Multiomic profiling of transcription factor binding and function in human brain.

Transcription factors (TFs) orchestrate gene expression programs crucial for brain function, but we lack detailed information about TF binding in human brain tissue. We generated a multiomic resource (ChIP-seq, ATAC-seq, RNA-seq, DNA methylation) on bulk tissues and sorted nuclei from several postmortem brain regions, including binding maps for more than 100 TFs. We demonstrate improved measurements of TF activity, including motif recognition and gene expression modeling, upon identification and removal of high TF occupancy regions. Further, predictive TF binding models demonstrate a bias for these high-occupancy sites. Neuronal TFs SATB2 and TBR1 bind unique regions depleted for such sites and promote neuronal gene expression. Binding sites for TFs, including TBR1 and PKNOX1, are enriched for risk variants associated with neuropsychiatric disorders, predominantly in neurons. This work, titled BrainTF, is a powerful resource for future studies seeking to understand the roles of specific TFs in regulating gene expression in the human brain.

Adolescent environmental enrichment induces social resilience and alters neural gene expression in a selectively bred rodent model with anxious phenotype.

Stress is a major influence on mental health status; the ways that individuals respond to or copes with stressors determine whether they are negatively affected in the future. Stress responses are established by an interplay between genetics, environment, and life experiences. Psychosocial stress is particularly impactful during adolescence, a critical period for the development of mood disorders. In this study we compared two established, selectively-bred Sprague Dawley rat lines, the "internalizing" bred Low Responder (bLR) line versus the "externalizing" bred High Responder (bHR) line, to investigate how genetic temperament and adolescent environment impact future responses to social interactions and psychosocial stress, and how these determinants of stress response interact. Male bLR and bHR rats were exposed to social and environmental enrichment in adolescence prior to experiencing social defeat and were then assessed for social interaction and anxiety-like behavior. Adolescent enrichment caused rats to display more social interaction, as well as nominally less social avoidance, less submission during defeat, and resilience to the effects of social stress on corticosterone, in a manner that seemed more notable in bLRs. For bHRs, enrichment also caused greater aggression during a neutral social encounter and nominally during defeat, and decreased anxiety-like behavior. To explore the neurobiology underlying the development of social resilience in the anxious phenotype bLRs, RNA-seq was conducted on the hippocampus and nucleus accumbens, two brain regions that mediate stress regulation and social behavior. Gene sets previously associated with stress, social behavior, aggression and exploratory activity were enriched with differential expression in both regions, with a particularly large effect on gene sets that regulate social behaviors. Our findings provide further evidence that adolescent enrichment can serve as an inoculating experience against future stressors. The ability to induce social resilience in a usually anxious line of animals by manipulating their environment has translational implications, as it underscores the feasibility of intervention strategies targeted at genetically vulnerable adolescent populations.

Resource: A Curated Database of Brain-Related Functional Gene Sets (Brain.GMT).

Transcriptional profiling has become a common tool for investigating the nervous system. During analysis, differential expression results are often compared to functional ontology databases, which contain curated gene sets representing well-studied pathways. This dependence can cause neuroscience studies to be interpreted in terms of functional pathways documented in better studied tissues (e.g., liver) and topics (e.g., cancer), and systematically emphasizes well-studied genes, leaving other findings in the obscurity of the brain "ignorome". To address this issue, we compiled a curated database of 918 gene sets related to nervous system function, tissue, and cell types ("Brain.GMT") that can be used within common analysis pipelines (GSEA, limma, edgeR) to interpret results from three species (rat, mouse, human). Brain.GMT includes brain-related gene sets curated from the Molecular Signatures Database (MSigDB) and extracted from public databases (GeneWeaver, Gemma, DropViz, BrainInABlender, HippoSeq) and published studies containing differential expression results. Although Brain.GMT is still undergoing development and currently only represents a fraction of available brain gene sets, "brain ignorome" genes are already better represented than in traditional Gene Ontology databases. Moreover, Brain.GMT substantially improves the quantity and quality of gene sets identified as enriched with differential expression in neuroscience studies, enhancing interpretation.

