The influence of sex difference on behavior and adult hippocampal neurogenesis in C57BL/6 mice.

Animal models have been used extensively in in vivo studies, especially within the biomedical field. Traditionally, single-sex studies, mostly males, are used to avoid any potential confounding variation caused by sex difference and the female estrous cycle. Historically, female animal subjects are believed to exhibit higher variability, and this could increase the statistical power needed to test a hypothesis. This study sets out to evaluate whether a sex difference does exist in mouse behavior, and whether female mice featured higher variability. We assessed the sensorimotor skills, anxiety-like behavior, depression-like behavior, and cognitive abilities of mice through a series of commonly used behavioral tests. Except for the stronger grip force and lower tactile sensory sensitivity detected in male mice, there was no significant difference between males and females in other tests. Furthermore, immunolabeling of neurogenesis markers suggested no significant difference between sexes in adult hippocampal neurogenesis. Within group variances were equivalent; females did not exhibit higher variability than males. However, the overall negative results could be due to the limitation of small sample size. In conclusion, our study provides evidence that sex difference in mice does not significantly influence these commonly used behavioral tests nor adult neurogenesis under basal conditions. We suggest that female mice could also be considered for test inclusion in future experiment design.

Radial stem astrocytes (aka neural stem cells): Identity, development, physio-pathology, and therapeutic potential.

Adult neurogenesis is a striking example of neuroplasticity, which enables adaptive network remodelling in response to all forms of environmental stimulation in physiological and pathological contexts. Dysregulation or cessation of adult neurogenesis contributes to neuropathology negatively affecting brain functions and hampering regeneration of the nervous tissue while targeting adult neurogenesis may provide the basis for potential therapeutic interventions. Neural stem cells in the adult mammalian brain are at the core and the entry point of adult neurogenesis. By their origin and properties, these cells belong to astroglia, and are represented by stem radial astrocytes (RSA) which exhibit multipotent "stemness". In the neurogenic niches, RSA interact with other cellular components, including protoplasmic astrocytes, which in turn regulate their neurogenic activity. In pathology, RSA become reactive, which affects their neurogenic capabilities, whereas reactive parenchymal astrocytes up-regulate stem cell hallmarks and are able to generate progeny that remain within astrocyte lineage. What makes RSA special is their multipotency, represented by self-renewing capacity capability to generate other cellular types as progeny. A broad understanding of the cellular features of RSA and parenchymal astrocytes provides an insight into the machinery that promotes/suppresses adult neurogenesis, clarifying principles of network remodelling. In this review, we discuss the cellular hallmarks, research tools, and models of RSA and astrocytes of the subventricular zone along the lateral ventricle and dentate gyrus of the hippocampus. We also discuss RSA in ageing, which has a great impact on the proliferative capacity of RSA, as well as the potential of RSA and astrocytes in therapeutic strategies aimed at cell replacement and regeneration.

Influence of diurnal phase on behavioral tests of sensorimotor performance, anxiety, learning and memory in mice.

Behavioral measurements in mice are critical tools used to evaluate the effects of interventions. Whilst mice are nocturnal animals, many studies conduct behavioral tests during the day. To better understand the effects of diurnal rhythm on mouse behaviors, we compared the results from behavioral tests conducted in the active and inactive phases. C57BL/6 mice were used in this study; we focus on sensorimotor performance, anxiety, learning and memory. Overall, our results show mice exhibit slightly higher cutaneous sensitivity, better long-term contextual memory, and a greater active avoidance escape response during the active phase. We did not observe significant differences in motor coordination, anxiety, or spatial learning and memory. Furthermore, apart from the elevated-O-maze, there was no remarkable sex effect among these tests. This study provides information on the effects of different diurnal phases on types of behavior and demonstrates the importance of the circadian cycle on learning and memory. Although we did not detect differences in anxiety and spatial learning/memory, diurnal rhythm may interact with other factors to influence these behaviors.

