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Microglia are increasingly recognized to contribute to brain health and disease. Preclinical studies using laboratory rodents are essential to advance our understanding of the physiological and pathophysiological roles of these cells in the central nervous system. Rodents are nocturnal animals, and they are mostly maintained in a defined light-dark cycle within animal facilities, with many laboratories investigating the molecular and functional profiles of microglia exclusively during the animals' light (sleep) phase. However, only a few studies have considered possible differences in microglial functions between the active and sleep phases. Based on initial evidence suggesting that microglial intrinsic clock genes can affect their phenotypes, we sought to investigate differences in transcriptional, proteotype and functional profiles of microglia between light (sleep) and dark (active) phases, and how these changes are affected in pathological models. We found marked transcriptional and proteotype differences between microglia harvested from male mice during the light or dark phase. Amongst others, these differences related to genes and proteins associated with immune responses, motility, and phagocytosis, which were reflected by functional alterations in microglial synaptic pruning and response to bacterial stimuli. Possibly accounting for such changes, we found RNA and protein regulation in SWI/SNF and NuRD chromatin remodeling complexes between light and dark phases. Importantly, we also show that the time of microglial sample collection influences the nature of microglial transcriptomic changes in a model of immune-mediated neurodevelopmental disorders. Our findings emphasize the importance of considering diurnal factors in studying microglial cells and indicate that implementing a circadian perspective is pivotal for advancing our understanding of their physiological and pathophysiological roles in brain health and disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Depression during pregnancy is detrimental for the wellbeing of the expectant mother and can exert long-term consequences on the offspring's development and mental health. In this context, both the gestational environment and the postpartum milieu may be negatively affected by the depressive pathology. It is, however, challenging to assess whether the contributions of prenatal and postnatal depression exposure are distinct, interactive, or cumulative, as it is unclear whether antenatal effects are due to direct effects on fetal development or because antenatal symptoms continue postnatally. Preclinical models have sought to answer this question by implementing stressors that induce a depressive-like state in the dams during pregnancy and studying the effects on the offspring. The aim of our present study was to disentangle the contribution of direct stress in utero from possible changes in maternal behavior in a novel model of preconceptional stress based on social isolation rearing (SIR). Using a cross-fostering paradigm in this model, we show that while SIR leads to subtle changes in maternal behavior, the behavioral changes observed in the offspring are driven by a complex interaction between sex, and prenatal and postnatal maternal factors. Indeed, male offspring are more sensitive to the prenatal environment, as demonstrated by behavioral and transcriptional changes driven by their birth mother, while females are likely affected by more complex interactions between the pre and the postpartum milieu, as suggested by the important impact of their surrogate foster mother. Taken together, our findings suggest that male and female offspring have different time-windows and behavioral domains of susceptibility to maternal preconceptional stress, and thus underscore the importance of including both sexes when investigating the mechanisms that mediate the negative consequences of exposure to such stressor., Competing Interests: None, (© 2024 The Authors.)

Antenatal psychopathology negatively affects obstetric outcomes and exerts long-term consequences on the offspring's wellbeing and mental health. However, the precise mechanisms underlying these associations remain largely unknown. Here, we present a novel model system in mice that allows for experimental investigations into the effects of antenatal depression-like psychopathology and for evaluating the influence of maternal pharmacological treatments on long-term outcomes in the offspring. This model system in based on rearing nulliparous female mice in social isolation prior to mating, leading to a depressive-like state that is initiated before and continued throughout pregnancy. Using this model, we show that the maternal depressive-like state induced by social isolation can be partially rescued by chronic treatment with the selective serotonin reuptake inhibitor, fluoxetine (FLX). Moreover, we identify numerous and partly sex-dependent behavioral and molecular abnormalities, including increased anxiety-like behavior, cognitive impairments and alterations of the amygdalar transcriptome, in offspring born to socially isolated mothers relative to offspring born to mothers that were maintained in social groups prior to conception. We also found that maternal FLX treatment was effective in preventing some of the behavioral and molecular abnormalities emerging in offspring born to socially isolated mothers. Taken together, our findings suggest that the presence of a depressive-like state during preconception and pregnancy has sex-dependent consequences on brain and behavioral functions in the offspring. At the same time, our study highlights that FLX treatment in dams with a depression-like state can prevent abnormal behavioral development in the offspring., (© 2021. The Author(s).)

BI 409306, a phosphodiesterase-9 inhibitor under development for treatment of schizophrenia and attenuated psychosis syndrome (APS), promotes synaptic plasticity and cognition. Here, we explored the effects of BI 409306 treatment in the polyriboinosinic-polyribocytidilic acid (poly[I:C])-based mouse model of maternal immune activation (MIA), which is relevant to schizophrenia and APS. In Study 1, adult offspring received BI 409306 0.2, 0.5, or 1 mg/kg or vehicle to establish an active dose. In Study 2, adult offspring received BI 409306 1 mg/kg and/or risperidone 0.025 mg/kg, risperidone 0.05 mg/kg, or vehicle, to evaluate BI 409306 as add-on to standard therapy for schizophrenia. In Study 3, offspring received BI 409306 1 mg/kg during adolescence only, or continually into adulthood to evaluate preventive effects of BI 409306. We found that BI 409306 significantly mitigated MIA-induced social interaction deficits and amphetamine-induced hyperlocomotion, but not prepulse inhibition impairments, in a dose-dependent manner (Study 1). Furthermore, BI 409306 1 mg/kg alone or in combination with risperidone 0.025 mg/kg significantly reversed social interaction deficits and attenuated amphetamine-induced hyperlocomotion in MIA offspring (Study 2). Finally, we revealed that BI 409306 1 mg/kg treatment restricted to adolescence prevented adult deficits in social interaction, whereas continued treatment into adulthood also significantly reduced amphetamine-induced hyperlocomotion (Study 3). Taken together, our findings suggest that symptomatic treatment with BI 409306 can restore social interaction deficits and dopaminergic dysfunctions in a MIA model of neurodevelopmental disruption, lending preclinical support to current clinical trials of BI 409306 in patients with schizophrenia. Moreover, BI 409306 given during adolescence has preventive effects on adult social interaction deficits in this model, supporting its use in people with APS.

