Molecular Psychiatry

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Schematic representation of participant selection process and demographics
Working memory was measured using an emotional N-back task, response inhibition (stop signal reaction time) and reaction time variability were measured with a stop signal task. ADHD traits were quantified as Bartlett factor scores derived from Child Behaviour Checklist Attention Problems items and Early Adolescent Temperament Questionnaire Revised Effortful Control items. Final samples size for each measure is in bold. Quality control procedures for each measure are described in section titled “Candidate Cognitive Endophenotypes”. QC quality control, EUR European, PRS polygenic risk score, ADHD attention-deficit/hyperactivity disorder.
Exploratory factor analysis and the associations between ADHD polygenic risk and ADHD traits scores
A Scree plot from an exploratory factor analysis of CBCL attention problems items and EATQ-R effortful control items indicates ‘essential unidimensionality’. Each dot indicates the amount of common variance of the observed variables each factor explains (i.e., eigenvalue). Any factor with an eigenvalue ≥1 explains more variance than a single observed variable (line y = 1), indicating it should be retained. However, due to the large drop in variance explained by the first factor to the second (and subsequent) factors, a one-factor solution was retained. The total sample used in the EFA, n = 5814. B Bar plot of the percentage of variance in ADHD trait scores explained by PRSs across a range of p-value thresholds (PT), where PT = 0.132 (retaining 51,165 most strongly associated SNPs) explained the most variance in ADHD traits (R² = 2.0%, p = 1.8 ×10⁻¹⁴). C Quantile plot demonstrating that as ADHD PRSs increased, ADHD traits increased (i.e., factor scores decreased). The total sample used in the PRS analyses, n = 2,847. CBCL child behaviour checklist, EATQ-R early adolescence temperament questionnaire revised, ADHD attention-deficit/hyperactivity disorder, PRS polygenic risk score.
Association between each candidate cognitive endophenotype and ADHD traits (column 1) and between ADHD PRSs and each candidate cognitive endophenotype (column 2)
A Higher working memory accuracy scores form the emotional n-back task were associated with less pronounced ADHD traits (β = 0.18, p = 1.4 × 10⁻¹⁶, n = 2221). B Higher ADHD PRS were associated with lower working memory response accuracy scores (β = −0.10, p = 2.3 × 10⁻⁶, n = 2221). C Higher stop signal reaction time scores from the stop signal task were associated with more pronounced ADHD traits (β = −0.13, p = 3.2 × 10⁻⁸, n = 2004). D No significant association was identified between ADHD PRS and stop signal reaction time scores (β = 0.05, p = 0.13, n = 2004). E Higher reaction time variability scores from the stop signal task were associated with more pronounced ADHD traits (β = −0.16, p = 1.2 × 10⁻¹³, n = 2122). F Higher ADHD PRS were associated with higher reaction time variability scores (β = 0.12, p = 3.5 × 10⁻⁸, n = 2122). ADHD attention-deficit/hyperactivity disorder, PRS polygenic risk score.
The relationship between ADHD PRS and ADHD traits is partially mediated by working memory and reaction time variability
Standardised regression coefficients and confidence intervals for the relationships between ADHD PRS and ADHD trait factor scores (higher scores=less pronounced ADHD traits) as mediated by candidate cognitive endophenotypes. A Working memory partially mediated the relationship between ADHD PRS and ADHD traits given both the indirect effect b [95% CI] = −378.70 [−557.6, −196.4], n = 2221) and the direct effect (i.e., c’ path; β = −0.11 [−0.15, −0.07], p = 2.2 × 10⁻⁷, n = 2221) were significant. B Reaction time variability partially mediated the relationship between ADHD PRS and ADHD traits given both the indirect effect (b [95% CI] = −412.17 [−594.7, −235.5], n = 2122) and the direct effect (i.e., c’ path; β = −0.10 [−0.14, −0.06], p = 2.1 × 10⁻⁶, n = 2122) were significant. ADHD attention-deficit/hyperactivity disorder, PRS polygenic risk score. Reported p-values have not been corrected for multiple comparisons.