PLA2G6 mutations cause motor dysfunction phenotypes of young-onset dystonia-parkinsonism type 14 and can be relieved by DHA treatment in animal models.

Parkinson's disease (PD), the most common neurodegenerative motor disorder, is currently incurable. Although many studies have provided insights on the substantial influence of genetic factors on the occurrence and development of PD, the molecular mechanism underlying the disease is largely unclear. Previous studies have shown that point mutations in the phospholipase A2 group VI gene (PLA2G6) correlate with young-onset dystonia-parkinsonism type 14 (PARK14). However, limited information is available regarding the pathogenic role of this gene and the mechanism underlying its function. To study the role of PLA2G6 mutations, we first used zebrafish larvae to screen six PLA2G6 mutations and revealed that injection of D331Y, T572I, and R741Q mutation constructs induced phenotypes such as motility defects and reduction in dopaminergic neurons. The motility defects could be alleviated by treatment with L-3, 4-dihydroxyphenylalanine (L-dopa), indicating that these mutations are pathological for PARK14 symptoms. Furthermore, the injection of D331Y and T572I mutation constructs reduced phospholipase activity of PLA2G6 and its lipid metabolites, which confirmed that these two mutations are loss-of-function mutations. Metabolomic analysis revealed that D331Y or T572I mutation led to higher phospholipid and lower docosahexaenoic acid (DHA) levels, indicating that reduced DHA levels are pathological for defective motor functions. Further, a dietary DHA supplement relieved the motility defects in PLA2G6D331Y/D331Y knock-in mice. This result revealed that the D331Y mutation caused defective PLA2G6 phospholipase activity and consequently reduced the DHA level, which is the pathogenic factor responsible for PARK14. The results of this study will facilitate the development of therapeutic strategies for PARK14.

Follistatin mediates learning and synaptic plasticity via regulation of Asic4 expression in the hippocampus.

The biological mechanisms underpinning learning are unclear. Mounting evidence has suggested that adult hippocampal neurogenesis is involved although a causal relationship has not been well defined. Here, using high-resolution genetic mapping of adult neurogenesis, combined with sequencing information, we identify follistatin (Fst) and demonstrate its involvement in learning and adult neurogenesis. We confirmed that brain-specific Fst knockout (KO) mice exhibited decreased hippocampal neurogenesis and demonstrated that FST is critical for learning. Fst KO mice exhibit deficits in spatial learning, working memory, and long-term potentiation (LTP). In contrast, hippocampal overexpression of Fst in KO mice reversed these impairments. By utilizing RNA sequencing and chromatin immunoprecipitation, we identified Asic4 as a target gene regulated by FST and show that Asic4 plays a critical role in learning deficits caused by Fst deletion. Long-term overexpression of hippocampal Fst in C57BL/6 wild-type mice alleviates age-related decline in cognition, neurogenesis, and LTP. Collectively, our study reveals the functions for FST in adult neurogenesis and learning behaviors.

Activation of a hippocampal CREB-pCREB-miRNA-MEF2 axis modulates individual variation of spatial learning and memory capability.

Phenotypic variation is a fundamental prerequisite for cell and organism evolution by natural selection. Whereas the role of stochastic gene expression in phenotypic diversity of genetically identical cells is well studied, not much is known regarding the relationship between stochastic gene expression and individual behavioral variation in animals. We demonstrate that a specific miRNA (miR-466f-3p) is upregulated in the hippocampus of a portion of individual inbred mice upon a Morris water maze task. Significantly, miR-466f-3p positively regulates the neuron morphology, function and spatial learning, and memory capability of mice. Mechanistically, miR-466f-3p represses translation of MEF2A, a negative regulator of learning/memory. Finally, we show that varied upregulation of hippocampal miR-466f-3p results from randomized phosphorylation of hippocampal cyclic AMP (cAMP)-response element binding (CREB) in individuals. This finding of modulation of spatial learning and memory via a randomized hippocampal signaling axis upon neuronal stimulation represents a demonstration of how variation in tissue gene expression lead to varied animal behavior.