The mitochondrial protein, translocator protein (TSPO), is a widely used biomarker of neuroinflammation, but its non-selective cellular expression pattern implies roles beyond inflammatory processes. In the present study, we investigated whether neuronal activity modifies TSPO levels in the adult central nervous system. First, we used single-cell RNA sequencing to generate a cellular landscape of basal TSPO gene expression in the hippocampus of adult (12 weeks old) C57BL6/N mice, followed by confocal laser scanning microscopy to verify TSPO protein in neuronal and non-neuronal cell populations. We then quantified TSPO mRNA and protein levels after stimulating neuronal activity with distinct stimuli, including designer receptors exclusively activated by designer drugs (DREADDs), exposure to a novel environment and acute treatment with the psychostimulant drug, amphetamine. Single-cell RNA sequencing demonstrated a non-selective and multi-cellular gene expression pattern of TSPO at basal conditions in the adult mouse hippocampus. Confocal laser scanning microscopy confirmed that TSPO protein is present in neuronal and non-neuronal (astrocytes, microglia, vascular endothelial cells) cells of cortical (medial prefrontal cortex) and subcortical (hippocampus) brain regions. Stimulating neuronal activity through chemogenetic (DREADDs), physiological (novel environment exposure) or psychopharmacological (amphetamine treatment) approaches led to consistent increases in TSPO gene and protein levels in neurons, but not in microglia or astrocytes. Taken together, our findings show that neuronal activity has the potential to modify TSPO levels in the adult central nervous system. These findings challenge the general assumption that altered TSPO expression or binding unequivocally mirrors ongoing neuroinflammation and emphasize the need to consider non-inflammatory interpretations in some physiological or pathological contexts., (© 2020. The Author(s).)

The designer receptor exclusively activated by designer drugs (DREADD) system is one of the most widely used chemogenetic techniques to modulate the activity of cell populations in the brains of behaving animals. DREADDs are activated by acute or chronic administration of their ligand, clozapine-N-oxide (CNO). There is, however, a current lack of a non-invasive CNO administration technique that can control for drug timing and dosing without inducing substantial distress for the animals. Here, we evaluated whether the recently developed micropipette-guided drug administration (MDA) method, which has been used as a non-invasive and minimally stressful alternative to oral gavages, may be applied to administer CNO orally to activate DREADDs in a dosing- and timing-controlled manner. Unlike standard intraperitoneal injections, administration of vehicle substances via MDA did not elevate plasma levels of the major stress hormone, corticosterone, and did not attenuate exploratory activity in the open field test. At the same time, however, administration of CNO via MDA or intraperitoneally was equally efficient in activating hM3D Gq -expressing neurons in the medial prefrontal cortex, as evident by time-dependent increases in mRNA levels of neuronal immediate early genes (cFos, Arc and Zif268) and cFos-immunoreactive neurons. Compared to vehicle given via MDA, oral administration of CNO via MDA was also found to potently increase locomotor activity in mice that express hM3D Gq in prefrontal neurons. Taken together, our study confirms the effectiveness of CNO given orally via MDA and provides a novel method for non-stressful, yet well controllable CNO treatments in mouse DREADD systems.

The pathophysiology of dopamine dysregulation in schizophrenia involves alterations at the ventral midbrain level. Given that inflammatory mediators such as cytokines influence the functional properties of midbrain dopamine neurons, midbrain inflammation may play a role in schizophrenia by contributing to presynaptic dopamine abnormalities. Thus, we quantified inflammatory markers in dopaminergic areas of the midbrain of people with schizophrenia and matched controls. We also measured these markers in midbrain of mice exposed to maternal immune activation (MIA) during pregnancy, an established risk factor for schizophrenia and other psychiatric disorders. We found diagnostic increases in SERPINA3, TNFα, IL1β, IL6, and IL6ST transcripts in schizophrenia compared with controls (p < 0.02-0.001). The diagnostic differences in these immune markers were accounted for by a subgroup of schizophrenia cases (~ 45%, 13/28) showing high immune status. Consistent with the human cohort, we identified increased expression of immune markers in the midbrain of adult MIA offspring (SERPINA3, TNFα, and IL1β mRNAs, all p ≤ 0.01), which was driven by a subset of MIA offspring (~ 40%, 13/32) with high immune status. There were no diagnostic (human cohort) or group-wise (mouse cohort) differences in cellular markers indexing the density and/or morphology of microglia or astrocytes, but an increase in the transcription of microglial and astrocytic markers in schizophrenia cases and MIA offspring with high inflammation. These data demonstrate that immune-related changes in schizophrenia extend to dopaminergic areas of the midbrain and exist in the absence of changes in microglial cell number, but with putative evidence of microglial and astrocytic activation in the high immune subgroup. MIA may be one of the contributing factors underlying persistent neuroimmune changes in the midbrain of people with schizophrenia.