High emotional reactivity is associated with activation of a molecularly distinct hippocampal-amygdala circuit modulated by the glucocorticoid receptor.

Emotions are characterized not only by their valence but also by whether they are stable or labile. Yet, we do not understand the molecular or circuit mechanisms that control the dynamic nature of emotional responses. We have shown that glucocorticoid receptor overexpression in the forebrain (GRov) leads to a highly reactive mouse with increased anxiety behavior coupled with greater swings in emotional responses. This phenotype is established early in development and persists into adulthood. However, the neural circuitry mediating this lifelong emotional lability remains unknown. In the present study, optogenetic stimulation in ventral dentate gyrus (vDG) of GRov mice led to a greater range and a prolonged duration of anxiety behavior. cFos expression analysis showed that the amplified behavioral response to vDG activation in GRov mice is coupled to increased neuronal activity in specific brain regions. Relative to wild type mice, GRov mice displayed glutamatergic/GABAergic activation imbalance in ventral CA1 (vCA1) and selectively increased glutamatergic activation in the basal posterior amygdaloid complex. Moreover, forebrain GR overexpression led to increased activation of molecularly distinct subpopulations of neurons within the hippocampus and the posterior basolateral amygdala (pBLA) as evident from the increased cFos co-labeling in the calbindin1+ glutamatergic neurons in vCA1 and in the DARPP-32/Ppp1r1b+ glutamatergic neurons in pBLA. We propose that a molecularly distinct hippocampal-amygdala circuit is shaped by stress early in life and tunes the dynamics of emotional responses.

The impact of COVID-19 on a college freshman sample reveals genetic and nongenetic forms of susceptibility and resilience to stress.

Using a longitudinal approach, we sought to define the interplay between genetic and nongenetic factors in shaping vulnerability or resilience to COVID-19 pandemic stress, as indexed by the emergence of symptoms of depression and/or anxiety. University of Michigan freshmen were characterized at baseline using multiple psychological instruments. Subjects were genotyped, and a polygenic risk score for depression (MDD-PRS) was calculated. Daily physical activity and sleep were captured. Subjects were sampled at multiple time points throughout the freshman year on clinical rating scales, including GAD-7 and PHQ-9 for anxiety and depression, respectively. Two cohorts (2019 to 2021) were compared to a pre-COVID-19 cohort to assess the impact of the pandemic. Across cohorts, 26 to 40% of freshmen developed symptoms of anxiety or depression (N = 331). Depression symptoms significantly increased in the pandemic years and became more chronic, especially in females. Physical activity was reduced, and sleep was increased by the pandemic, and this correlated with the emergence of mood symptoms. While low MDD-PRS predicted lower risk for depression during a typical freshman year, this genetic advantage vanished during the pandemic. Indeed, females with lower genetic risk accounted for the majority of the pandemic-induced rise in depression. We developed a model that explained approximately half of the variance in follow-up depression scores based on psychological trait and state characteristics at baseline and contributed to resilience in genetically vulnerable subjects. We discuss the concept of multiple types of resilience, and the interplay between genetic, sex, and psychological factors in shaping the affective response to different types of stressors.

Allele biased transcription factor binding across human brain regions gives mechanistic insight into eQTLs.

Transcription Factors (TFs) influence gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Because genomic localization of TFs is in part driven by TF recognition of DNA sequence, variation in TF binding sites can disrupt TF-DNA associations and affect gene regulation. To identify variants that impact TF binding in human brain tissues, we quantified allele bias for 93 TFs analyzed with ChIP-seq experiments of multiple structural brain regions from two donors. Using graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signal between alleles at heterozygous variants within each tissue sample from each donor. Comparison of results from different brain regions within donors and the same regions between donors provided measures of allele bias reproducibility. We identified thousands of DNA variants that show reproducible bias in ChIP-seq for at least one TF. We found that alleles that are rarer in the general population were more likely than common alleles to exhibit large biases, and more frequently led to reduced TF binding. Combining ChIP-seq with RNA-seq, we identified TF-allele interaction biases with RNA bias in a phased allele linked to 6,709 eQTL variants identified in GTEx data, 3,309 of which were found in neural contexts. Our results provide insights into the effects of both common and rare variation on gene regulation in the brain. These findings can facilitate mechanistic understanding of cis-regulatory variation associated with biological traits, including disease.