Enterovirus A71 Induces Neurological Diseases and Dynamic Variants in Oral Infection of Human SCARB2-Transgenic Weaned Mice.

Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae, poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

The effect of nerve growth factor on supporting spatial memory depends upon hippocampal cholinergic innervation.

Nerve growth factor (NGF) gene therapy has been used in clinical trials of Alzheimer's disease. Understanding the underlying mechanisms of how NGF influences memory may help develop new strategies for treatment. Both NGF and the cholinergic system play important roles in learning and memory. NGF is essential for maintaining cholinergic innervation of the hippocampus, but it is unclear whether the supportive effect of NGF on learning and memory is specifically dependent upon intact hippocampal cholinergic innervation. Here we characterize the behavior and hippocampal measurements of volume, neurogenesis, long-term potentiation, and cholinergic innervation, in brain-specific Ngf-deficient mice. Our results show that knockout mice exhibit increased anxiety, impaired spatial learning and memory, decreased adult hippocampal volume, neurogenesis, short-term potentiation, and cholinergic innervation. Overexpression of Ngf in the hippocampus of Ngf gene knockout mice rescued spatial memory and partially restored cholinergic innervations, but not anxiety. Selective depletion of hippocampal cholinergic innervation resulted in impaired spatial memory. However, Ngf overexpression in the hippocampus failed to rescue spatial memory in mice with hippocampal-selective cholinergic fiber depletion. In conclusion, we demonstrate the impact of Ngf deficiency in the brain and provide evidence that the effect of NGF on spatial memory is reliant on intact cholinergic innervations in the hippocampus. These results suggest that adequate cholinergic targeting may be a critical requirement for successful use of NGF gene therapy of Alzheimer's disease.

Snake venom proteome of Protobothrops mucrosquamatus in Taiwan: Delaying venom-induced lethality in a rodent model by inhibition of phospholipase A2 activity with varespladib.

Protobothrops mucrosquamatus, also known as the brown spotted pit viper or Taiwanese habu, is a medically significant venomous snake in Taiwan, especially in the northern area. To more fully understand the proteome profile of P. mucrosquamatus, we characterized its venom composition using a bottom-up proteomic approach. Whole venom components were fractionated by RP-HPLC and then analyzed by SDS-PAGE. Each protein band in gels was excised and subjected to protein identification by LC-MS/MS. A subsequent proteomic analysis revealed the presence of 61 distinct proteins belonging to 19 families in P. mucrosquamatus venom. Snake venom metalloproteinase (SVMP; 29.4%), C-type lectin (CLEC; 21.1%), snake venom serine protease (SVSP; 17.6%) and phospholipase A2 (PLA2; 15.9%) were the most abundant protein families, whereas several low-abundance proteins, categorized into eight protein families, were demonstrated in P. mucrosquamatus venom for the first time. Because PLA2 is known to make a major contribution to venom lethality, we evaluated whether the known PLA2 inhibitor, varespladib, was capable of preventing the toxic effects of P. mucrosquamatus venom. This small-molecule drug demonstrated the ability to inhibit PLA2 activity in vitro (IC50 = 101.3 nM). It also blunted lethality in vivo, prolonging survival following venom injection in a mouse model, but it showed limited potency against venom-induced local hemorrhage in this model. Our findings provide essential biological and pathophysiological insights into the composition of P. mucrosquamatus venom and suggest PLA2 inhibition as an adjunctive or alternative therapeutic strategy in the clinical management of P. mucrosquamatus envenoming in emergency medicine. SIGNIFICANCE: P. mucrosquamatus envenomation is a significant medical concern in Taiwan, especially in the northern region. Although antivenom is commonly used for rescuing P. mucrosquamatus envenoming, severe clinical events still occur, with more than 20% of cases requiring surgical intervention. Small-molecule therapy offers several advantages as a potential adjunctive, or even alternative, to antivenom treatment, such as heat stability, low antigenicity and ease of administration, among others. A deeper understanding of the venom proteome of P. mucrosquamatus would aid in the discovery of small-molecule drugs that could be repurposed to target specific venom proteins. Here, we applied a bottom-up proteomic approach to characterize the protein profile of P. mucrosquamatus venom. Varespladib, a small-molecule drug used to treat inflammatory disease, was repurposed to inhibit the toxicity of P. mucrosquamatus venom, and was shown to reduce the lethal effects of P. mucrosquamatus envenomation in a rodent model. Varespladib might be used as a first-aid therapeutic against P. mucrosquamatus envenoming in the pre-referral period and/or as an adjunctive agent administered together with anti-P. mucrosquamatus antivenom.