Epigenetic modifications are increasingly recognized to play a role in the etiology and pathophysiology of schizophrenia and other psychiatric disorders with developmental origins. Here, we summarize clinical and preclinical findings of epigenetic alterations in schizophrenia and relevant disease models and discuss their putative origin. Recent findings suggest that certain schizophrenia risk loci can influence stochastic variation in gene expression through epigenetic processes, highlighting the intricate interaction between genetic and epigenetic control of neurodevelopmental trajectories. In addition, a substantial portion of epigenetic alterations in schizophrenia and related disorders may be acquired through environmental factors and may be manifested as molecular "scars." Some of these scars can influence brain functions throughout the entire lifespan and may even be transmitted across generations via epigenetic germline inheritance. Epigenetic modifications, whether caused by genetic or environmental factors, are plausible molecular sources of phenotypic heterogeneity and offer a target for therapeutic interventions. The further elucidation of epigenetic modifications thus may increase our knowledge regarding schizophrenia's heterogeneous etiology and pathophysiology and, in the long term, may advance personalized treatments through the use of biomarker-guided epigenetic interventions., (Copyright © 2020 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Infectious or noninfectious maternal immune activation (MIA) is an environmental risk factor for psychiatric and neurological disorders with neurodevelopmental etiologies. Whilst there is increasing evidence for significant health consequences, the effects of MIA on the offspring appear to be variable. Here, we aimed to identify and characterize subgroups of isogenic mouse offspring exposed to identical MIA, which was induced in C57BL6/N mice by administration of the viral mimetic, poly(I:C), on gestation day 12. Cluster analysis of behavioral data obtained from a first cohort containing >150 MIA and control offspring revealed that MIA offspring could be stratified into distinct subgroups that were characterized by the presence or absence of multiple behavioral dysfunctions. The two subgroups also differed in terms of their transcriptional profiles in cortical and subcortical brain regions and brain networks of structural covariance, as measured by ex vivo structural magnetic resonance imaging (MRI). In a second, independent cohort containing 50 MIA and control offspring, we identified a subgroup of MIA offspring that displayed elevated peripheral production of innate inflammatory cytokines, including IL-1β, IL-6, and TNF-α, in adulthood. This subgroup also showed significant impairments in social approach behavior and sensorimotor gating, whereas MIA offspring with a low inflammatory cytokine status did not. Taken together, our results highlight the existence of subgroups of MIA-exposed offspring that show dissociable behavioral, transcriptional, brain network, and immunological profiles even under conditions of genetic homogeneity. These data have relevance for advancing our understanding of the variable neurodevelopmental effects induced by MIA and for biomarker-guided approaches in preclinical psychiatric research.

Prenatal exposure to infectious and/or inflammatory insults is increasingly recognized to contribute to the etiology of psychiatric disorders with neurodevelopmental components. Recent research using animal models suggests that maternal immune activation (MIA) can induce transgenerational effects on brain and behavior, possibly through epigenetic mechanisms. Using a mouse model of MIA that is based on gestational treatment with the viral mimeticpoly(I:C) (= polyriboinosinic-polyribocytidilic acid), the present study explored whether the transgenerational effects of MIA are extendable to dopaminergic dysfunctions. We show that the direct descendants born to poly(I:C)-treated mothers display signs of hyperdopaminergia, as manifested by a potentiated sensitivity to the locomotor-stimulating effects of amphetamine (Amph) and increased expression of tyrosine hydroxylase (Th) in the adult ventral midbrain. In stark contrast, second- and third-generation offspring of MIA-exposed ancestors displayed blunted locomotor responses to Amph and reduced expression of Th. Furthermore, we found increased DNA methylation at the promoter region of the dopamine-specifying factor, nuclear receptor-related 1 protein (Nurr1), in the sperm of first-generation MIA offspring and in the ventral midbrain of second-generation offspring of MIA-exposed ancestors. The latter effect was further accompanied by reduced mRNA levels of Nurr1 in this brain region. Together, our results suggest that MIA has the potential to modify dopaminergic functions across multiple generations with opposite effects in the direct descendants and their progeny. The presence of altered DNA methylation in the sperm of MIA-exposed offspring highlights the possibility that epigenetic processes in the male germline play a role in the transgenerational effects of MIA.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

