Evaluation of neurological behavior alterations and metabolic changes in mice under chronic glyphosate exposure.

Glyphosate is a widely used active ingredient in agricultural herbicides, inhibiting the biosynthesis of aromatic amino acids in plants by targeting their shikimate pathway. Our gut microbiota also facilitates the shikimate pathway, making it a vulnerable target when encountering glyphosate. Dysbiosis in the gut microbiota may impair the gut-brain axis, bringing neurological outcomes. To evaluate the neurotoxicity and biochemical changes attributed to glyphosate, we exposed mice with the reference dose (RfD) set by the U.S. EPA (1.75 mg/Kg-BW/day) and its hundred-time-equivalence (175 mg/Kg-BW/day) chronically via drinking water, then compared a series of neurobehaviors and their fecal/serum metabolomic profile against the non-exposed vehicles (n = 10/dosing group). There was little alteration in the neurobehavior, including motor activities, social approach, and conditioned fear, under glyphosate exposure. Metabolomic differences attributed to glyphosate were observed in the feces, corresponding to 68 and 29 identified metabolites with dysregulation in the higher and lower dose groups, respectively, compared to the vehicle-control. There were less alterations observed in the serum metabolome. Under 175 mg/Kg-BW/day of glyphosate exposure, the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were reduced in the feces but not in the serum of mice. We further focused on how tryptophan metabolism was dysregulated based on the pathway analysis, and identified the indole-derivatives were more altered compared to the serotonin and kynurenine derivatives. Together, we obtained a three-dimensional data set that records neurobehavioral, fecal metabolic, and serum biomolecular dynamics caused by glyphosate exposure at two different doses. Our data showed that even under the high dose of glyphosate irrelevant to human exposure, there were little evidence that supported the impairment of the gut-brain axis.

Amygdala Arginine Vasopressin Modulates Chronic Ethanol Withdrawal Anxiety-Like Behavior in the Social Interaction Task.

BACKGROUND: Chronic ethanol (EtOH) exposure induces neurobehavioral maladaptations in the brain though the precise changes have not been fully explored. The central nucleus of the amygdala (CEA) regulates anxiety-like behavior induced by withdrawal from chronic intermittent EtOH (CIE) exposure, and the arginine vasopressin (AVP) system within the CEA regulates many anxiety-like behaviors. Thus, adaptations occur in the CEA AVP system due to chronic EtOH exposure, which lead to anxiety-like behaviors in rats. METHODS: Chronic exposure to a low-dose EtOH (4.5% wt/vol) induces anxiety-like behavior in rats. Wistar or Sprague Dawley rats were exposed to a modified CIE or CIE, while intra-CEA microinjections of AVP or a V1b receptor antagonist were used to elicit or block withdrawal-induced anxiety. Additionally, AVP microinjections into the CEA were given 24 hours following 15 days of continuous high-dose EtOH (7% wt/vol), a time period when rats no longer express anxiety. Chemogenetics was also used to activate the basolateral amygdala (BLA) or deactivate the dorsal periaqueductal gray=(dm/dlPAG) therefore PAG=periaqueductal gray to elicit or block withdrawal-induced anxiety. RESULTS: AVP microinjected into the CEA in lieu of exposure to the first 2 cycles of CIE was sufficient to induce anxiety-like behavior in these commonly used rat strains. The V1b receptor antagonist, but not an oxytocin receptor agonist, into the CEA during the first 2 withdrawal cycles suppressed anxiety. However, activation of the BLA in lieu of exposure to the first 2 cycles of CIE was insufficient to induce anxiety-like behavior. AVP microinjection into the CEA 24 hours into withdrawal reelicited anxiety-like behavior, and deactivation of the dm/dlPAG reduced this effect of CEA AVP. CONCLUSIONS: Taken together, this study demonstrates a role of CEA AVP and a CEA-dm/dlPAG circuit in the development of anxiety induced by CIE. Such information is valuable for identifying novel therapeutic targets for alcohol- and anxiety-associated disorders.

Thyroxine administration prevents matrilineal intergenerational consequences of in utero ethanol exposure in rats.