Neurotransmission-related gene expression in the frontal pole is altered in subjects with bipolar disorder and schizophrenia.

The frontal pole (Brodmann area 10, BA10) is the largest cytoarchitectonic region of the human cortex, performing complex integrative functions. BA10 undergoes intensive adolescent grey matter pruning prior to the age of onset for bipolar disorder (BP) and schizophrenia (SCHIZ), and its dysfunction is likely to underly aspects of their shared symptomology. In this study, we investigated the role of BA10 neurotransmission-related gene expression in BP and SCHIZ. We performed qPCR to measure the expression of 115 neurotransmission-related targets in control, BP, and SCHIZ postmortem samples (n = 72). We chose this method for its high sensitivity to detect low-level expression. We then strengthened our findings by performing a meta-analysis of publicly released BA10 microarray data (n = 101) and identified sources of convergence with our qPCR results. To improve interpretation, we leveraged the unusually large database of clinical metadata accompanying our samples to explore the relationship between BA10 gene expression, therapeutics, substances of abuse, and symptom profiles, and validated these findings with publicly available datasets. Using these convergent sources of evidence, we identified 20 neurotransmission-related genes that were differentially expressed in BP and SCHIZ in BA10. These results included a large diagnosis-related decrease in two important therapeutic targets with low levels of expression, HTR2B and DRD4, as well as other findings related to dopaminergic, GABAergic and astrocytic function. We also observed that therapeutics may produce a differential expression that opposes diagnosis effects. In contrast, substances of abuse showed similar effects on BA10 gene expression as BP and SCHIZ, potentially amplifying diagnosis-related dysregulation.

Single nucleus multiomics identifies ZEB1 and MAFB as candidate regulators of Alzheimer's disease-specific cis-regulatory elements.

Cell type-specific transcriptional differences between brain tissues from donors with Alzheimer's disease (AD) and unaffected controls have been well documented, but few studies have rigorously interrogated the regulatory mechanisms responsible for these alterations. We performed single nucleus multiomics (snRNA-seq plus snATAC-seq) on 105,332 nuclei isolated from cortical tissues from 7 AD and 8 unaffected donors to identify candidate cis-regulatory elements (CREs) involved in AD-associated transcriptional changes. We detected 319,861 significant correlations, or links, between gene expression and cell type-specific transposase accessible regions enriched for active CREs. Among these, 40,831 were unique to AD tissues. Validation experiments confirmed the activity of many regions, including several candidate regulators of APP expression. We identified ZEB1 and MAFB as candidate transcription factors playing important roles in AD-specific gene regulation in neurons and microglia, respectively. Microglia links were globally enriched for heritability of AD risk and previously identified active regulatory regions.

Large Common Mitochondrial DNA Deletions Are Associated with a Mitochondrial SNP T14798C Near the 3' Breakpoints.

Introduction: Large somatic deletions of mitochondrial DNA (mtDNA) accumulate with aging in metabolically active tissues such as the brain. We have cataloged the breakpoints and frequencies of large mtDNA deletions in the human brain. Methods: We quantified 112 high-frequency mtDNA somatic deletions across four human brain regions with the Splice-Break2 pipeline. In addition, we utilized PLINK/Seq to test the association of mitochondrial genotypes with the abundance of these high-frequency mtDNA deletions. A conservative p value threshold of 5E-08 was used to find the significant loci. Results: One mtDNA SNP (T14798C) was significantly associated with mtDNA deletions in two brain regions, the dorsolateral prefrontal cortex (DLPFC) and the superior temporal gyrus. Since the DLPFC showed the most robust association between T14798C and two deletion breakpoints (7816-14807 and 5462-14807), this association was tested in the DLPFC of a replication sample and validated the first results. Incorporating the C allele at 14,798 bp increased the perfect/imperfect length of the repeat at the 3' breakpoint of the two associated deletions. Conclusion: This is the first study to identify the association of mtDNA SNP with large mtDNA deletions in the human brain. The T14798C allele located in the MT-CYB gene is a common polymorphism that occurs in several mitochondrial haplogroups. We hypothesize that the T14798C association with two deletions occurs by extending the repeat length around the 3' deletion breakpoints. This simple mechanism suggests that mtDNA SNPs can affect the mitochondrial genome structure, especially in brain where high levels of reactive oxygen species lead to deletion accumulation with aging.

Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

Adolescent cocaine differentially impacts psychomotor sensitization and epigenetic profiles in adult male rats with divergent affective phenotypes.

Adolescent drug use reliably predicts increased addiction liability in adulthood, but not all individuals are equally impacted. To explore the biological bases of this differential reactivity to early life drug experience, we used a genetic rat model of temperament and evaluated the impact of adolescent cocaine exposure on adult psychomotor sensitization. Relative to adult bred low-responder (bLR) rats, bred high-responders (bHR) are more sensitive to the psychomotor-activating effects of cocaine and reinstate drug-seeking behavior more readily following prolonged cocaine exposure and/or abstinence. We found that a 7-day sensitizing cocaine regimen (15 mg/kg/day) during either adolescence or adulthood produced psychomotor sensitization in bHRs only, while a dual cocaine exposure prevented further sensitization, suggesting limits on neuroplasticity. By contrast, adolescent cocaine in bLRs shifted their resilient phenotype, rendering them more responsive to cocaine in adulthood following adolescent cocaine. To begin to explore the neural correlates of these behavioral phenotypes, we assessed two functionally opposite epigenetic chromatin modifications implicated in addiction liability, permissive acetylation (ac) and repressive tri-methylation (me3) on Histone 3 Lysine 9 (H3K9), in four striatal sub-regions. In bHRs, decreased H3K9me3 and increased acH3K9 in the nucleus accumbens (NAc) core associated with cocaine sensitization. In bLRs, the combination of cocaine exposure in adolescence and adulthood, which lead to an increased response to a cocaine challenge, also increased acH3K9 in the core. Thus, adolescent cocaine experience interacts with genetic background to elicit different behavioral profiles relevant to addiction in adulthood, with concurrent modifications in the epigenetic histone profiles in the NAc that associate with cocaine sensitization and with metaplasticity.

Mitochondria DNA copy number, mitochondria DNA total somatic deletions, Complex I activity, synapse number, and synaptic mitochondria number are altered in schizophrenia and bipolar disorder.

Mitochondrial dysfunction is a neurobiological phenomenon implicated in the pathophysiology of schizophrenia and bipolar disorder that can synergistically affect synaptic neurotransmission. We hypothesized that schizophrenia and bipolar disorder share molecular alterations at the mitochondrial and synaptic levels. Mitochondria DNA (mtDNA) copy number (CN), mtDNA common deletion (CD), mtDNA total deletion, complex I activity, synapse number, and synaptic mitochondria number were studied in the postmortem human dorsolateral prefrontal cortex (DLPFC), superior temporal gyrus (STG), primary visual cortex (V1), and nucleus accumbens (NAc) of controls (CON), and subjects with schizophrenia (SZ), and bipolar disorder (BD). The results showed (i) the mtDNA CN is significantly higher in DLPFC of both SZ and BD, decreased in the STG of BD, and unaltered in V1 and NAc of both SZ and BD; (ii) the mtDNA CD is significantly higher in DLPFC of BD while unaltered in STG, V1, and NAc of both SZ and BD; (iii) The total deletion burden is significantly higher in DLPFC in both SZ and BD while unaltered in STG, V1, and NAc of SZ and BD; (iv) Complex I activity is significantly lower in DLPFC of both SZ and BD, which is driven by the presence of medications, with no alteration in STG, V1, and NAc. In addition, complex I protein concentration, by ELISA, was decreased across three cortical regions of SZ and BD subjects; (v) The number of synapses is decreased in DLPFC of both SZ and BD, while the synaptic mitochondria number was significantly lower in female SZ and female BD compared to female controls. Overall, these findings will pave the way to understand better the pathophysiology of schizophrenia and bipolar disorder for therapeutic interventions.