  • Mia MosesMia Moses
  • Jeggan TiegoJeggan Tiego
  • Ditte DemontisDitte Demontis
  • [...]
  • Mark A. BellgroveMark A. Bellgrove
Endophenotypes are heritable and quantifiable traits indexing genetic liability for a disorder. Here, we examined three potential endophenotypes, working memory function, response inhibition, and reaction time variability, for attention-deficit hyperactivity disorder (ADHD) measured as a dimensional latent trait in a large general population sample derived from the Adolescent Brain Cognitive DevelopmentSM Study. The genetic risk for ADHD was estimated using polygenic risk scores (PRS) whereas ADHD traits were quantified as a dimensional continuum using Bartlett factor score estimates, derived from Attention Problems items from the Child Behaviour Checklist and Effortful Control items from the Early Adolescent Temperament Questionnaire-Revised. The three candidate cognitive endophenotypes were quantified using task-based performance measures. Higher ADHD PRSs were associated with higher ADHD traits, as well as poorer working memory performance and increased reaction time variability. Lower working memory performance, poorer response inhibition, and increased reaction time variability were associated with more pronounced ADHD traits. Working memory and reaction time variability partially statistically mediated the relationship between ADHD PRS and ADHD traits, explaining 14% and 16% of the association, respectively. The mediation effect was specific to the genetic risk for ADHD and did not generalise to genetic risk for four other major psychiatric disorders. Together, these findings provide robust evidence from a large general population sample that working memory and reaction time variability can be considered endophenotypes for ADHD that mediate the relationship between ADHD PRS and ADHD traits.
Social anhedonia, a loss of interest and pleasure in social interactions, is a common symptom of major depression as well as other psychiatric disorders. Depression can occur at any age, but typically emerges in adolescence or early adulthood, which represents a sensitive period for social interaction that is vulnerable to stress. In this study, we evaluated social interaction reward using a conditioned place preference (CPP) paradigm in adolescent male and female mice. Adolescent mice of both sexes exhibited a preference for the social interaction-associated context. Chronic unpredictable stress (CUS) impaired the development of CPP for social interaction, mimicking social anhedonia in depressed adolescents. Conversely, administration of leptin, an adipocyte-derived hormone, enhanced social interaction-induced CPP in non-stressed control mice and reversed social anhedonia in CUS mice. By dissecting the motivational processes of social CPP into social approach and isolation avoidance components, we demonstrated that leptin treatment increased isolation aversion without overt social reward effect. Further mechanistic exploration revealed that leptin stimulated oxytocin gene transcription in the paraventricular nucleus of the hypothalamus, while oxytocin receptor blockade abolished the leptin-induced enhancement of socially-induced CPP. These results establish that chronic unpredictable stress can be used to study social anhedonia in adolescent mice and provide evidence that leptin modulates social motivation possibly via increasing oxytocin synthesis and oxytocin receptor activation.
EV mRNA Communication is Disrupted in PPD
We performed differential expression analysis separately on PPD cases and unaffected controls to determine how EV mRNA levels change during pregnancy and the postpartum period. EV mRNAs with altered levels are plotted for PPD and unaffected controls. Each line represents the trajectory of a single EV mRNA during pregnancy and the postpartum period. We standardized for high and low expressing EV mRNAs by dividing each sample with the mean read counts for the corresponding EV mRNA.
EV mRNA Changes in PPD during Pregnancy and the Postpartum Period
A Histogram of p-values that remain significant following adjustment for multiple testing. The low p-values demonstrate that the majority of the identified differences are highly significant B Histogram of the changes in PPD EV mRNA levels. The dashed lines are at −1 and 1 log2 fold change. CP-values for PPD vs control EV level comparison for each time period. The dashed line demarcates the threshold of p = 0.1 following multiple testing correction via the Benjamini–Hochberg method.