The neurodevelopmental fetal alcohol spectrum disorder (FASD) is characterized by cognitive and behavioral deficits in the offspring. Conferring the deficits to the next generation would increase overall FASD disease burden and prevention of this transmission could be highly significant. Prior studies showed the reversal of these behavioral deficits by low dose thyroxine (T4) supplementation to the ethanol-consuming mothers. Here we aim to identify whether prenatal ethanol (PE) exposure impairs hippocampus-dependent learning and memory in the second-generation (F2) progeny, and whether T4 administration to the ethanol-consuming dam can prevent it. Sprague-Dawley (S) dams received control diets (ad libitum and nutritional control) or ethanol containing liquid diet with and without simultaneous T4 (0.3mg/L diet) administration. Their offspring (SS F1) were mated with naive Brown Norway (B) males and females generating the SB F2 and BS F2 progeny. Hippocampus-dependent contextual fear memory and hippocampal expression of the thyroid hormone-regulated type 3 deiodinase, (Dio3) and neurogranin (Nrgn) were assessed. SS F1 PE-exposed females and their SB F2 progeny exhibited fear memory deficits. T4 administration to the mothers of F1 females reversed these deficits. Although SS F1 PE-exposed males also experienced fear memory deficit, this was neither transmitted to their BS F2 offspring nor reversed by prenatal T4 treatment. Hippocampal Dio3 and Nrgn expression showed similar pattern of changes. Grandmaternal ethanol consumption during pregnancy affects fear memory of the matrilineal second-generation progeny. Low dose T4 supplementation prevents this process likely via altering allele-specific and total expression of Dio3 in the hippocampus.

Withdrawal from Chronic Alcohol Induces a Unique CCL2 mRNA Increase in Adolescent But Not Adult Brain--Relationship to Blood Alcohol Levels and Seizures.

BACKGROUND: The role of neuroimmune activation in withdrawal from chronic alcohol (ethanol) has been established in both adolescent and adult models, but direct comparisons across age are sparse. Studies need to elucidate age-dependent neuroimmune effects of alcohol and to focus research attention on age-dependent mechanisms and outcomes. METHODS: Adult and adolescent rats from 2 commonly used strains, Wistar and Sprague Dawley (SD), were maintained on continuous 7%, 5.35%, 4.5% alcohol diet (CAD) or cycled 7% w/v alcohol diet for 15 days. Cortical tissue was collected at 0, 8, 16, and 24 hours postwithdrawal followed by measurement of chemokine (C-C motif) ligand 2 (CCL2), tumor necrosis factor alpha, and interleukin 1 beta mRNA with quantitative real-time polymerase chain reaction. RESULTS: Both age groups and strains showed a strong cytokine mRNA response at 7% CAD. Further, a greater increase in CCL2 mRNA was observed in the cortex of adolescents at 7% CAD, which correlated with higher blood alcohol levels (BALs). Adolescents exposed to 5.35% CAD exhibited similar blood levels and cytokine responses as adults exposed to 7% CAD. Substantial variability in CCL2 mRNA responses was found only in adolescent rats exposed to 7% CAD. In this group, data could be segregated into high-responding and low-responding groups. Moreover, the data from the high-responding group were associated with seizures. CONCLUSIONS: Relative to other cytokine mRNAs, CCL2 exhibits a unique response profile during withdrawal from CAD. This profile is shown in adolescents, where CCL2 is uniquely influenced by the effects of seizures. Additionally, this profile is shown by the fact that only CCL2 expression correlated with BAL that transcended age groups. These data emphasize the importance of BALs and treatment regimen on developmental neuroimmune responses and suggest that select components of the neuroimmune system are more responsive to CAD withdrawal and that neurobiological mechanisms differentiating these responses should be further explored.

Intergenerational effects of prenatal ethanol on glucose tolerance and insulin response.