Identification of potential blood biomarkers associated with suicide in major depressive disorder.

Suicides have increased to over 48,000 deaths yearly in the United States. Major depressive disorder (MDD) is the most common diagnosis among suicides, and identifying those at the highest risk for suicide is a pressing challenge. The objective of this study is to identify changes in gene expression associated with suicide in brain and blood for the development of biomarkers for suicide. Blood and brain were available for 45 subjects (53 blood samples and 69 dorsolateral prefrontal cortex (DLPFC) samples in total). Samples were collected from MDD patients who died by suicide (MDD-S), MDDs who died by other means (MDD-NS) and non-psychiatric controls. We analyzed gene expression using RNA and the NanoString platform. In blood, we identified 14 genes which significantly differentiated MDD-S versus MDD-NS. The top six genes differentially expressed in blood were: PER3, MTPAP, SLC25A26, CD19, SOX9, and GAR1. Additionally, four genes showed significant changes in brain and blood between MDD-S and MDD-NS; SOX9 was decreased and PER3 was increased in MDD-S in both tissues, while CD19 and TERF1 were increased in blood but decreased in DLPFC. To our knowledge, this is the first study to analyze matched blood and brain samples in a well-defined population of MDDs demonstrating significant differences in gene expression associated with completed suicide. Our results strongly suggest that blood gene expression is highly informative to understand molecular changes in suicide. Developing a suicide biomarker signature in blood could help health care professionals to identify subjects at high risk for suicide.

Characterizing the behavioral and neuroendocrine features of susceptibility and resilience to social stress.

Evaluating and coping with stressful social events as they unfold is a critical strategy in overcoming them without long-lasting detrimental effects. Individuals display a wide range of responses to stress, which can manifest in a variety of outcomes for the brain as well as subsequent behavior. Chronic Social Defeat Stress (CSDS) in mice has been widely used to model individual variation following a social stressor. Following a course of repeated intermittent psychological and physical stress, mice diverge into separate populations of social reactivity: resilient (socially interactive) and susceptible (socially avoidant) animals. A rich body of work reveals distinct neurobiological and behavioral consequences of this experience that map onto the resilient and susceptible groups. However, the range of factors that emerge over the course of defeat have not been fully described. Therefore, in the current study, we focused on characterizing behavioral, physiological, and neuroendocrine profiles of mice in three separate phases: before, during, and following CSDS. We found that following CSDS, traditional read-outs of anxiety-like and depression-like behaviors do not map on to the resilient and susceptible groups. By contrast, behavioral coping strategies used during the initial social stress encounter better predict which mice will eventually become resilient or susceptible. In particular, mice that will emerge as susceptible display greater escape behavior on Day 1 of social defeat than those that will emerge as resilient, indicating early differences in coping mechanisms used between the two groups. We further show that the social avoidance phenotype in susceptible mice is specific to the aggressor strain and does not generalize to conspecifics or other strains, indicating that there may be features of threat discrimination that are specific to the susceptible mice. Our findings suggest that there are costs and benefits to both the resilient and susceptible outcomes, reflected in their ability to cope and adapt to the social stressor.

Differences in microglia morphological profiles reflect divergent emotional temperaments: insights from a selective breeding model.

