Ventral zona incerta parvalbumin neurons modulate sensory-induced and stress-induced self-grooming via input-dependent mechanisms in mice.

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.

Imbalance between hippocampal projection cell and parvalbumin interneuron architecture increases epileptic susceptibility in mouse model of methyl CpG binding protein 2 duplication syndrome.

OBJECTIVE: Methyl CpG-binding protein 2 (MECP2) duplication syndrome is a rare X-linked genomic disorder affecting predominantly males, which is usually manifested as epilepsy and autism spectrum disorder (ASD) comorbidity. The transgenic line MeCP2Tg1 was used for mimicking MECP2 duplication syndrome and showed autism-epilepsy co-occurrence. Previous works suggested that the excitatory/inhibitory (E/I) imbalance is a potential common mechanism for both epilepsy and ASD. The projection neurons and parvalbumin (PV) interneurons account for the majority of E/I balance in the hippocampus. Therefore, we explored how structural changes of projection and PV+ neurons occur in the hippocampus of MeCP2Tg1 mice and whether these morphological changes contribute to epilepsy susceptibility. METHODS: We used the interneuron Designer receptors exclusively activated by designer drugs mouse model to inhibit inhibitory neurons in the hippocampus to verify the epilepsy susceptibility of MeCP2Tg1 (FVB, an inbred strain named as sensitivity to Friend leukemia virus) mice. Electroencephalograms were recorded for the definition of seizure. We performed retro-orbital injection of virus in MeCP2Tg1 (FVB):CaMKIIα-Cre (C57BL/6) mice or MeCP2Tg1:PV-Cre (C57BL/6) mice and their littermate controls to specifically label projection and PV+ neurons for structural analysis. RESULTS: Epilepsy susceptibility was increased in MeCP2Tg1 mice. There was a reduced number of PV neurons and reduced dendritic complexity in the hippocampus of MeCP2Tg1 mice. The dendritic complexity in MeCP2Tg1 mice was increased compared to wild-type mice, and total dendritic spine density in dentate gyrus of MeCP2Tg1 mice was also increased. Total dendritic spine density was increased in CA1 of MeCP2Tg1 mice. SIGNIFICANCE: Overexpression of MeCP2 may disrupt crucial signaling pathways, resulting in decreased dendritic complexity of PV interneurons and increased dendritic spine density of projection neurons. This reciprocal modulation of excitatory and inhibitory neuronal structures associated with MeCP2 implies its significance as a potential target in the development of epilepsy and offers a novel perspective on the co-occurrence of autism and epilepsy.

Restoring thalamocortical circuit dysfunction by correcting HCN channelopathy in Shank3 mutant mice.

Thalamocortical (TC) circuits are essential for sensory information processing. Clinical and preclinical studies of autism spectrum disorders (ASDs) have highlighted abnormal thalamic development and TC circuit dysfunction. However, mechanistic understanding of how TC dysfunction contributes to behavioral abnormalities in ASDs is limited. Here, our study on a Shank3 mouse model of ASD reveals TC neuron hyperexcitability with excessive burst firing and a temporal mismatch relationship with slow cortical rhythms during sleep. These TC electrophysiological alterations and the consequent sensory hypersensitivity and sleep fragmentation in Shank3 mutant mice are causally linked to HCN2 channelopathy. Restoring HCN2 function early in postnatal development via a viral approach or lamotrigine (LTG) ameliorates sensory and sleep problems. A retrospective case series also supports beneficial effects of LTG treatment on sensory behavior in ASD patients. Our study identifies a clinically relevant circuit mechanism and proposes a targeted molecular intervention for ASD-related behavioral impairments.

CB1R dysfunction of inhibitory synapses in the ACC drives chronic social isolation stress-induced social impairments in male mice.

Social isolation is a risk factor for multiple mood disorders. Specifically, social isolation can remodel the brain, causing behavioral abnormalities, including sociability impairments. Here, we investigated social behavior impairment in mice following chronic social isolation stress (CSIS) and conducted a screening of susceptible brain regions using functional readouts. CSIS enhanced synaptic inhibition in the anterior cingulate cortex (ACC), particularly at inhibitory synapses of cholecystokinin (CCK)-expressing interneurons. This enhanced synaptic inhibition in the ACC was characterized by CSIS-induced loss of presynaptic cannabinoid type-1 receptors (CB1Rs), resulting in excessive axonal calcium influx. Activation of CCK-expressing interneurons or conditional knockdown of CB1R expression in CCK-expressing interneurons specifically reproduced social impairment. In contrast, optogenetic activation of CB1R or administration of CB1R agonists restored sociability in CSIS mice. These results suggest that the CB1R may be an effective therapeutic target for preventing CSIS-induced social impairments by restoring synaptic inhibition in the ACC.