When considering neurodevelopmental disorders (NDDs), Schizophrenia (SZ) and Autism Spectrum Disorder (ASD) are considered to be among the most severe in term of prevalence, morbidity and impact on the society. Similar features and overlapping symptoms have been observed at multiple levels, suggesting common pathophysiological bases. Indeed, recent genome-wide association studies (GWAS) and epidemiological data report shared vulnerability genes and environmental triggers across the two disorders. In this review, we will discuss the possible biological mechanisms, including glutamatergic and GABAergic neurotransmissions, inflammatory signals and oxidative stress related systems, which are targeted by adverse environmental exposures and that have been associated with the development of SZ and ASD. We will also discuss the emerging role of the gut microbiome as possible interplay between environment, immune system and brain development. Finally, we will describe the involvement of epigenetic mechanisms in the maintenance of long-lasting effects of adverse environments early in life. This will allow us to better understand the pathophysiology of these NDDs, and also to identify novel targets for future treatment strategies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pharmacological treatments in laboratory rodents remain a cornerstone of preclinical psychopharmacological research and drug development. There are numerous ways in which acute or chronic pharmacological treatments can be implemented, with each method having certain advantages and drawbacks. Here, we describe and validate a novel treatment method in mice, which we refer to as the micropipette-guided drug administration (MDA) procedure. This administration method is based on a sweetened condensed milk solution as a vehicle for pharmacological substances, which motivates the animals to consume vehicle and/or drug solutions voluntarily in the presence of the experimenter. In a proof-of-concept study, we show that the pharmacokinetic profiles of the atypical antipsychotic drug, risperidone, were similar whether administered via the MDA procedure or via the conventional oral gavage method. Unlike the latter, however, MDA did not induce the stress hormone, corticosterone. Furthermore, we assessed the suitability and validity of the MDA method in a mouse model of maternal immune activation, which is frequently used as a model of immune-mediated neurodevelopmental disorders. Using this model, we found that chronic treatment (>4 weeks, once per day) with risperidone via MDA led to a dose-dependent mitigation of MIA-induced social interaction deficits and amphetamine hypersensitivity. Taken together, the MDA procedure described herein represents a novel pharmacological administration method for per os treatments in mice that is easy to implement, cost effective, non-invasive, and less stressful for the animals than conventional oral gavage methods., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Maternal immune activation (MIA) models that are based on administration of the viral mimetic, poly(I:C), are widely used as experimental tools to study neuronal and behavioral dysfunctions in relation to immune-mediated neurodevelopmental disorders and mental illnesses. Evidence from investigations in non-pregnant rodents suggests that different poly(I:C) products can vary in terms of their immunogenicity, even if they are obtained from the same vendor. The present study aimed at extending these findings to pregnant mice, while also controlling various poly(I:C) products for potential contamination with lipopolysaccharide (LPS). We found significant variability between different batches of poly(I:C) potassium salt obtained from the same vendor (Sigma-Aldrich) in terms of the relative amount of dsRNA fragments in the high molecular weight range (1000-6000 nucleotides long) and with regards to their effects on maternal thermoregulation and immune responses in maternal plasma, placenta and fetal brain. Batches of poly(I:C) potassium salt containing larger amounts of high molecular weight fragments induced more extensive effects on thermoregulation and immune responses compared to batches with minimal amounts of high molecular weight fragments. Consistent with these findings, poly(I:C) enriched for high molecular weight dsRNA (HMW) caused larger maternal and placental immune responses compared to low molecular weight (LMW) poly(I:C). These variable effects were unrelated to possible LPS contamination. Finally, we found marked variability between different batches of the poly(I:C) potassium salt in terms of their effects on spontaneous abortion rates. This batch-to-batch variability was confirmed by three independent research groups using distinct poly(I:C) administration protocols in mice. Taken together, the present data confirm that different poly(I:C) products can induce varying immune responses and can differentially affect maternal physiology and pregnancy outcomes. It is therefore pivotal that researchers working with poly(I:C)-based MIA models ascertain and consider the precise molecular composition and immunogenicity of the product in use. We recommend the establishment of reference databases that combine phenotype data with empirically acquired quality information, which can aid the design, implementation and interpretation of poly(I:C)-based MIA models., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Brain calcifications are commonly detected in aged individuals and accompany numerous brain diseases, but their functional importance is not understood. In cases of primary familial brain calcification, an autosomally inherited neuropsychiatric disorder, the presence of bilateral brain calcifications in the absence of secondary causes of brain calcification is a diagnostic criterion. To date, mutations in five genes including solute carrier 20 member 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), myogenesis regulating glycosidase (MYORG), platelet-derived growth factor B (PDGFB) and platelet-derived growth factor receptor β (PDGFRB), are considered causal. Previously, we have reported that mutations in PDGFB in humans are associated with primary familial brain calcification, and mice hypomorphic for PDGFB (Pdgfbret/ret) present with brain vessel calcifications in the deep regions of the brain that increase with age, mimicking the pathology observed in human mutation carriers. In this study, we characterize the cellular environment surrounding calcifications in Pdgfbret/ret animals and show that cells around vessel-associated calcifications express markers for osteoblasts, osteoclasts and osteocytes, and that bone matrix proteins are present in vessel-associated calcifications. Additionally, we also demonstrate the osteogenic environment around brain calcifications in genetically confirmed primary familial brain calcification cases. We show that calcifications cause oxidative stress in astrocytes and evoke expression of neurotoxic astrocyte markers. Similar to previously reported human primary familial brain calcification cases, we describe high interindividual variation in calcification load in Pdgfbret/ret animals, as assessed by ex vivo and in vivo quantification of calcifications. We also report that serum of Pdgfbret/ret animals does not differ in calcification propensity from control animals and that vessel calcification occurs only in the brains of Pdgfbret/ret animals. Notably, ossification of vessels and astrocytic neurotoxic response is associated with specific behavioural and cognitive alterations, some of which are associated with primary familial brain calcification in a subset of patients., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Hemizygous microdeletion at the chromosomal locus 22q11.2 is a copy number variation with strong genetic linkage to schizophrenia and related disorders. This association, along with its phenotypic overlap with the 22q11.2 microdeletion syndrome, has motivated the establishment of Df[h22q11]/+ mice, in which the human 22q11.2 orthologous region is deleted. Previous investigations using this model showed the presence of reduced prepulse inhibition (PPI) of the acoustic startle reflex, a form of sensorimotor gating known to be impaired in a number of psychiatric disorders. Concomitantly to reduced PPI, however, Df[h22q11]/+ mice are also characterized by a robust increase in baseline startle reactivity, which may complicate or confound the interpretation of PPI. Therefore, the present study re-examined the relationship between acoustic startle reactivity and PPI in this mouse model. We found that while PPI is reduced in Df[h22q11]/+ mice when using its relative indexation (ie, % PPI), this deficit is no longer apparent when using the absolute quantification, that is, the direct comparison between pulse-alone and prepulse-plus-pulse conditions with successively increasing prepulse intensities. We further identified marked negative correlations between % PPI and startle reactivity in Df[h22q11]/+ mice. Moreover, when stratifying Df[h22q11]/+ mice into subgroups displaying low- and high-startle reactivity, only the latter subgroup displayed a significant reduction in % PPI. Collectively, our data suggest that alterations in baseline startle reactivity can confound the outcomes and interpretation of PPI in this mouse model of the human 22q11.2 microdeletion syndrome., (© 2018 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Different timing and light phases are critical factors in behavioural neuroscience, which can greatly affect the experimental outcomes of the performed tests. Despite the fact that time of testing is one of the most common factors that varies across behavioural laboratories, knowledge about the consequences of testing time on behavioural readouts is limited. Thus, in this study we systematically assessed the effect of this factor on the readout of a variety of elementary and recurrent behavioural paradigms in C57Bl/6 mice. Furthermore, we investigated potential neuronal correlates of this phenomenon by analysing how testing time influences the expression pattern of genes relevant for neuronal activation functions and the control of brain circadian rhythms. We show that animals tested in the light phase display reduced social approach behaviour and sensorimotor gating and increased locomotor activity, whereas anxiety-related behaviour and working memory are not affected. In addition, animals tested in the light phase also exhibit increased locomotor response to systemic amphetamine treatment, which is paralleled by alterations in the expression patterns of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the Nucleus Accumbens (NAc) and/or Midbrain (Mid). Lastly, we observed that neuronal activation, indexed by the gene expression levels of cFos, was increased in the NAc and Mid of animals tested during the light phase. Our data thus suggest that daily alterations in gene expression in mesolimbic brain structures might be involved in the different behavioural responses of mice tested in the light- versus the dark-phase. At the same time, our study adds further weight to the notion that the specific timing of testing can indeed strongly affect the readout of a given test. As comparison and reproducibility of findings is pivotal in science, experimental protocols should be clarified in detail to allow appropriate data comparison across different laboratories., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rodent models of maternal immune activation (MIA) are increasingly used as experimental tools to study neuronal and behavioral dysfunctions in relation to infection-mediated neurodevelopmental disorders. One of the most widely used MIA models is based on gestational administration of poly(I:C) (= polyriboinosinic-polyribocytdilic acid), a synthetic analog of double-stranded RNA that induces a cytokine-associated viral-like acute phase response. The effects of poly(I:C)-induced MIA on phenotypic changes in the offspring are known to be influenced by various factors, including the precise prenatal timing, genetic background, and immune stimulus intensity. Thus far, however, it has been largely ignored whether differences in the basic type of laboratory housing can similarly affect the outcomes of MIA models. Here, we examined this possibility by comparing the poly(I:C)-based MIA model in two housing systems that are commonly used in preclinical mouse research, namely the open cage (OC) and individually ventilated cage (IVC) systems. Pregnant C57BL6/N mice were kept in OCs or IVCs and treated with a low (1 mg/kg, i.v.) or high (5 mg/kg, i.v.) dose of poly(I:C), or with control vehicle solution. MIA or control treatment was induced on gestation day (GD) 9 or 12, and the resulting offspring were raised and maintained in OCs or IVCs until adulthood for behavioral testing. An additional cohort of dams was used to assess the influence of the different caging systems on poly(I:C)-induced cytokine and stress responses in the maternal plasma. Maternal poly(I:C) administration on GD9 caused a dose-dependent increase in spontaneous abortion in IVCs but not in OCs, whereas MIA in IVC systems during a later gestational time-point (GD12) did not affect pregnancy outcomes. Moreover, the precise type of caging system markedly affected maternal cytokines and chemokines at basal states and in response to poly(I:C) and further influenced the maternal levels of the stress hormone, corticosterone. The efficacy of MIA to induce deficits in working memory, social interaction, and sensorimotor gating in the adult offspring was influenced by the different housing conditions, the dosing of poly(I:C), and the precise prenatal timing. Taken together, the present study identifies the basic type of caging system as a novel factor that can confound the outcomes of MIA in mice. Our findings thus urge the need to consider and report the kind of laboratory housing systems used to implement MIA models. Providing this information seems pivotal to yield reproducible results in these models., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The medial prefrontal cortex (mPFC), master regulator of higher-order cognitive functions, is the only brain region that matures until late adolescence. During this period, the mPFC is sensitive to stressful events or suboptimal nutrition. For instance, high-fat diet (HFD) feeding during adolescence markedly impairs prefrontal-dependent cognition. It also provokes multiple changes at the cellular and synaptic scales within the mPFC, suggesting that major transcriptional events are elicited by HFD during this maturational period. The nature of this transcriptional reprogramming remains unknown, but may include epigenetic processes, in particular microRNAs, known to directly regulate synaptic functions. We used high-throughput screening in the adolescent mouse mPFC and identified 38 microRNAs differentially regulated by HFD, in particular mir-30e-5p. We used a luciferase assay to confirm the functional effect of mir-30e-5p on a chosen target: Ephrin-A3. Using global pathway analyses of predicted microRNA targets, we identified biological pathways putatively affected by HFD. Axon guidance was the top-1 pathway, validated by identifying gene expression changes of axon guidance molecules following HFD. Our findings delineate major microRNA transcriptional reprogramming within the mPFC induced by adolescent HFD. These results will help understanding the contribution of microRNAs in the emergence of cognitive deficits following early-life environmental events.