Consequences of prenatal exposure to ethanol (E) include morphological, physiological, and cognitive deficits and are collectively classified as fetal alcohol spectrum disorders. Adult prenatal E exposed offspring show insulin resistance, and given that in utero hyperglycemic environment can cause metabolic disorders in subsequent generations; we investigated the effects of grandmaternal E on functional glucose and insulin responses of the second generation. Sprague-Dawley (S) rat dams, mated with S males, received E-containing liquid diet and two different control diets between gestational days 8 and 20. Additionally, because prenatal E-induced behavioral deficits can be reversed by simultaneous thyroxine (T4) treatment, another group of dams received 0.3 mg/l T4 in their E diet. Their first-generation (F1) offspring were mated with control Brown Norway (B) males or females to produce SB and BS F2 progeny. Dams consuming E during pregnancy were hyperglycemic, and their F1 offspring showed insulin resistance in the glucose tolerance test (GTT). However, F2 responses to GTT varied based on the sex of prenatal E-exposed parent. BS F2 females, and both male and female SB F2 progeny, displayed hypoglycemic and hyperinsulinemic GTT response patterns. Although administering T4 to E dams normalized thyroid function of the F1 generation, it did not reverse their prenatal E-induced metabolic dysfunction. In contrast, administration of T4 to the alcohol-consuming grandmother reversed or alleviated the aberrant GTT responses of the F2 progeny. Prenatal E-induced dysregulation of glucose metabolism can affect the next generation, possibly via ethanol effects on the germline of the F1 fetus.

Alterations of social interaction through genetic and environmental manipulation of the 22q11.2 gene Sept5 in the mouse brain.

Social behavior dysfunction is a symptomatic element of schizophrenia and autism spectrum disorder (ASD). Although altered activities in numerous brain regions are associated with defective social cognition and perception, the causative relationship between these altered activities and social cognition and perception-and their genetic underpinnings-are not known in humans. To address these issues, we took advantage of the link between hemizygous deletion of human chromosome 22q11.2 and high rates of social behavior dysfunction, schizophrenia and ASD. We genetically manipulated Sept5, a 22q11.2 gene, and evaluated its role in social interaction in mice. Sept5 deficiency, against a high degree of homogeneity in a congenic genetic background, selectively impaired active affiliative social interaction in mice. Conversely, virally guided overexpression of Sept5 in the hippocampus or, to a lesser extent, the amygdala elevated levels of active affiliative social interaction in C57BL/6J mice. Congenic knockout mice and mice overexpressing Sept5 in the hippocampus or amygdala were indistinguishable from control mice in novelty and olfactory responses, anxiety or motor activity. Moreover, post-weaning individual housing, an environmental condition designed to reduce stress in male mice, selectively raised levels of Sept5 protein in the amygdala and increased active affiliative social interaction in C57BL/6J mice. These findings identify this 22q11.2 gene in the hippocampus and amygdala as a determinant of social interaction and suggest that defective social interaction seen in 22q11.2-associated schizophrenia and ASD can be genetically and environmentally modified by altering this 22q11.2 gene.

Sleep Disruption Precedes Forebrain Synaptic Tau Burden and Contributes to Cognitive Decline in a Sex-Dependent Manner in the P301S Tau Transgenic Mouse Model.

Sleep disruption and impaired synaptic processes are common features in neurodegenerative diseases, including Alzheimer's disease (AD). Hyperphosphorylated Tau is known to accumulate at neuronal synapses in AD, contributing to synapse dysfunction. However, it remains unclear how sleep disruption and synapse pathology interact to contribute to cognitive decline. Here, we examined sex-specific onset and consequences of sleep loss in AD/tauopathy model PS19 mice. Using a piezoelectric home-cage monitoring system, we showed PS19 mice exhibited early-onset and progressive hyperarousal, a selective dark-phase sleep disruption, apparent at 3 months in females and 6 months in males. Using the Morris water maze test, we report that chronic sleep disruption (CSD) accelerated the onset of decline of hippocampal spatial memory in PS19 males only. Hyperarousal occurs well in advance of robust forebrain synaptic Tau burden that becomes apparent at 6-9 months. To determine whether a causal link exists between sleep disruption and synaptic Tau hyperphosphorylation, we examined the correlation between sleep behavior and synaptic Tau, or exposed mice to acute or chronic sleep disruption at 6 months. While we confirm that sleep disruption is a driver of Tau hyperphosphorylation in neurons of the locus ceruleus, we were unable to show any causal link between sleep loss and Tau burden in forebrain synapses. Despite the finding that hyperarousal appears earlier in females, female cognition was resilient to the effects of sleep disruption. We conclude sleep disruption interacts with the synaptic Tau burden to accelerate the onset of cognitive decline with greater vulnerability in males.

Evaluating Fatty Acid Amide Hydrolase as a Suitable Target for Sleep Promotion in a Transgenic TauP301S Mouse Model of Neurodegeneration.