Axin2 coupled excessive Wnt-glycolysis signaling mediates social defect in autism spectrum disorders.

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 ß-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.

Reversal of hyperactive higher-order thalamus attenuates defensiveness in a mouse model of PTSD.

Posttraumatic stress disorder (PTSD) is a highly prevalent and debilitating psychiatric disease often accompanied by severe defensive behaviors, preventing individuals from integrating into society. However, the neural mechanisms of defensiveness in PTSD remain largely unknown. Here, we identified that the higher-order thalamus, the posteromedial complex of the thalamus (PoM), was overactivated in a mouse model of PTSD, and suppressing PoM activity alleviated excessive defensive behaviors. Moreover, we found that diminished thalamic inhibition derived from the thalamic reticular nucleus was the major cause of thalamic hyperactivity in PTSD mice. Overloaded thalamic innervation to the downstream cortical area, frontal association cortex, drove abnormal defensiveness. Overall, our study revealed that the malfunction of the higher-order thalamus mediates defensive behaviors and highlighted the thalamocortical circuit as a potential target for treating PTSD-related overreactivity symptoms.

IRES-mediated Wnt2 translation in apoptotic neurons triggers astrocyte dedifferentiation.

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 ß-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.

Excitatory and Inhibitory Synaptic Imbalance Caused by Brain-Derived Neurotrophic Factor Deficits During Development in a Valproic Acid Mouse Model of Autism.

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.

TNF signaling pathway-mediated microglial activation in the PFC underlies acute paradoxical sleep deprivation-induced anxiety-like behaviors in mice.

Acute sleep deprivation is a common condition in modern life and increases anxiety symptoms in healthy individuals. The neuroinflammatory response induced by microglial activation could be an important contributing factor, but its underlying molecular mechanisms are still unclear. In the present study, we first found that acute paradoxical sleep deprivation (PSD) induced by the modified multiple platform method (MMPM) for 6 h led to anxiety-like behavior in mice, as verified by the open field test, elevated plus maze test, light-dark box test, and marble burying test. In addition, bioinformatic analysis suggested an important relationship between acute sleep deprivation and brain inflammatory signaling pathways. Key genes enriched in the TNF signaling pathway were confirmed to be altered during acute PSD by qPCR and Western blot analyses, including the upregulation of the prostaglandin-endoperoxide synthase 2 (Ptgs2) and suppressor of cytokine signaling 3 protein (Socs3) genes and the downregulation of the cysteine-aspartic acid protease 3 (Casp3) gene. Furthermore, we found that microglial cells in the prefrontal cortex (PFC) were activated with significant branch structure changes and that the cell body area was increased in the PSD model. Finally, we found that minocycline, a tetracycline with anti-inflammatory properties, may ameliorate the anxiogenic effect and microglial activation. Our study reveals significant correlations of anxiety-like behavior, microglial activation, and inflammation during acute PSD.

Dissection of the relationship between anxiety and stereotyped self-grooming using the Shank3B mutant autistic model, acute stress model and chronic pain 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.

Translational relevance of behavioral, neural, and electroencephalographic profiles in a mouse model of post-traumatic stress disorder.

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.

Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain.

Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. Here, we identify a genome-wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. We also show that TET1 is required for remyelination in adulthood. Transcriptomic, genomic occupancy, and 5-hydroxymethylcytosine (5hmC) profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2, impairs the onset of OPC differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice.

Extracellular matrix protein laminin ß1 regulates pain sensitivity and anxiodepression-like behaviors in mice.

Patients with neuropathic pain often experience comorbid psychiatric disorders. Cellular plasticity in the anterior cingulate cortex (ACC) is assumed to be a critical interface for pain perception and emotion. However, substantial efforts have thus far been focused on the intracellular mechanisms of plasticity rather than the extracellular alterations that might trigger and facilitate intracellular changes. Laminin, a key element of the extracellular matrix (ECM), consists of one α-, one ß-, and one γ-chain and is implicated in several pathophysiological processes. Here, we showed in mice that laminin ß1 (LAMB1) in the ACC was significantly downregulated upon peripheral neuropathy. Knockdown of LAMB1 in the ACC exacerbated pain sensitivity and induced anxiety and depression. Mechanistic analysis revealed that loss of LAMB1 caused actin dysregulation via interaction with integrin ß1 and the subsequent Src-dependent RhoA/LIMK/cofilin pathway, leading to increased presynaptic transmitter release probability and abnormal postsynaptic spine remodeling, which in turn orchestrated the structural and functional plasticity of pyramidal neurons and eventually resulted in pain hypersensitivity and anxiodepression. This study sheds new light on the functional capability of ECM LAMB1 in modulating pain plasticity and identifies a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified cingulate LAMB1/integrin ß1 signaling as a promising therapeutic target for the treatment of neuropathic pain and associated anxiodepression.