Reduced activity of vagal efferents has long been implicated in schizophrenia and appears to be responsible for diminished parasympathetic activity and associated peripheral symptoms such as low heart rate variability and cardiovascular complications in affected individuals. In contrast, only little attention has been paid to the possibility that impaired afferent vagal signaling may be relevant for the disorder's pathophysiology as well. The present study explored this hypothesis using a model of subdiaphragmatic vagal deafferentation (SDA) in male rats. SDA represents the most complete and selective vagal deafferentation method existing to date as it leads to complete disconnection of all abdominal vagal afferents while sparing half of the abdominal vagal efferents. Using next-generation mRNA sequencing, we show that SDA leads to brain transcriptional changes in functional networks annotating with schizophrenia. We further demonstrate that SDA induces a hyperdopaminergic state, which manifests itself as increased sensitivity to acute amphetamine treatment and elevated accumbal levels of dopamine and its major metabolite, 3,4-dihydroxyphenylacetic acid. Our study also shows that SDA impairs sensorimotor gating and the attentional control of associative learning, which were assessed using the paradigms of prepulse inhibition and latent inhibition, respectively. These data provide converging evidence suggesting that the brain transcriptome, dopamine neurochemistry, and behavioral functions implicated in schizophrenia are subject to visceral modulation through abdominal vagal afferents. Our findings may encourage the further establishment and use of therapies for schizophrenia that are based on vagal interventions. SIGNIFICANCE STATEMENT The present work provides a better understanding of how disrupted vagal afferent signaling can contribute to schizophrenia-related brain and behavioral abnormalities. More specifically, it shows that subdiaphragmatic vagal deafferentation (SDA) in rats leads to (1) brain transcriptional changes in functional networks related to schizophrenia, (2) increased sensitivity to dopamine-stimulating drugs and elevated dopamine levels in the nucleus accumbens, and (3) impairments in sensorimotor gating and the attentional control of associative learning. These findings may encourage the further establishment of novel therapies for schizophrenia that are based on vagal interventions., (Copyright © 2018 the authors 0270-6474/18/381634-14$15.00/0.)