Pogz deficiency leads to transcription dysregulation and impaired cerebellar activity underlying autism-like behavior in mice.

Several genes implicated in autism spectrum disorder (ASD) are chromatin regulators, including POGZ. The cellular and molecular mechanisms leading to ASD impaired social and cognitive behavior are unclear. Animal models are crucial for studying the effects of mutations on brain function and behavior as well as unveiling the underlying mechanisms. Here, we generate a brain specific conditional knockout mouse model deficient for Pogz, an ASD risk gene. We demonstrate that Pogz deficient mice show microcephaly, growth impairment, increased sociability, learning and motor deficits, mimicking several of the human symptoms. At the molecular level, luciferase reporter assay indicates that POGZ is a negative regulator of transcription. In accordance, in Pogz deficient mice we find a significant upregulation of gene expression, most notably in the cerebellum. Gene set enrichment analysis revealed that the transcriptional changes encompass genes and pathways disrupted in ASD, including neurogenesis and synaptic processes, underlying the observed behavioral phenotype in mice. Physiologically, Pogz deficiency is associated with a reduction in the firing frequency of simple and complex spikes and an increase in amplitude of the inhibitory synaptic input in cerebellar Purkinje cells. Our findings support a mechanism linking heterochromatin dysregulation to cerebellar circuit dysfunction and behavioral abnormalities in ASD.

Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models.

Naja atra envenomation is one of the most significant clinical snakebite concerns in Taiwan. Taiwanese freeze-dried neurotoxic antivenom (FNAV) is currently used clinically for the treatment of cobra snakebite, and has been shown to limit the mortality of cobra envenomation to less than 1%. However, more than half of victims (60%) require surgery because of local tissue necrosis, a major problem in patients with cobra envenomation. Although the importance of evaluating the neutralizing effect of FNAV on this pathology is recognized, whether FNAV is able to prevent the local necrosis extension induced by N. atra venom has not been investigated in detail. Cytotoxins (CTXs) are considered as the major components of N. atra venom that cause necrosis. In the current study, we isolated CTXs from whole cobra venom and used both whole venom and purified CTXs to develop animal models for assessing the neutralization potential of FNAV against venom necrotizing activity. Local necrotic lesions were successfully produced in mice using CTXs in place of whole N. atra venom. FNAV was able to rescue mice from a subcutaneously injected lethal dose of cobra venom; however, it was unable to prevent CTX-induced dermo-necrosis. Furthermore, using the minimal necrosis dose (MND) of CTXs and venom proteome data, we found a dose of whole N. atra venom suitable for FNAV and developed a workable protocol for inducing local necrosis in rodent models that successfully imitated the clinical circumstance of cobra envenoming. This information provides a more comprehensive understanding of the pathophysiology of N. atra envenomation, and serves as a guide for improving current antivenom strategies and advancing clinical snakebite management in Taiwan.

Upregulation of Cisd2 attenuates Alzheimer's-related neuronal loss in mice.