Microglia play critical roles in healthy brain development and function, as well as the neuropathology underlying a range of brain diseases. Despite evidence for a role of microglia in affective regulation and mood disorders, little is known regarding how variation in microglia status relates to individual differences in emotionality. Using a selective breeding model, we have generated rat lines with unique temperamental phenotypes that reflect broad emotional traits: bred low responder rats (bLRs) are novelty-averse and model a passive coping style, whereas bred high responder rats (bHRs) are highly exploratory and model an active coping style. To identify a functional role of microglia in these phenotypes, we administered minocycline, an antibiotic with potent microglia inhibiting properties and observed shifts in forced swim, sucrose preference, and social interaction behaviors in bLRs. Using detailed anatomical analyses, we compared hippocampal microglia profiles of bHRs and bLRs and found that although the lines had similar numbers of microglia, selective breeding was associated with a shift in the morphological features of these cells. Specifically, microglia from bLRs were characterized by a hyper-ramified morphology, with longer processes and more complicated branching patterns than microglia from bHRs. This morphology is thought to reflect an early stage of microglia activation and suggests that bLR microglia are in a reactive state even when animals are not overtly challenged. Taken together, our results provide novel evidence linking variation in inborn temperament with differences in the baseline status of microglia and implicate a role for microglia in shaping enduring emotional characteristics.

Genome-wide association study in a rat model of temperament identifies multiple loci for exploratory locomotion and anxiety-like traits.

Common genetic factors likely contribute to multiple psychiatric diseases including mood and substance use disorders. Certain stable, heritable traits reflecting temperament, termed externalizing or internalizing, play a large role in modulating vulnerability to these disorders. To model these heritable tendencies, we selectively bred rats for high and low exploration in a novel environment [bred High Responders (bHR) vs. Low Responders (bLR)]. To identify genes underlying the response to selection, we phenotyped and genotyped 538 rats from an F2 cross between bHR and bLR. Several behavioral traits show high heritability, including the selection trait: exploratory locomotion (EL) in a novel environment. There were significant phenotypic and genetic correlations between tests that capture facets of EL and anxiety. There were also correlations with Pavlovian conditioned approach (PavCA) behavior despite the lower heritability of that trait. Ten significant and conditionally independent loci for six behavioral traits were identified. Five of the six traits reflect different facets of EL that were captured by three behavioral tests. Distance traveled measures from the open field and the elevated plus maze map onto different loci, thus may represent different aspects of novelty-induced locomotor activity. The sixth behavioral trait, number of fecal boli, is the only anxiety-related trait mapping to a significant locus on chromosome 18 within which the Pik3c3 gene is located. There were no significant loci for PavCA. We identified a missense variant in the Plekhf1 gene on the chromosome 1:95 Mb QTL and Fancf and Gas2 as potential candidate genes that may drive the chromosome 1:107 Mb QTL for EL traits. The identification of a locomotor activity-related QTL on chromosome 7 encompassing the Pkhd1l1 and Trhr genes is consistent with our previous finding of these genes being differentially expressed in the hippocampus of bHR vs. bLR rats. The strong heritability coupled with identification of several loci associated with exploratory locomotion and emotionality provide compelling support for this selectively bred rat model in discovering relatively large effect causal variants tied to elements of internalizing and externalizing behaviors inherent to psychiatric and substance use disorders.

Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues.

Transcript labeling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, large tissue labeling in non-perfused or fresh-frozen rodent and postmortem human samples, which provide more flexible utilization than perfused tissues, is largely unexplored. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labeling between two clearing methods, CLARITY and iDISCO+. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO+ resulted in better tissue clearing, greater imaging depth and a more uniform labeling of larger samples. Based on these results, we used iDISCO+-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labeling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods, especially iDISCO+, may find broad application in the transcript analysis in rodent studies, but its limited use in postmortem human tissues can be improved by further optimizations.

Genetic Liability for Internalizing Versus Externalizing Behavior Manifests in the Developing and Adult Hippocampus: Insight From a Meta-analysis of Transcriptional Profiling Studies in a Selectively Bred Rat Model.