Prenatal exposure to maternal infection increases the risk of neurodevelopmental disorders, including schizophrenia and autism. The molecular processes underlying this pathological association, however, are only partially understood. Here, we combined unbiased genome-wide transcriptional profiling with follow-up epigenetic analyses and structural magnetic resonance imaging to explore convergent molecular and neuromorphological alterations in corticostriatal areas of adult offspring exposed to prenatal immune activation. Genome-wide transcriptional profiling revealed that prenatal immune activation caused a differential expression of 116 and 251 genes in the medial prefrontal cortex and nucleus accumbens, respectively. A large part of genes that were commonly affected in both brain areas were related to myelin functionality and stability. Subsequent epigenetic analyses indicated that altered DNA methylation of promoter regions might contribute to the differential expression of myelin-related genes. Quantitative relaxometry comparing T1, T2, and myelin water fraction revealed sparse increases in T1 relaxation times and consistent reductions in T2 relaxation times. Together, our multi-system approach demonstrates that prenatal viral-like immune activation causes myelin-related transcriptional and epigenetic changes in corticostriatal areas. Even though these abnormalities do not seem to be associated with overt white matter reduction, they may provide a molecular mechanism whereby prenatal infection can impair myelin functionality and stability., (© The Author 2017. Published by Oxford University Press.)