CDGSH iron-sulfur domain-containing protein 2 (Cisd2), a protein that declines in an age-dependent manner, mediates lifespan in mammals. Cisd2 deficiency causes accelerated aging and shortened lifespan, whereas persistent expression of Cisd2 promotes longevity in mice. Alzheimer's disease (AD) is the most prevalent form of senile dementia and is without an effective therapeutic strategy. We investigated whether Cisd2 upregulation is able to ameliorate amyloid ß (Aß) toxicity and prevent neuronal loss using an AD mouse model. Our study makes three major discoveries. First, using the AD mouse model (APP/PS1 double transgenic mice), the dosage of Cisd2 appears to modulate the severity of AD phenotypes. Cisd2 overexpression (∼two-fold) significantly promoted survival and alleviated the pathological defects associated with AD. Conversely, Cisd2 deficiency accelerated AD pathogenesis. Secondly, Cisd2 overexpression protected against Aß-mediated mitochondrial damage and attenuated loss of neurons and neuronal progenitor cells. Finally, an increase in Cisd2 shifted the expression profiles of a panel of genes that are dysregulated by AD toward the patterns observed in wild-type mice. These findings highlight Cisd2-based therapies as a potential disease-modifying strategy for AD. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Large Scale Calcium Imaging of the Cerebellar Vermis During Sensory Stimulus Unravels Two Response's Components That Differ in Their Spatiotemporal Properties.

The well documented precision of the cerebellar sagittal organization is commonly used to compose a comprehensive view on principles of cerebellar function. However, the physiological manifestation of this organization is either limited to information derived from single unit recordings or from imaging of a small group of closely located neurons. Here we used large scale imaging to monitor calcium concentration changes in the entire vermal area of folia V and VI in anesthetized mice. We found that the response to a strong auditory input or electrical shock to the tail area is composed of an early and a late component that differ in their spatiotemporal properties. The early component occurs throughout the scanned area whereas the late component reflects synchronous activation of Purkinje cells located along symmetric parasagittal bands that correspond well to sagittal band 2+ (Sugihara and Shinoda, 2004). Similar organization was found in the rigorously disorganized cerebellum of Cxcr4 KO mice, suggesting that the sagittal organization is determined by the climbing fiber inputs to the cerebellar cortex. The responses for both stimuli are followed by a prolonged recovery period but the rate of recovery from auditory stimulus is much longer, reflecting a different site for the adapting process. We suggest that these sensory inputs, which are commonly used to evoke startle response, activate two sets of climbing fiber inputs that differ in their spatiotemporal properties and contribute to the motor organization and habituation of the startle response. Significance Statement: The ensemble activity of neurons in the brain is one of the current challenges of neuroscience. Here we use a fast and large-scale calcium imaging system to monitor ensemble activity in the cerebellar cortex following auditory stimuli or electric shocks to the tail. The system, which enables the detection of the response to a single trail, reveals the robustness of the functional organization of the olivo-cerebellar system in sagittal bands that is preserved in genetically induced disorganized cerebellar cortex. Furthermore, the response, which represents the activation of two sets of climbing fibers inputs, is followed by a prolonged recovery process that indicates the cerebellar involvement in startle response.

Altered hippocampal gene expression and structure in transgenic mice overexpressing neuregulin 1 (Nrg1) type I.

Transgenic mice overexpressing the type I isoform of neuregulin 1 (Nrg1; NRG1) have alterations in hippocampal gamma oscillations and an age-emergent deficit in hippocampus-dependent spatial working memory. Here, we examined the molecular and morphological correlates of these findings. Microarrays showed over 100 hippocampal transcripts differentially expressed in Nrg1tg-type I mice, with enrichment of genes related to neuromodulation and, in older mice, of genes involved in inflammation and immunity. Nrg1tg-type I mice had an enlarged hippocampus with a widened dentate gyrus. The results show that Nrg1 type I impacts on hippocampal gene expression and structure in a multifaceted and partly age-related way, complementing the evidence implicating Nrg1 signaling in aspects of hippocampal function. The findings are also relevant to the possible role of NRG1 signaling in the pathophysiology of schizophrenia or other disorders affecting this brain region.

NPTX2 is a key component in the regulation of anxiety.