Sleep disruption is an expected component of aging and neurodegenerative conditions, including Alzheimer's disease (AD). Sleep disruption has been demonstrated as a driver of AD pathology and cognitive decline. Therefore, treatments designed to maintain sleep may be effective in slowing or halting AD progression. However, commonly used sleep aid medications are associated with an increased risk of AD, highlighting the need for sleep aids with novel mechanisms of action. The endocannabinoid system holds promise as a potentially effective and novel sleep-enhancing target. By using pharmacology and genetic knockout strategies, we evaluated fatty acid amide hydrolase (FAAH) as a therapeutic target to improve sleep and halt disease progression in a transgenic Tau P301S (PS19) model of Tauopathy and AD. We have recently shown that PS19 mice exhibit sleep disruption in the form of dark phase hyperarousal as an early symptom that precedes robust Tau pathology and cognitive decline. Acute FAAH inhibition with PF3845 resulted in immediate improvements in sleep behaviors in male and female PS19 mice, supporting FAAH as a potentially suitable sleep-promoting target. Moreover, sustained drug dosing for 5-10 days resulted in maintained improvements in sleep. To evaluate the effect of chronic FAAH inhibition as a possible therapeutic strategy, we generated FAAH-/- PS19 mice models. Counter to our expectations, FAAH knockout did not protect PS19 mice from progressive sleep loss, neuroinflammation, or cognitive decline. Our results provide support for FAAH as a novel target for sleep-promoting therapies but further indicate that the complete loss of FAAH activity may be detrimental.

Prenatal stress unmasks behavioral phenotypes in genetic mouse models of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are complex conditions characterized by heterogeneous clinical profiles and symptoms that arise in infancy and childhood. NDDs are often attributed to a complicated interaction between genetic risk and environmental factors, suggesting a need for preclinical models reflecting the combined impact of heritable susceptibility and environmental effects. A notable advantage of "two-hit" models is the power to reveal underlying vulnerability that may not be detected in studies employing only genetic or environmental alterations. In this review, we summarize existing literature that investigates detrimental interactions between prenatal stress (PNS) and genes associated with NDDs, with a focus on behavioral phenotyping approaches in mouse models. A challenge in determining the overall role of PNS exposure in genetic models is the diversity of approaches for inducing stress, variability in developmental timepoints for exposure, and differences in phenotyping regimens across laboratories. Identification of optimal stress protocols and critical windows for developmental effects would greatly improve the use of PNS in gene × environment mouse models of NDDs.

Neonatal Behavioral Screen for Mouse Models of Neurodevelopmental Disorders.

Behavioral phenotyping approaches for neonatal mice are important for investigating early alterations in brain development and function, relevant to neurodevelopmental disorders in humans. This chapter describes a behavioral screen that can provide an overall profile of function across the neonatal and preweaning period while also minimizing pup stress and disturbance of the maternal environment. Testing begins when mice are between 6 and 8 days in age, with additional evaluations at discrete time points until postnatal day (PD) 20-21, using tests for negative geotaxis, surface righting reflex, activity in an open field, acoustic startle responses and sensorimotor gating, and limb clasp.

Sleep disruption precedes forebrain synaptic Tau burden and contributes to cognitive decline in a sex-dependent manner in the P301S Tau transgenic mouse model.

Background: Sleep is an essential process that supports brain health and cognitive function in part through the modification of neuronal synapses. Sleep disruption, and impaired synaptic processes, are common features in neurodegenerative diseases, including Alzheimer's disease (AD). However, the casual role of sleep disruption in disease progression is not clear. Neurofibrillary tangles, made from hyperphosphorylated and aggregated Tau protein, form one of the major hallmark pathologies seen in AD and contribute to cognitive decline, synapse loss and neuronal death.Tau has been shown to aggregate in synapses which may impair restorative synapse processes occurring during sleep. However, it remains unclear how sleep disruption and synaptic Tau pathology interact to drive cognitive decline. It is also unclear whether the sexes show differential vulnerability to the effects of sleep loss in the context of neurodegeneration. Methods: We used a piezoelectric home-cage monitoring system to measure sleep behavior in 3-11month-old transgenic hTau P301S Tauopathy model mice (PS19) and littermate controls of both sexes. Subcellular fractionation and Western blot was used to examine Tau pathology in mouse forebrain synapse fractions. To examine the role of sleep disruption in disease progression, mice were exposed to acute or chronic sleep disruption. The Morris water maze test was used to measure spatial learning and memory performance. Results: PS19 mice exhibited a selective loss of sleep during the dark phase, referred to as hyperarousal, as an early symptom with an onset of 3months in females and 6months in males. At 6months, forebrain synaptic Tau burden did not correlate with sleep measures and was not affected by acute or chronic sleep disruption. Chronic sleep disruption accelerated the onset of decline of hippocampal spatial memory in PS19 males, but not females. Conclusions: Dark phase hyperarousal is an early symptom in PS19 mice that precedes robust Tau aggregation. We find no evidence that sleep disruption is a direct driver of Tau pathology in the forebrain synapse. However, sleep disruption synergized with Tau pathology to accelerate the onset of cognitive decline in males. Despite the finding that hyperarousal appears earlier in females, female cognition was resilient to the effects of sleep disruption.