Cellular Deconvolution Analysis of EV mRNA
Fraction of cell types predicted by CIBERSORTx based on EV mRNA levels are plotted for T2) 2nd Trimester, T3) 3rd Trimester and 2 W) 2 weeks, 6 W) 6 weeks, 3 M) 3 months and 6 M) 6 months postpartum. Grouping is by time-point. The red stars denote statistically significant differences between PPD and controls as determined by the Mann–Whitney U test.
Quantitative PCR Validation of EV mRNA Levels in an Additional Cohort
A sequencing and quantitative PCR results B 2nd trimester (p = 0.05) and C 3rd trimester (p = 0.03) for the SET gene. D Sequencing and quantitative PCR results E trimester 2 (p = 0.6 – not significant) and F trimester 3 (p = 0.02) for the COX6C gene. The RNA sequencing and the quantitative PCR validation was performed on different samples.
We investigated whether extracellular RNA communication, which is a recently discovered mode of intercellular communication that is involved in a variety of important biological processes including pregnancy, is associated with postpartum depression (PPD). Extracellular RNA communication is increased during pregnancy and is involved in embryo implantation, uterine spiral artery remodeling, parturition, preterm birth, immunity, and the inflammatory response. Since immune anomalies are associated with PPD, we characterized the mRNA content of extracellular vesicles (EV) in a cohort of prospectively collected blood plasma samples at six time-points throughout pregnancy and the postpartum (2nd trimester, 3rd trimester, 2 weeks postpartum, 6 weeks postpartum, 3 months postpartum, and 6 months postpartum) in an academic medical setting from women who went on to develop PPD (N = 7, defined as euthymic in pregnancy with postpartum-onset depressive symptoms assessed by Edinburgh Postnatal Depression Scale ≥13 at any postpartum time point) and matched unaffected controls (N = 7, defined as euthymic throughout pregnancy and postpartum). Blood samples were available for all participants at the T2 and W6 timepoints, with fewer samples available at other time points. This analysis revealed that EV mRNA levels during pregnancy and the postpartum period were extensively altered in women who went on to develop PPD. Gene set enrichment analysis revealed that mRNAs associated with autophagy were decreased in PPD cases. In contrast, EV mRNAs from ribosomes and mitochondria, two organelles that are selectively targeted by autophagy, were elevated in PPD cases. Cellular deconvolution analysis discovered that EV mRNAs associated with PPD originated from monocytes and macrophages. Quantitative PCR analysis for four relevant genes in another cohort replicated these findings and confirmed that extracellular RNA levels are altered in PPD. We demonstrate that EV mRNA communication is robustly altered during pregnancy and the postpartum period in women who go on to develop PPD. Our work also establishes a direct link between reduced autophagy and PPD in patient samples. These data warrant investigating the feasibility of developing EV mRNA based biomarkers and therapeutic agents for PPD.
As a prime mover in Alzheimer’s disease (AD), microglial activation requires membrane translocation, integration, and activation of the metamorphic protein chloride intracellular channel 1 (CLIC1), which is primarily cytoplasmic under physiological conditions. However, the formation and activation mechanisms of functional CLIC1 are unknown. Here, we found that the human antimicrobial peptide (AMP) LL-37 promoted CLIC1 membrane translocation and integration. It also activates CLIC1 to cause microglial hyperactivation, neuroinflammation, and excitotoxicity. In mouse and monkey models, LL-37 caused significant pathological phenotypes linked to AD, including elevated amyloid-β, increased neurofibrillary tangles, enhanced neuronal death and brain atrophy, enlargement of lateral ventricles, and impairment of synaptic plasticity and cognition, while Clic1 knockout and blockade of LL-37-CLIC1 interactions inhibited these phenotypes. Given AD’s association with infection and that overloading AMP may exacerbate AD, this study suggests that LL-37, which is up-regulated upon infection, may be a driving force behind AD by acting as an endogenous agonist of CLIC1.