Anxiety disorders significantly impair quality of life. However, limited knowledge of the underlying mechanisms impedes the development of effective therapeutics. Previous studies have suggested that the expression of the Nptx2 gene is associated with anxiety, but the neurobiological processes underlying this association remain unclear. We generated multiple mouse models with knockout or overexpression of Nptx2 in specific brain regions and during different developmental stages to assess anxiety, adult neurogenesis, and glucocorticoid-related gene expression. Our results provide evidence that Nptx2 expression in the adult hippocampus regulates anxiety in mice. Eliminating Nptx2 expression in either the developing mouse brain or in adulthood leads to increased anxiety levels. The increase in anxiety was evident in hippocampus-specific Nptx2 knockout mice, but not in an amygdala specific knockouts. Gene expression analysis revealed increased expression of glucocorticoid receptor target genes in Nptx2 knockout mice after acute stress. Overexpression of Nptx2 in the hippocampus alleviates stress-induced anxious behaviors and reverses the changes in expression of glucocorticoid receptor related genes. In conclusion, we demonstrate that Nptx2 in the hippocampus performs a critical role in modulating anxiety, hippocampal cell proliferation, and glucocorticoid receptor related gene expression. Our results suggest Nptx2 may be a potential target for anxiolytic therapeutics.

Analysis of the efficacy of Taiwanese freeze-dried neurotoxic antivenom against Naja kaouthia, Naja siamensis and Ophiophagus hannah through proteomics and animal model approaches.

In Southeast Asia, envenoming resulting from cobra snakebites is an important public health issue in many regions, and antivenom therapy is the standard treatment for the snakebite. Because these cobras share a close evolutionary history, the amino acid sequences of major venom components in different snakes are very similar. Therefore, either monovalent or polyvalent antivenoms may offer paraspecific protection against envenomation of humans by several different snakes. In Taiwan, a bivalent antivenom-freeze-dried neurotoxic antivenom (FNAV)-against Bungarus multicinctus and Naja atra is available. However, whether this antivenom is also capable of neutralizing the venom of other species of snakes is not known. Here, to expand the clinical application of Taiwanese FNAV, we used an animal model to evaluate the neutralizing ability of FNAV against the venoms of three common snakes in Southeast Asia, including two 'true' cobras Naja kaouthia (Thailand) and Naja siamensis (Thailand), and the king cobra Ophiophagus hannah (Indonesia). We further applied mass spectrometry (MS)-based proteomic techniques to characterize venom proteomes and identify FNAV-recognizable antigens in the venoms of these Asian snakes. Neutralization assays in a mouse model showed that FNAV effectively neutralized the lethality of N. kaouthia and N. siamensis venoms, but not O. hannah venom. MS-based venom protein identification results further revealed that FNAV strongly recognized three-finger toxin and phospholipase A2, the major protein components of N. kaouthia and N. siamensis venoms. The characterization of venom proteomes and identification of FNAV-recognizable venom antigens may help researchers to further develop more effective antivenom designed to block the toxicity of dominant toxic proteins, with the ultimate goal of achieving broadly therapeutic effects against these cobra snakebites.

Neuropsin Inactivation Has Protective Effects against Depressive-Like Behaviours and Memory Impairment Induced by Chronic Stress.

Mounting evidence suggests the interaction between stress and genetics contribute to the development of depressive symptoms. Currently, the molecular mechanisms mediating this process are poorly understood, hindering the development of new clinical interventions. Here, we investigate the interaction between neuropsin, a serine protease, and chronic stress on the development of depressive-like behaviours in mice. We found no difference in baseline behaviour between neuropsin knockout and wild-type mice. However, our results show that neuropsin knockout mice are protected against the development of depressive-like behaviours and memory impairment following chronic stress. We hypothesised that this difference in behaviour may be due to an interaction between neuropsin and elevated plasma corticosterone. To test this, we subjected mice to chronic corticosterone injections. These injections resulted in changes to hippocampal structure similar to that observed following chronic stress. We found that inactivation of neuropsin limits the extent of these anatomical changes in both the chronic stress and the corticosterone injection exposed cohorts. We next used viral vectors to knockdown or overexpress neuropsin in the hippocampus to confirm the results of the KO study. Additionally, we found that inactivation of neuropsin limited glutamate dysregulation, associated with increased generation of reactive oxygen species, resulting from prolonged elevated plasma corticosterone. In this study, we demonstrate that neuropsin inactivation protects against the impairment of hippocampal functions and the depressive-like behaviour induced by chronic stress or high levels of corticosterone. Consequently, we suggest neuropsin is a potential target for clinical interventions for the management of stress disorders.