Background: Prenatal exposure to infectious or inflammatory insults increases the risk of neurodevelopmental disorders. Using a well-established mouse model of prenatal viral-like immune activation, we examined whether this pathological association involves genome-wide DNA methylation differences at single nucleotide resolution., Methods: Prenatal immune activation was induced by maternal treatment with the viral mimetic polyriboinosinic-polyribocytidylic acid in middle or late gestation. Following behavioral and cognitive characterization of the adult offspring (n = 12 per group), unbiased capture array bisulfite sequencing was combined with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitative real-time polymerase chain reaction analyses to quantify DNA methylation changes and transcriptional abnormalities in the medial prefrontal cortex of immune-challenged and control offspring. Gene ontology term enrichment analysis was used to explore shared functional pathways of genes with differential DNA methylation., Results: Adult offspring of immune-challenged mothers displayed hyper- and hypomethylated CpGs at numerous loci and at distinct genomic regions, including genes relevant for gamma-aminobutyric acidergic differentiation and signaling (e.g., Dlx1, Lhx5, Lhx8), Wnt signaling (Wnt3, Wnt8a, Wnt7b), and neural development (e.g., Efnb3, Mid1, Nlgn1, Nrxn2). Altered DNA methylation was associated with transcriptional changes of the corresponding genes. The epigenetic and transcriptional effects were dependent on the offspring's age and were markedly influenced by the precise timing of prenatal immune activation., Conclusions: Prenatal viral-like immune activation is capable of inducing stable DNA methylation changes in the medial prefrontal cortex. These long-term epigenetic modifications are a plausible mechanism underlying the disruption of prefrontal gene transcription and behavioral functions in subjects with prenatal infectious histories., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The translational assessment of mechanisms underlying cognitive functions using touchscreen-based approaches for rodents is growing in popularity. In these paradigms, daily training is usually accompanied by extended food restriction to maintain animals' motivation to respond for rewards. Here, we show a transient elevation in stress hormone levels due to food restriction and touchscreen training, with subsequent adaptation effects, in fecal corticosterone metabolite concentrations, indicating effective coping in response to physical and psychological stressors. Corticosterone concentrations of experienced but training-deprived mice revealed a potential anticipation of task exposure, indicating a possible temporary environmental enrichment-like effect caused by cognitive challenge. Furthermore, the analyses of immediate early gene (IEG) immunoreactivity in the hippocampus revealed alterations in Arc, c-Fos and zif268 expression immediately following training. In addition, BDNF expression was altered as a function of satiation state during food restriction. These findings suggest that standard protocols for touchscreen-based training induce changes in hippocampal neuronal activity related to satiation and learning that should be considered when using this paradigm., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Prenatal exposure to infection and/or inflammation is increasingly recognized to play an important role in neurodevelopmental brain disorders. It has recently been postulated that prenatal immune activation, especially when occurring during late gestational stages, may also induce pathological brain aging via sustained effects on systemic and central inflammation. Here, we tested this hypothesis using an established mouse model of exposure to viral-like immune activation in late pregnancy., Methods: Pregnant C57BL6/J mice on gestation day 17 were treated with the viral mimetic polyriboinosinic-polyribocytidilic acid (poly(I:C)) or control vehicle solution. The resulting offspring were first tested using cognitive and behavioral paradigms known to be sensitive to hippocampal damage, after which they were assigned to quantitative analyses of inflammatory cytokines, microglia density and morphology, astrocyte density, presynaptic markers, and neurotrophin expression in the hippocampus throughout aging (1, 5, and 22 months of age)., Results: Maternal poly(I:C) treatment led to a robust increase in inflammatory cytokine levels in late gestation but did not cause persistent systemic or hippocampal inflammation in the offspring. The late prenatal manipulation also failed to cause long-term changes in microglia density, morphology, or activation, and did not induce signs of astrogliosis in pubescent, adult, or aged offspring. Despite the lack of persistent inflammatory or glial anomalies, offspring of poly(I:C)-exposed mothers showed marked and partly age-dependent deficits in hippocampus-regulated cognitive functions as well as impaired hippocampal synaptophysin and brain-derived neurotrophic factor (BDNF) expression., Conclusions: Late prenatal exposure to viral-like immune activation in mice causes hippocampus-related cognitive and synaptic deficits in the absence of chronic inflammation across aging. These findings do not support the hypothesis that this form of prenatal immune activation may induce pathological brain aging via sustained effects on systemic and central inflammation. We further conclude that poly(I:C)-based prenatal immune activation models are reliable in their effectiveness to induce (hippocampal) neuropathology across aging, but they appear unsuited for studying the role of chronic systemic or central inflammation in brain aging.

Background: Impaired γ-aminobutyric acid (GABA) signaling may contribute to the emergence of cognitive deficits and subcortical dopaminergic hyperactivity in patients with schizophrenia and related psychotic disorders. Against this background, it has been proposed that pharmacological interventions targeting GABAergic dysfunctions may prove useful in correcting such cognitive impairments and dopaminergic imbalances., Methods: Here, we explored possible beneficial effects of the benzodiazepine-positive allosteric modulator SH-053-2'F-S-CH₃, with partial selectivity at the α2, α3, and α5 subunits of the GABAA receptor in an immune-mediated neurodevelopmental disruption model. The model is based on prenatal administration of the viral mimetic polyriboinosinic-polyribocytidilic acid [poly(I:C)] in mice, which is known to capture various GABAergic, dopamine-related, and cognitive abnormalities implicated in schizophrenia and related disorders., Results: Real-time polymerase chain reaction analyses confirmed the expected alterations in GABAA receptor α subunit gene expression in the medial prefrontal cortices and ventral hippocampi of adult poly(I:C) offspring relative to control offspring. Systemic administration of SH-053-2'F-S-CH₃ failed to normalize the poly(I:C)-induced deficits in working memory and social interaction, but instead impaired performance in these cognitive and behavioral domains both in control and poly(I:C) offspring. In contrast, SH-053-2'F-S-CH₃ was highly effective in mitigating the poly(I:C)-induced amphetamine hypersensitivity phenotype without causing side effects in control offspring., Conclusions: Our preclinical data suggest that benzodiazepine-like positive allosteric modulators with activity at the α2, α3, and α5 subunits of the GABAA receptor may be particularly useful in correcting pathological overactivity of the dopaminergic system, but they may be ineffective in targeting multiple pathological domains that involve the co-existence of psychotic, social, and cognitive dysfunctions., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

The impact of early physical and social environments on life-long pathological phenotypes is well known and there is now compelling evidence that stressful experiences during gestation or early in life can lead to enhanced susceptibility to mental illness. Here, we discuss the data from preclinical studies aimed at investigating the molecular consequences of the exposure to stressful events during prenatal or early postnatal life that might contribute to later psychopathology. Particularly, we will discuss the existence of age windows of vulnerability to environmental conditions during brain maturation using as examples several studies performed with different animal models. Specifically, major deviations from normative neurobehavioural trajectories have been reported in animal models obtained following exposure to severe stress (maternal separation) ea rly in infancy or with rodent models of difficult and/or stressful pregnancies, including obstetric complications (e.g. prenatal restrain stress) and gestational exposure to infection (e.g prenatal immune challenge). These models have been associated with profound long-lasting deficits in the offspring's emotional and social behaviour, and with molecular changes associated with neuroplasticity.