Objective: People with mood disorders have increased risk of comorbid medical diseases versus the general population. It is paramount to identify interventions to improve physical health in this population. Methods: Umbrella review of meta-analyses of randomised controlled trials (RCTs) on pharmacological/non-pharmacological interventions for physical health outcomes/intolerability-related discontinuation in mood disorders (any age). Results: Ninety-seven meta-analyses were included. Among youths, against placebo, in depression, antidepressants/antipsychotics had higher discontinuation rates; in bipolar depression, olanzapine+fluoxetine worsened total cholesterol (TC)/triglycerides/weight gain (WG) (large ES). In adults with bipolar disorder, olanzapine worsened HbA1c/TC/WG (moderate/large ES); asenapine increased fasting glucose (small ES); quetiapine/cariprazine/risperidone induced WG (small/moderate ES). In bipolar depression, lurasidone was metabolically neutral. In depression, psychological interventions improved physical health-related quality of life (PHQoL) (small ES), fasting glucose/HbA1c (medium/large ES); SSRIs improved fasting glucose/HbA1c, readmission for coronary disease, pain (small ES); quetiapine/aripiprazole/olanzapine induced WG (small to large ES). Exercise improved cardiorespiratory fitness (moderate ES). In the elderly, fluoxetine yielded more detrimental cardiovascular effects than sertraline/escitalopram (large ES); antidepressants were neutral on exercise tolerance and PHQoL. In mixed age groups, in bipolar disorder aripiprazole was metabolically neutral; in depression, SSRIs lowered blood pressure versus placebo and serotonin-noradrenaline reuptake inhibitors (small ES); brexpiprazole augmentation caused WG and was less tolerated (small ES); exercise improved PHQoL (moderate ES). Conclusions: Some interventions (psychological therapies, exercise and SSRIs) improve certain physical health outcomes in mood disorders, few are neutral, but various pharmacological interventions are associated with negative effects. Evidence from this umbrella review has limitations, should consider evidence from other disorders and should be integrated with recent evidence from individual RCTs, and observational evidence. Effective treatments with either beneficial or physically neutral profiles should be prioritized.
Workflow analysis
Panel a shows a schematic representation of the concept proposed in this paper, showing the reciprocal interaction between mitochondria, NMDAR, neuro-immune system, dopamine on one hand and the complex redox regulation/oxidative stress on the other. We focused on five pathways (glutamate, oxidative stress, GABA/interneurons (hereafter called interneurons), neuroimmune/neuroinflammation (hereafter called neuroinflammation) and myelin). Panel b summarizes the experimental set up used for five different pathways. For each pathway, we defined the corresponding biological mechanisms and used them as keywords that were entered into the GSEA platform to retrieve the corresponding gene sets. The gene sets were then manually parsed to keep only those more pertinent to each pathway. For each gene set we defined (1) those SNPs that mapped to the gene set and (2) those SNPs that were eQTLs at least for one gene of the set.
Results for the TIPP study and polygenic risk scores (GW-PRSs and pathway-PRSs) analyses at pt ≤ 1
Early psychosis status (dependent variable) was regressed on the polygenic risk scores using logistic regressions and including the first five ancestry-informative genetic principal components as covariates. Horizontal bars show the Odds Ratio estimates (OR), and error bars indicate 95% confidence intervals (95% CI).
Results for the PAFIP study and polygenic risk scores (GW-PRSs and pathway-PRSs) analyses at pt ≤ 1
Early psychosis status (dependent variable) was regressed on the polygenic risk scores using logistic regressions and including the first five ancestry-informative genetic principal components as covariates. Horizontal bars show the Odds Ratio estimates (OR), and error bars indicate 95% confidence intervals (95% CI).