Neuronal Splicing Regulator RBFOX3 (NeuN) Regulates Adult Hippocampal Neurogenesis and Synaptogenesis.

Dysfunction of RBFOX3 has been identified in neurodevelopmental disorders such as autism spectrum disorder, cognitive impairments and epilepsy and a causal relationship with these diseases has been previously demonstrated with Rbfox3 homozygous knockout mice. Despite the importance of RBFOX3 during neurodevelopment, the function of RBFOX3 regarding neurogenesis and synaptogenesis remains unclear. To address this critical question, we profiled the developmental expression pattern of Rbfox3 in the brain of wild-type mice and analyzed brain volume, disease-relevant behaviors, neurogenesis, synaptic plasticity, and synaptogenesis in Rbfox3 homozygous knockout mice and their corresponding wild-type counterparts. Here we report that expression of Rbfox3 differs developmentally for distinct brain regions. Moreover, Rbfox3 homozygous knockout mice exhibited cold hyperalgesia and impaired cognitive abilities. Focusing on hippocampal phenotypes, we found Rbfox3 homozygous knockout mice displayed deficits in neurogenesis, which was correlated with cognitive impairments. Furthermore, RBFOX3 regulates the exons of genes with synapse-related function. Synaptic plasticity and density, which are related to cognitive behaviors, were altered in the hippocampal dentate gyrus of Rbfox3 homozygous knockout mice; synaptic plasticity decreased and the density of synapses increased. Taken together, our results demonstrate the important role of RBFOX3 during neural development and maturation. In addition, abnormalities in synaptic structure and function occur in Rbfox3 homozygous knockout mice. Our findings may offer mechanistic explanations for human brain diseases associated with dysfunctional RBFOX3.

Chronic activation of NPFFR2 stimulates the stress-related depressive behaviors through HPA axis modulation.

Neuropeptide FF (NPFF) is a morphine-modulating peptide that regulates the analgesic effect of opioids, and also controls food consumption and cardiovascular function through its interaction with two cognate receptors, NPFFR1 and NPFFR2. In the present study, we explore a novel modulatory role for NPFF-NPFFR2 in stress-related depressive behaviors. In a mouse model of chronic mild stress (CMS)-induced depression, the expression of NPFF significantly increased in the hypothalamus, hippocampus, medial prefrontal cortex (mPFC) and amygdala. In addition, transgenic (Tg) mice over-expressing NPFFR2 displayed clear depression and anxiety-like behaviors with hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis, reduced expression of glucocorticoid receptor (GR) and neurogenesis in the hippocampus. Furthermore, acute treatment of NPFFR2 agonists in wild-type (WT) mice enhanced the activity of the HPA axis, and chronic administration resulted in depressive and anxiety-like behaviors. Chronic stimulation of NPFFR2 also decreased the expression of hippocampal GR and led to persistent activation of the HPA axis. Strikingly, bilateral intra-paraventricular nucleus (PVN) injection of NPFFR2 shRNA predominately inhibits the depressive-like behavior in CMS-exposed mice. Antidepressants, fluoxetine and ketamine, effectively relieved the depressive behaviors of NPFFR2-Tg mice. We speculate that persistent NPFFR2 activation, in particular in the hypothalamus, up-regulates the HPA axis and results in long-lasting increases in circulating corticosterone (CORT), consequently damaging hippocampal function. This novel role of NPFFR2 in regulating the HPA axis and hippocampal function provides a new avenue for combating depression and anxiety-like disorder.