Different environmental factors acting during sensitive prenatal periods can have a negative impact on neurodevelopment and predispose the individual to the development of various psychiatric conditions that often share cognitive impairments as a common component. As cognitive symptoms remain one of the most challenging and resistant aspects of mental illness to be treated pharmacologically, it is important to investigate the mechanisms underlying such cognitive deficits, with particular focus on the impact of early life adverse events that predispose the individual to mental disorders. Multiple clinical studies have, in fact, repeatedly confirmed that prenatal maternal factors, such as infection, stress or malnutrition, are pivotal in shaping behavioral and cognitive functions of the offspring, and in the past decade many preclinical studies have investigated this hypothesis. The purpose of this review is to describe recent preclinical studies aimed at dissecting the relative impact of various prenatal maternal factors on the development of cognitive impairments in offspring, focusing on animal models of prenatal stress and prenatal infection. These recent studies point to the pivotal role of prenatal stressful experiences in shaping memory and learning functions associated with specific brain structures, such as the hippocampus and the prefrontal cortex. More importantly, such experimental evidence suggests that different insults converge on similar downstream functional targets, such as cognition, which may therefore represent an endophenotype for several pathological conditions. Future studies should thus focus on investigating the mechanisms contributing to the convergent action of different prenatal insults in order to identify targets for novel therapeutic intervention., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Neuronal dysfunctions in the cortical GABAergic system have been widely documented in neuropsychiatric disorders with prenatal infectious etiologies, including schizophrenia. At least some of these abnormalities may stem from transcriptional impairments in the GABAergic transcriptome. However, the extent to which prenatal exposure to immune challenge can induce long-term alterations in GABAergic gene transcription remains largely elusive. Here, we use an established mouse model of prenatal immune activation induced by maternal gestational administration of the viral mimetic poly(I:C) (= polyriboinosinic-polyribocytidilic acid) to demonstrate that prenatal immune activation causes maturation-dependent alterations in prefrontal GABAergic gene expression. The spectrum of abnormalities included altered mRNA expression levels of enzymes regulating γ-aminobutyric acid (GABA) biosynthesis (glutamic acid decarboxylase 65-kDa [GAD65] and GAD67), vesicular GABA transporter (VGAT), alpha-subunits of the GABA(A) receptor (α2, α3, α4, and α5), and the chloride transporters sodium-potassium-chloride cotransporter 1 and potassium-chloride cotransporter 2. Additional western blot analyses confirmed the deficits in prefrontal GAD65/GAD67 and VGAT expression at the protein level. Intriguingly, the prefrontal GABAergic transcriptome was found to be more strongly affected in adult compared with peripubertal offspring born to immune-challenged mothers, and these age-dependent changes in GABAergic gene expression were paralleled by an adult onset of working memory deficiency. Collectively, our data emphasize a critical impact of prenatal immune-related insults on long-term GABAergic changes relevant to neuropsychiatric disorders with prenatal infectious etiologies, especially for those with delayed onset in early adulthood.

Prenatal maternal infection is an environmental risk factor for neurodevelopmental psychiatric illness and disease-associated cognitive impairments. Modeling this epidemiological link in animals shows that prenatal immune challenge is capable of inducing long-lasting deficits in numerous cognitive domains. Here, we combined a neonatal cross-fostering design with a mouse model of prenatal immune challenge induced by maternal gestational treatment with the viral mimetic poly(I:C) to dissect the relative contribution of prenatal and postnatal maternal effects on the offspring. We show that offspring prenatally exposed to poly(I:C) display significant impairments in spatial matching-to-position working memory and spatial novelty presence regardless of whether they are raised by gestationally immune-challenged or non-challenged control surrogate mothers. Likewise, prenatally immune challenged offspring exhibit reduced glutamic acid decarboxylase 65-kDa (GAD65) and 67-kDa (GAD67) gene expression in the adult medial prefrontal cortex and dorsal hippocampus largely independently of the postnatal rearing conditions. In addition, we confirm that being raised by a gestationally immune-challenged surrogate mother is sufficient to increase the offspring's locomotor response to systemic amphetamine treatment. Our data thus suggest that prenatal infection-induced deficits in spatial short-term memory are mediated by prenatal maternal effects on the offspring. At the same time, our study adds further weight to the notion that being reared by a surrogate mother that experienced immune activation during pregnancy may constitute a risk factor for specific dopaminergic abnormalities., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Repeated administration of the indirect dopamine receptor agonist amphetamine (AMPH) produces robust locomotor sensitization and additional behavioral abnormalities. Accumulating evidence suggests that the developmental timing of drug exposure can critically influence this effect. The present study compared the consequences of withdrawal from repeated AMPH exposure in adolescence and adulthood on information processing and locomotor sensitization in C57BL/6 mice. Animals were injected daily with AMPH (1 or 2.5 mg/kg) or vehicle on 7 consecutive days starting either from postnatal day 35 to 42, or from postnatal day 70 to 77, following which they were given a 4 week withdrawal period before behavioral and pharmacological testing commenced. We found that withdrawal from the higher dose of AMPH (2.5 mg/kg/day) given either in adolescence or adulthood similarly disrupted selective associative learning as measured by the latent inhibition paradigm. None of the AMPH withdrawal groups displayed alterations in sensorimotor gating in the form of prepulse inhibition. Withdrawal from adult AMPH exposure at both doses induced marked locomotor sensitization, whereas adolescent pre-treatment with the higher (2.5 mg/kg/day) but not lower (1 mg/kg/day) dose of AMPH potentiated the locomotor-enhancing effects of acute AMPH re-challenge. Our study suggests that withdrawal from repeated AMPH exposure in adolescence and adulthood has similar consequences on selective associative learning, but the two manipulations differ with respect to their efficacy to induce long-term locomotor sensitization to the drug. The latter finding supports the hypothesis that the precise developmental timing determines, at least in part, the impact on long-term dopamine-associated sensitization processes., (Copyright © 2012 Elsevier B.V. and ECNP. All rights reserved.)