Polygenic risk prediction remains an important aim of genetic association studies. Currently, the predictive power of schizophrenia polygenic risk scores (PRSs) is not large enough to allow highly accurate discrimination between cases and controls and thus is not adequate for clinical integration. Since PRSs are rarely used to reveal biological functions or to validate candidate pathways, to fill this gap, we investigated whether their predictive ability could be improved by building genome-wide (GW-PRSs) and pathway-specific PRSs, using distance- or expression quantitative trait loci (eQTLs)- based mapping between genetic variants and genes. We focused on five pathways (glutamate, oxidative stress, GABA/interneurons, neuroimmune/neuroinflammation and myelin) which belong to a critical hub of schizophrenia pathophysiology, centred on redox dysregulation/oxidative stress. Analyses were first performed in the Lausanne Treatment and Early Intervention in Psychosis Program (TIPP) study (n = 340, cases/controls: 208/132), a sample of first-episode of psychosis patients and matched controls, and then validated in an independent study, the epidemiological and longitudinal intervention program of First-Episode Psychosis in Cantabria (PAFIP) (n = 352, 224/128). Our results highlighted two main findings. First, GW-PRSs for schizophrenia were significantly associated with early psychosis status. Second, oxidative stress was the only significantly associated pathway that showed an enrichment in both the TIPP (p = 0.03) and PAFIP samples (p = 0.002), and exclusively when gene-variant linking was done using eQTLs. The results suggest that the predictive accuracy of polygenic risk scores could be improved with the inclusion of information from functional annotations, and through a focus on specific pathways, emphasizing the need to build and study functionally informed risk scores.
PCA scores plots of NMR serum metabolomics
a The NMR metabolite signature spontaneously separated the psychosis patient cohort into two groups: the ‘predominant biochemical signature group’ and the ‘distinct biochemical signature group’. b Psychosis patients who tested positive for serum antibodies against VGKC (red, n = 13) or GlyR (orange, n = 7) spontaneously separated from those who tested positive for C2 (pink, n = 9), LGI1 (purple, n = 9), or NMDAR (blue, n = 35) and antibody negative Control (green, n = 70) samples.
OPLS-DA models discriminating VGKC/GlyR (red square, n = 20) samples from NMDAR/LGI1/CASPR2 (blue triangle, n = 53) samples and Control (green circle, n = 70) samples using NMR serum metabolomic data
a, c, e OPLS-DA scores plots of NMDAR/LGI1/CASPR2 v. Control, VGKC&GlyR v. Control, VGKC/GlyR v. NMDAR/LGI1/CASPR2. b, d, f Predictive accuracy of the ensemble of the OPLS-DA models compared with that of the randomly permutated null distribution. Kolmogorov-Smirnov test. ***p < 0.001.
Levels of discriminatory serum metabolites selected by the OPLS-DA models in VGKC/GlyR (red, n = 20), control (green, n = 70), and NMDAR/LGI1/CASPR2(blue, n = 53) groups
a–e Decreased levels of several fatty acid resonances within serum lipoproteins (−CH3, (−CH2-)n, -N(CH3)3, unsaturated fatty acid, =CH-CH2-CH2−) in the VGKC/GlyR antibody group. f–k Increased levels of several amino acids (leucine, isoleucine, lysine, and valine), choline and glucose in the VGKC/GlyR antibody group. Error bars: ±SEM. One-way ANOVA with Tukey’s post-hoc corrections. ***p  <  0.001.
A range of studies suggest that a proportion of psychosis may have an autoimmune basis, but this has not translated through into clinical practice—there is no biochemical test able to accurately identify psychosis resulting from an underlying inflammatory cause. Such a test would be an important step towards identifying who might require different treatments and have the potential to improve outcomes for patients. To identify novel subgroups within patients with acute psychosis we measured the serum nuclear magnetic resonance (NMR) metabolite profiles of 75 patients who had identified antibodies (anti-glycine receptor [GlyR], voltage-gated potassium channel [VGKC], Contactin-associated protein-like 2 [CASPR2], leucine-rich glioma inactivated 1 [LGI1], N-methyl-D-aspartate receptor [NMDAR] antibody) and 70 antibody negative patients matched for age, gender, and ethnicity. Clinical symptoms were assessed using the positive and negative syndrome scale (PANSS). Unsupervised principal component analysis identified two distinct biochemical signatures within the cohort. Orthogonal partial least squared discriminatory analysis revealed that the serum metabolomes of NMDAR, LGI1, and CASPR2 antibody psychosis patients were indistinct from the antibody negative control group while VGKC and GlyR antibody patients had significantly decreased lipoprotein fatty acids and increased amino acid concentrations. Furthermore, these patients had more severe presentation with higher PANSS scores than either the antibody negative controls or the NMDAR, LGI1, and CASPR2 antibody groups. These results suggest that a proportion of patients with acute psychosis have a distinct clinical and biochemical phenotype that may indicate an inflammatory subtype.