RNA-binding deficient TDP-43 drives cognitive decline in a mouse model of TDP-43 proteinopathy.

TDP-43 proteinopathies including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by aggregation and mislocalization of the nucleic acid-binding protein TDP-43 and subsequent neuronal dysfunction. Here, we developed endogenous models of sporadic TDP-43 proteinopathy based on the principle that disease-associated TDP-43 acetylation at lysine 145 (K145) alters TDP-43 conformation, impairs RNA-binding capacity, and induces downstream mis-regulation of target genes. Expression of acetylation-mimic TDP-43K145Q resulted in stress-induced nuclear TDP-43 foci and loss of TDP-43 function in primary mouse and human-induced pluripotent stem cell (hiPSC)-derived cortical neurons. Mice harboring the TDP-43K145Q mutation recapitulated key hallmarks of FTLD, including progressive TDP-43 phosphorylation and insolubility, TDP-43 mis-localization, transcriptomic and splicing alterations, and cognitive dysfunction. Our study supports a model in which TDP-43 acetylation drives neuronal dysfunction and cognitive decline through aberrant splicing and transcription of critical genes that regulate synaptic plasticity and stress response signaling. The neurodegenerative cascade initiated by TDP-43 acetylation recapitulates many aspects of human FTLD and provides a new paradigm to further interrogate TDP-43 proteinopathies.

Developmental pyrethroid exposure and age influence phenotypes in a Chd8 haploinsufficient autism mouse model.

Hundreds of genes have been associated with autism spectrum disorder (ASD), including loss-of-function mutations in chromodomain helicase DNA binding protein 8 (Chd8). Environmental factors also are implicated in autism risk and have the potential to exacerbate phenotypes in genetically sensitized backgrounds. Here we investigate transcriptional and behavioral phenotypes in a Chd8 haploinsufficient (Chd8V986*/+) mouse line exposed to the pesticide deltamethrin (DM) from conception to postnatal day 22. Vehicle-exposed Chd8V986*/+ mice displayed ASD-associated phenotypes, including anxiety-like behavior and altered sociability, replicating a previous study with this mouse line. A core set of genes was altered in Chd8V986*/+ mice at multiple ages, including Usp11, Wars2, Crlf2, and Eglf6, and proximity ligation data indicated direct binding of CHD8 to the 5' region of these genes. Moreover, oligodendrocyte and neurodegenerative transcriptional phenotypes were apparent in 12 and 18 month old Chd8V986*/+ mice. Following DM exposure, the mutant mice displayed an exacerbated phenotype in the elevated plus maze, and genes associated with vascular endothelial cells were downregulated in the cerebral cortex of older Chd8V986*/+ animals. Our study reveals a gene x environment interaction with a Chd8 haploinsufficient mouse line and points to the importance of investigating phenotypes in ASD animal models across the lifespan.

Early life sleep disruption potentiates lasting sex-specific changes in behavior in genetically vulnerable Shank3 heterozygous autism model mice.