BACKGROUND: For more than 16 years, we have selectively bred rats for either high or low levels of exploratory activity within a novel environment. These bred high-responder (bHR) and bred low-responder (bLR) rats model temperamental extremes, exhibiting large differences in internalizing and externalizing behaviors relevant to mood and substance use disorders. METHODS: We characterized persistent differences in gene expression related to bHR/bLR phenotype across development and adulthood in the hippocampus, a region critical for emotional regulation, by meta-analyzing 8 transcriptional profiling datasets (microarray and RNA sequencing) spanning 43 generations of selective breeding (postnatal day 7: n = 22; postnatal day 14: n = 49; postnatal day 21: n = 21; adult: n = 46; all male). We cross-referenced expression differences with exome sequencing within our colony to pinpoint candidates likely to mediate the effect of selective breeding on behavioral phenotype. The results were compared with hippocampal profiling from other bred rat models. RESULTS: Genetic and transcriptional profiling results converged to implicate multiple candidate genes, including two previously associated with metabolism and mood: Trhr and Ucp2. Results also highlighted bHR/bLR functional differences in the hippocampus, including a network essential for neurodevelopmental programming, proliferation, and differentiation, centering on Bmp4 and Mki67. Finally, we observed differential expression related to microglial activation, which is important for synaptic pruning, including 2 genes within implicated chromosomal regions: C1qa and Mfge8. CONCLUSIONS: These candidate genes and functional pathways may direct bHR/bLR rats along divergent developmental trajectories and promote a widely different reactivity to the environment.

Sepsis survivor mice exhibit a behavioral endocrine syndrome with ventral hippocampal dysfunction.

Severe acute stressors are known to trigger mood disorders in humans. Sepsis represents one such stressor, and survivors often suffer long term from psychiatric morbidity. We hypothesized that sepsis leads to lasting changes in neural circuits involved in stress integration, altering affective behavior and the stress response. To investigate this hypothesis, sepsis was induced in male C57Bl/6 mice using cecal ligation and puncture (CLP), and control mice underwent sham surgery. Mice recovered from acute illness within 2 weeks, after which they exhibited increased avoidance behavior and behavioral despair compared with sham, with behavioral changes observed more than 5 weeks after recovery. Sepsis survivors also showed evidence of enhanced hypothalamic-pituitary-adrenal (HPA) axis activity, with increased corticosterone after a novel stressor and increased adrenal weight. In the brain, sepsis survivor mice showed decreased stress-induced cfos mRNA and increased glucocorticoid receptor immunoreactivity specifically in the ventral hippocampus, a brain region known to coordinate emotional behavior and HPA axis activity. We conclude that murine sepsis survivors exhibit a behavioral neuroendocrine syndrome of negative affective behavior and HPA axis hyperactivity, which could be explained by ventral hippocampal dysfunction. These findings could contribute to our understanding of the human post-intensive care syndrome.

Blockade of the cholecystokinin CCK-2 receptor prevents the normalization of anxiety levels in the rat.

Cholecystokinin (CCK), through the CCK-2 receptor, exerts complex effects on anxiety. While CCK agonists are panicogenic, CCK-2 antagonists fail to alleviate human anxiety. Preclinical studies with CCK-2 antagonists are also inconsistent because their anxiolytic effects largely depend on the behavioral paradigm and antecedent stress. The controversy might be accounted by the neuromodulatory role for CCK in anxiety which is ill-defined. If this is its actual role, blocking CCK-2 will have carry-over effects on the anxiety baseline over time. To test this hypothesis, the consequences of acute administration of the CCK-2 antagonist Ly225.910 (0.1 mg Kg-1) was evaluated in the temporal expression of aversion toward exploration-conflicting tasks. Ly225.910 effects were evaluated in rats exposed to the elevated plus-maze (EPM) twice, an approach-avoidance anxiety-like test. While LY225.910-treated rats had less anxiety than vehicle-treated rats, the difference was reversed during the EPM retest 24 h later without drug. Moreover, Ly225.910 effects in stress-induced cognitive impairment was measured giving the novel-object discrimination (NOD) test to rats not habituated to the exploration apparatus to elicit neophobia. After a first encounter with objects ("old"), Ly225.910-treated rats did not recognize the "novel" object introduced 6 h later. Ly225.910-exposed rats did not discriminate the new location of the "novel object" when it was repositioned in the arena 24 h later. Ly225.910-treated rats also failed to explore objects. In line with its neuromodulatory role, aversive carry-over effects of Ly225.910 suggest that CCK-2 activation by endogenous CCK, rather than triggering anxiety, may return the anxiety state to its normal level.