Genome-wide CNV burden association
The bar plot depicts regression beta coefficients as effect sizes (on the x-axis) of genome-wide CNV burden on PTSD, including overall burden, overlapping neurodevelopmental regions only, and genome-wide with neurodevelopmental regions excluded (on the y-axis). Data are shown stratified by CNV type, both CNV types (colored black), duplications only (colored red), and deletions only (colored blue). Effect sizes are shown in terms of megabases of the genome spanned by CNV.
Association of individual NDD CNVs with PTSD
The bar plot depicts regression beta coefficients as effect sizes (on the x-axis) of NDD CNVs (on the y-axis) on PTSD. Data are colored by CNV type, with deletions in blue and duplications in red. Effect sizes are shown in terms of megabases of the genome spanned by CNV. A star indicates an FDR significant CNVs.
Posttraumatic stress disorder (PTSD) is a heritable (h² = 24–71%) psychiatric illness. Copy number variation (CNV) is a form of rare genetic variation that has been implicated in the etiology of psychiatric disorders, but no large-scale investigation of CNV in PTSD has been performed. We present an association study of CNV burden and PTSD symptoms in a sample of 114,383 participants (13,036 cases and 101,347 controls) of European ancestry. CNVs were called using two calling algorithms and intersected to a consensus set. Quality control was performed to remove strong outlier samples. CNVs were examined for association with PTSD within each cohort using linear or logistic regression analysis adjusted for population structure and CNV quality metrics, then inverse variance weighted meta-analyzed across cohorts. We examined the genome-wide total span of CNVs, enrichment of CNVs within specified gene-sets, and CNVs overlapping individual genes and implicated neurodevelopmental regions. The total distance covered by deletions crossing over known neurodevelopmental CNV regions was significant (beta = 0.029, SE = 0.005, P = 6.3 × 10⁻⁸). The genome-wide neurodevelopmental CNV burden identified explains 0.034% of the variation in PTSD symptoms. The 15q11.2 BP1-BP2 microdeletion region was significantly associated with PTSD (beta = 0.0206, SE = 0.0056, P = 0.0002). No individual significant genes interrupted by CNV were identified. 22 gene pathways related to the function of the nervous system and brain were significant in pathway analysis (FDR q < 0.05), but these associations were not significant once NDD regions were removed. A larger sample size, better detection methods, and annotated resources of CNV are needed to explore this relationship further.
Although the link of white matter to pathophysiology of schizophrenia is documented, loss of myelin is not detected in patients at the early stages of the disease, suggesting that pathological evolution of schizophrenia may occur before significant myelin loss. Disrupted-in-schizophrenia-1 (DISC1) protein is highly expressed in oligodendrocyte precursor cells (OPCs) and regulates their maturation. Recently, DISC1-Δ3, a major DISC1 variant that lacks exon 3, has been identified in schizophrenia patients, although its pathological significance remains unknown. In this study, we detected in schizophrenia patients a previously unidentified pathological phenotype of OPCs exhibiting excessive branching. We replicated this phenotype by generating a mouse strain expressing DISC1-Δ3 gene in OPCs. We further demonstrated that pathological OPCs, rather than myelin defects, drive the onset of schizophrenic phenotype by hyperactivating OPCs' Wnt/β-catenin pathway, which consequently upregulates Wnt Inhibitory Factor 1 (Wif1), leading to the aberrant synaptic formation and neuronal activity. Suppressing Wif1 in OPCs rescues synaptic loss and behavioral disorders in DISC1-Δ3 mice. Our findings reveal the pathogenetic role of OPC-specific DISC1-Δ3 variant in the onset of schizophrenia and highlight the therapeutic potential of Wif1 as an alternative target for the treatment of this disease.
