autism spectrum disorders (ASD), the mechanisms remain unclear. Here, we used a Shank3 gene complete knockout mouse model (Shank3B−/−) to explore the change in mGluR5 in the brain. To assess whether deletion of Shank3 in mice results in ASD-like behavior, we conducted a battery of behavioral experiments to characterize Shank3B−/− mice, including repetitive grooming behavior tests, three-chamber tests and resident-intruder tests. Wild-type C57/BL6 and Shank3B−/− mice underwent PET scans with [18F]FPEB, which was highly specific to mGluR5. Mouse brains were extracted post-scan, and mGluR5 protein levels were assessed by immunoblotting. The binding potential (BPnd) of mGluR5 was rich in the hippocampus, thalamus, striatum, and amygdala. More importantly, Shank3B−/− mice showed significantly increased BPnd compared to the control mice in these brain regions. Immunoblotting revealed elevated mGluR5 levels in the hippocampus, thalamus, and amygdala but not in the striatum compared with control mice. These findings indicated that [18F]FPEB could visualize mGluR5 in the mouse brain. The deficiency of Shank3 can impair mGluR5 expression in multiple brain regions. Future work is also needed to understand the reasons for different results between in vivo PET and ex vivo immunoblotting.

Chronic pain usually results in persistent anxiety, which worsens the life quality of patients and complicates the treatment of pain. Hippocampus is one of the few brain regions in many mammalians species which harbors adult neural stem cells (NSCs), and plays a key role in the development and maintenance of chronic anxiety. Recent studies have suggested a potential involvement of hippocampal neurogenesis in modulating chronic pain. Whether and how hippocampal NSCs are involved in the pain-associated anxiety remains unclear. Here, we report that mice suffering persistent neuropathic pain showed a quick reduction of active NSCs in the ventral dentate gyrus (vDG), which was followed by the decrease of neurogenesis and appearance of anxiety. Wnt/β-catenin signaling, a key pathway in sustaining the active status of NSCs was suppressed in the vDG of mice suffering chronic pain. Depleting β-catenin by inducible Nestin-Cre significantly reduced the number of active NSCs and facilitated anxiety development, while expressing stabilized β-catenin amplified active NSCs and alleviated anxiety, indicating that Wnt activated NSCs is required for anxiety development under chronic pain. Treatment with Fluoxetine, the most widely used anxiolytic in clinic, significantly increased the proliferation of active NSCs and enhanced Wnt signaling. Interestingly, both β-catenin manipulation and Fluoxetine treatment had no significant effects on the pain thresholds. Therefore, our data demonstrated an anxiety-specific response and contribution of activated NSCs to chronic pain through Wnt/β-catenin signaling, which may be targeted for treating chronic pain- or other diseases-associated anxiety.

As the main input nucleus of the basal ganglion, the striatum executes different functions, including motivation, reward and attention. The functions of the striatum highly rely on its subregions that receive projections from various cortical areas and the distribution of striatonigral neurons that express D1 dopamine (DA) receptors (or D1 medium-sized spiny neurons, D1 MSNs) and striatopallidal neurons that express D2 DA receptors (or D2 MSNs). Using bacterial artificial chromosome (BAC) transgenic mice, several studies have recently been performed on the spatial distribution of D1 and D2 MSNs. However, these studies mainly focused on enumeration of either D1-enhanced fluorescent protein (eGFP) or D2-eGFP in mice. In the present work, we used Drd1a-tdTamato and Drd2-eGFP double BAC transgenic mice to evaluate the spatial pattern of D1 MSNs (red fluorescence) and D2 MSNs (green fluorescence) along the rostro-caudal axis of the dorsal striatum. The dorsal striatum was divided into three subregions: rostral caudoputamen (CPr), intermediate CP (CPi), and caudal CP (CPc) across the rostral–caudal extent of the striatum. The results demonstrate that D1 and D2 MSNs were intermingled with each other in most of these regions. The cell density of D1 MSNs was slightly higher than D2 MSNs through CPr, CPi, and CPc, though it did not reach significance. However, in CPi, the ratio of D1/D2 in the ventromedial CPi group was significantly higher than those in dorsolateral, dorsomedial, and ventrolateral CPi. There was similar proportion of cells that co-expressed D1 and D2 receptors. Moreover, we demonstrated a pathway-specific activation pattern of D1 MSNs and D2 MSNs in a manic like mouse model induced by D-Amphetamine by utilizing this double transgenic mice and c-fos immunoreactivity. Our results may provide a morphological basis for the function or pathophysiology of striatonigral and striatopallidal neurons with diverse cortical inputs to the dorsal striatum.

The inhibitory receptor programmed death-1 (PD-1) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (FH) has yet to be described. Here, we investigated the functional mechanisms of PD-1 as related to FH pathogenesis induced by the murine hepatitis virus strain-3 (MHV-3). High levels of PD-1-positive CD4(+), CD8(+) T cells, NK cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following MHV-3 infection. PD-1-deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein 2 (FGL2), than did their wild-type (WT) littermates. As a result, more severe tissue damage was produced and mortality rates were higher. Fluorescence double-staining revealed that FGL2 and PD-1 were not co-expressed on the same cells, while quantitative RT-PCR demonstrated that higher levels of IFN-γ and TNF-α mRNA transcription occurred in PD-1-deficient mice in response to MHV-3 infection. Conversely, in vivo blockade of IFN-γ and TNF-α led to efficient inhibition of FGL2 expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. Thus, the up-regulation of FGL2 in PD-1-deficient mice was determined to be mediated by IFN-γ and TNF-α. Taken together, our results suggest that PD-1 signaling plays an essential role in decreasing the immunopathological damage induced by MHV-3 and that manipulation of this signal might be a useful strategy for FH immunotherapy.

Emerging research implicates the participation of spinal dorsal horn (SDH) neurons and astrocytes in nerve injury-induced neuropathic pain. However, the crosstalk between spinal astrocytes and neurons in neuropathic pain is not clear. Using a lumbar 5 (L5) spinal nerve ligation (SNL) pain model, we testified our hypothesis that SDH neurons and astrocytes reciprocally regulate each other to maintain the persistent neuropathic pain states. Glial fibrillary acidic protein (GFAP) was used as the astrocytic specific marker and Fos, protein of the protooncogene c-fos, was used as a marker for activated neurons. SNL induced a significant mechanical allodynia as well as activated SDH neurons indicated by the Fos expression at the early phase and activated astrocytes with the increased expression of GFAP during the late phase of pain, respectively. Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or astroglial toxin L-alpha-aminoadipate (L-AA) reversed the mechanical allodynia, respectively. Immunofluorescent histochemistry revealed that intrathecal administration of c-fos ASO significantly suppressed activation of not only neurons but also astrocytes induced by SNL. Meanwhile, L-AA shortened the duration of neuronal activation by SNL. Our data offers evidence that neuronal and astrocytic activations are closely related with the maintenance of neuropathic pain through a reciprocal "crosstalk". The current study suggests that neuronal and non-neuronal elements should be taken integrally into consideration for nociceptive transmission, and that the intervention of such interaction may offer some novel pain therapeutic strategies.