BACKGROUND: Patients with autism spectrum disorder (ASD) experience high rates of sleep disruption beginning early in life; however, the developmental consequences of this disruption are not understood. We examined sleep behavior and the consequences of sleep disruption in developing mice bearing C-terminal truncation mutation in the high-confidence ASD risk gene SHANK3 (Shank3ΔC). We hypothesized that sleep disruption may be an early sign of developmental divergence, and that clinically relevant Shank3WT/ΔC mice may be at increased risk of lasting deleterious outcomes following early life sleep disruption. METHODS: We recorded sleep behavior in developing Shank3ΔC/ΔC, Shank3WT/ΔC, and wild-type siblings of both sexes using a noninvasive home-cage monitoring system. Separately, litters of Shank3WT/ΔC and wild-type littermates were exposed to automated mechanical sleep disruption for 7 days prior to weaning (early life sleep disruption: ELSD) or post-adolescence (PASD) or undisturbed control (CON) conditions. All groups underwent standard behavioral testing as adults. RESULTS: Male and female Shank3ΔC/ΔC mice slept significantly less than wild-type and Shank3WT/ΔC siblings shortly after weaning, with increasing sleep fragmentation in adolescence, indicating that sleep disruption has a developmental onset in this ASD model. ELSD treatment interacted with genetic vulnerability in Shank3WT/ΔC mice, resulting in lasting, sex-specific changes in behavior, whereas wild-type siblings were largely resilient to these effects. Male ELSD Shank3WT/ΔC subjects demonstrated significant changes in sociability, sensory processing, and locomotion, while female ELSD Shank3WT/ΔC subjects had a significant reduction in risk aversion. CON Shank3WT/ΔC mice, PASD mice, and all wild-type mice demonstrated typical behavioral responses in most tests. LIMITATIONS: This study tested the interaction between developmental sleep disruption and genetic vulnerability using a single ASD mouse model: Shank3ΔC (deletion of exon 21). The broader implications of this work should be supported by additional studies using ASD model mice with distinct genetic vulnerabilities. CONCLUSION: Our study shows that sleep disruption during sensitive periods of early life interacts with underlying genetic vulnerability to drive lasting and sex-specific changes in behavior. As individuals progress through maturation, they gain resilience to the lasting effects of sleep disruption. This work highlights developmental sleep disruption as an important vulnerability in ASD susceptibility.

Loss of GABA co-transmission from cholinergic neurons impairs behaviors related to hippocampal, striatal, and medial prefrontal cortex functions.

Introduction: Altered signaling or function of acetylcholine (ACh) has been reported in various neurological diseases, including Alzheimer's disease, Tourette syndrome, epilepsy among others. Many neurons that release ACh also co-transmit the neurotransmitter gamma-aminobutyrate (GABA) at synapses in the hippocampus, striatum, substantia nigra, and medial prefrontal cortex (mPFC). Although ACh transmission is crucial for higher brain functions such as learning and memory, the role of co-transmitted GABA from ACh neurons in brain function remains unknown. Thus, the overarching goal of this study was to investigate how a systemic loss of GABA co-transmission from ACh neurons affected the behavioral performance of mice. Methods: To do this, we used a conditional knock-out mouse of the vesicular GABA transporter (vGAT) crossed with the ChAT-Cre driver line to selectively ablate GABA co-transmission at ACh synapses. In a comprehensive series of standardized behavioral assays, we compared Cre-negative control mice with Cre-positive vGAT knock-out mice of both sexes. Results: Loss of GABA co-transmission from ACh neurons did not disrupt the animal's sociability, motor skills or sensation. However, in the absence of GABA co-transmission, we found significant alterations in social, spatial and fear memory as well as a reduced reliance on striatum-dependent response strategies in a T-maze. In addition, male conditional knockout (CKO) mice showed increased locomotion. Discussion: Taken together, the loss of GABA co-transmission leads to deficits in higher brain functions and behaviors. Therefore, we propose that ACh/GABA co-transmission modulates neural circuitry involved in the affected behaviors.

Sept5 deficiency exerts pleiotropic influence on affective behaviors and cognitive functions in mice.

Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression.

Over-expression of a human chromosome 22q11.2 segment including TXNRD2, COMT and ARVCF developmentally affects incentive learning and working memory in mice.