Quercetin relieves D-amphetamine-induced manic-like behaviour through activating TREK-1 potassium channels in mice.

BACKGROUND AND PURPOSE: Quercetin is a well-known plant flavonoid with neuroprotective properties. Earlier work suggested it may relieve psychiatric disorders, cognition deficits and memory dysfunction through anti-oxidant and/or radical scavenging mechanisms. In addition, quercetin modulated the physiological function of some ion channels. However, the detailed ionic mechanisms of the bioeffects of quercetin remain unknown. EXPERIMENTAL APPROACH: Effects of quercetin on neuronal activities in the prefrontal cortex (PFC) and its ionic mechanisms were analysed by calcium imaging using mice bearing a green fluorescent protein, calmodulin, and M13 fusion protein and patch clamp in acute brain slices from C57BL/6 J mice and in HEK 293 cells. The possible ionic mechanism of action of quercetin on D-amphetamine-induced manic-like effects in mice was explored with c-fos staining and the open field behaviour test. KEY RESULTS: Quercetin reduced calcium influx triggered by PFC pyramidal neuronal activity. This effect involved increasing the rheobase of neuronal firing through decreasing membrane resistance following quercetin treatment. Spadin, a blocker of TREK-1 potassium channels, also blocked the effect of quercetin on the membrane resistance and neuronal firing. Further, spadin blocked the neuroprotective effects of quercetin. The effects of quercetin on TREK-1 channels could be mimicked by GF109203X, a protein kinase C inhibitor. In vivo, injection of quercetin relieved the manic hyperlocomotion in mice, induced by D-amphetamine. This action was partly alleviated by spadin. CONCLUSION AND IMPLICATIONS: TREK-1 channels are a novel target for quercetin, by inhibiting PKC. This action could contribute to both the neuroprotective and anti-manic-like effects.

A Whole-Brain Cell-Type-Specific Sparse Neuron Labeling Method and Its Application in a Shank3 Autistic Mouse Model.

Single neurons, as the basic unit of the brain, consist of a cell body and processes, including dendrites and axons. Even neurons of the same type show various subtle process characteristics to fit into the diverse neural circuits. Different cell types of neurons form complicated circuits in the brain. Therefore, detailed neuronal morphology is required to understand normal neuronal function and pathological mechanisms, such as those that occur in autism. Here, we developed a strategy to sparsely label the same type of neurons throughout the whole brain and tested its application in an autistic animal model-Shank3 knockout (KO) mice. To achieve this, we designed an adeno-associated virus (AAV) that expresses Cre recombinase-dependent regular and membrane-targeted enhanced green fluorescent protein (EGFP) under a human synapsin 1 promoter and verified it in several Cre transgenic mice. We could sparsely label the projection neurons in multiple brain areas by retro-ocular injection of the virus into CaMKIIα-Cre mice. Then, we analyzed the morphology of the projection neurons in Shank3 KO mice with this method. We found differential dendritic complexity and dendritic spine changes in projection neurons in Shank3 KO mice crossed with CaMKIIα-Cre mice compared with littermate control mice in the striatum, cortex, and hippocampus. By combining this method with various Cre mouse lines crossed with mouse models of disease, we can screen the morphological traits of distinct types of neurons throughout the whole brain that will help us to understand the exact role of the specific cell types of neurons not only in autism spectrum disorder (ASD) mouse models but also in other psychiatric disorder mouse models.

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund's adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Anterior cingulate cortex dysfunction underlies social deficits in Shank3 mutant mice.

Social deficit is a core clinical feature of autism spectrum disorder (ASD) but the underlying neural mechanisms remain largely unclear. We demonstrate that structural and functional impairments occur in glutamatergic synapses in the pyramidal neurons of the anterior cingulate cortex (ACC) in mice with a mutation in Shank3, a high-confidence candidate ASD gene. Conditional knockout of Shank3 in the ACC was sufficient to generate excitatory synaptic dysfunction and social interaction deficits, whereas selective enhancement of ACC activity, restoration of SHANK3 expression in the ACC, or systemic administration of an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-positive modulator improved social behavior in Shank3 mutant mice. Our findings provide direct evidence for the notion that the ACC has a role in the regulation of social behavior in mice and indicate that ACC dysfunction may be involved in social impairments in ASD.