Duplication of human chromosome 22q11.2 is associated with elevated rates of mental retardation, autism and many other behavioral phenotypes. However, because duplications cover 1.5-6 Mb, the precise manner in which segments of 22q11.2 causally affect behavior is not known in humans. We have now determined the developmental impact of over-expression of an approximately 190 kb segment of human 22q11.2, which includes the genes TXNRD2, COMT and ARVCF, on behaviors in bacterial artificial chromosome (BAC) transgenic (TG) mice. BAC TG mice and wild-type (WT) mice were tested for their cognitive capacities, affect- and stress-related behaviors and motor activity at 1 and 2 months of age. An enzymatic assay determined the impact of BAC over-expression on the activity level of COMT. BAC TG mice approached a rewarded goal faster (i.e. incentive learning), but were impaired in delayed rewarded alternation during development. In contrast, BAC TG and WT mice were indistinguishable in rewarded alternation without delays, spontaneous alternation, prepulse inhibition, social interaction, anxiety-, stress- and fear-related behaviors and motor activity. Compared with WT mice, BAC TG mice had an approximately 2-fold higher level of COMT activity in the prefrontal cortex, striatum and hippocampus. These data suggest that over-expression of this 22q11.2 segment enhances incentive learning and impairs the prolonged maintenance of working memory, but has no apparent effect on working memory per se, affect- and stress-related behaviors or motor capacity. High copy numbers of this 22q11.2 segment might contribute to a highly selective set of phenotypes in learning and cognition during development.

Aplnr knockout mice display sex-specific changes in conditioned fear.

The G-protein-coupled receptor APLNR and its ligands apelin and ELABELA/TODDLER/apela comprise the apelinergic system, a signaling pathway that is critical during development and physiological homeostasis. Targeted regulation of the receptor has been proposed to treat several important diseases including heart failure, pulmonary arterial hypertension and metabolic syndrome. The apelinergic system is widely expressed within the central nervous system (CNS). However, the role of this system in the CNS has not been completely elucidated. Utilizing an Aplnr knockout mouse model, we report here results from tests of sensory ability, locomotion, reward preference, social preference, learning and memory, and anxiety. We find that knockout of Aplnr leads to significant effects on acoustic startle response and sex-specific effects on conditioned fear responses without significant changes in baseline anxiety. In particular, male Aplnr knockout mice display enhanced context- and cue-dependent fear responses. Our results complement previous reports that exogenous Apelin administration reduced conditioned fear and freezing responses in rodent models, and future studies will explore the therapeutic benefit of APLNR-targeted drugs in rodent models of PTSD.

Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome.

SPTBN1 encodes ßII-spectrin, the ubiquitously expressed ß-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal ßII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect ßII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of ßII-spectrin in the central nervous system.

Phenotyping CCL2 Containing Central Amygdala Neurons Controlling Alcohol Withdrawal-Induced Anxiety.

Chemokines such as chemokine (C-C motif) ligand 2 (CCL2) play a role in several behaviors, including anxiety-like behavior, but whether neurons are an important source of CCL2 for behavior and how neuronal CCL2 may work to affect behavior are still debated. When a herpes simplex virus (HSV) vector was used to knockdown CCL2 mRNA in neurons of the central nucleus of the amygdala (CeA) in rats experiencing multiple withdrawals from low dose ethanol, anxiety-like behavior appeared in the social interaction task. To examine this finding further Fractalkine (CX3CL1), a chemokine that is often found to have an opposing function to CCL2 was measured in these rats. Both alcohol withdrawal and CCL2 knockdown increased the levels of the anti-inflammatory protein CX3CL1. The combination of alcohol withdrawal and CCL2 knockdown decreased CX3CL1 and may alter pro-inflammatory/anti-inflammatory balance, and thus highlights the potential importance of CCL2 and CCL2/CX3CL1 balance in anxiety. To find a mechanism by which neuronal chemokines like CCL2 could affect behavior, retrograde tracing with fluorescent nanobeads was done in two brain regions associated with anxiety the bed nucleus of the stria terminalis (BNST) and the ventral periaqueductal gray (VPAG). These studies identified CeA projection neurons to these brain regions that contain CCL2. To demonstrate that CCL2 can be transported via axons to downstream brain regions, the axonal transport blocker, colchicine, was given and 24 h later, the accumulation of CCL2 in CeA neuronal cell bodies was found. Finally, CCL2 in CeA neurons was localized to the synapse using confocal microscopy with enhanced resolution following deconvolution and electron microscopy, which along with the other evidence suggests that CCL2 may be transported down axons in CeA neurons and released from nerve terminals perhaps into brain regions like the BNST and VPAG to affect behaviors such as anxiety. These results suggest that neurons are an important target for chemokine research related to behavior.