Mitochondrial DNA variants have previously associated with disease, but the underlying mechanisms have been largely elusive. Here, we report that mitochondrial SNP rs2853499 associated with Alzheimer’s disease (AD), neuroimaging, and transcriptomics. We mapped rs2853499 to a novel mitochondrial small open reading frame called SHMOOSE with microprotein encoding potential. Indeed, we detected two unique SHMOOSE-derived peptide fragments in mitochondria by using mass spectrometry—the first unique mass spectrometry-based detection of a mitochondrial-encoded microprotein to date. Furthermore, cerebrospinal fluid (CSF) SHMOOSE levels in humans correlated with age, CSF tau, and brain white matter volume. We followed up on these genetic and biochemical findings by carrying out a series of functional experiments. SHMOOSE acted on the brain following intracerebroventricular administration, differentiated mitochondrial gene expression in multiple models, localized to mitochondria, bound the inner mitochondrial membrane protein mitofilin, and boosted mitochondrial oxygen consumption. Altogether, SHMOOSE has vast implications for the fields of neurobiology, Alzheimer’s disease, and microproteins.
Knock-down (KD) of Prdm2 in the dorsomedial prefrontal cortex (dmPFC) causes a lasting increase in fear expression
A Representative tile scan of virus injection site and spread. B, C Representative images used for RNAscope quantification. D KD of Prdm2 induces a significant downregulation of Prdm2 in the dmPFC. E Experimental timeline: On day 1, rats received bilateral infusion of an AAV9 containing either a shRNA-Prdm2 or a scrambled control. Rats were then tested for fear acquisition (day 31), fear expression (day 32), and fear extinction (day 33–34). F KD of Prdm2 did not affect the acquisition of fear memory (acquisition of fear memory is presented as an average of tones 2–6 during the conditioning session), but G significantly increased fear expression 24 h after conditioning (indicated by the % freezing ±SEM; N = 17–20/ group). H Average of the first 2 tones from the fear expression test. IPrdm2 KD did not affect the rate of extinction, indicated by the interaction for time x group not being significant (i.e., similar slopes). J In a separate batch (N = 12/group), Prdm2 KD was found to increase fear expression also 1w after conditioning. K When Prdm2 was knocked down 1 week after the acquisition of fear memory (N = 18–20/group), no effect was observed on fear expression measured 1 month later. *p < 0.05; **p < 0.01; p < 0.001.
Prdm2 knock-down (KD) in the dorsomedial prefrontal cortex (dmPFC) does not affect foot shock sensitivity, basal anxiety-like behavior, or associative learning
Prdm2 KD does not affect shock sensitivity (A), locomotor activity (B), novel object recognition (C) and basal anxiety (D).
Prdm2 knock-down (KD) in the dmPFC-BLA projecting neurons increases fear expression
A Schematic representing the dual viral approach. B Tile scans showing the viral spread in the dmPFC and BLA and representative images showing dmPFC neurons infected by AAV9-DIO-shRNA-PRDM2-ZS green (green), dmPFC neurons infected by rAAV2 retro Cre-mCherry (red) and dmPFC neurons showing co-infection of AAV9-DIO-shRNA-PRDM2-ZS green and rAAV2 retro Cre-mCherry (yellow; scale bar, 50 µm). C KD of Prdm2 in neurons projecting from the dmPFC to the BLA did not affect fear acquisition measured as % freezing ± SEM. DPrdm2 KD in dmPFC-BLA was sufficient to increase fear expression 24 h after conditioning (N = 19–20). *p < 0.05. BLA basolateral amygdala, dmPFC dorsomedial prefrontal cortex.
Prdm2 knock-down (KD) in dmPFC-BLA neurons regulates genes involved in synaptogenesis
Prdm2 KD in dmPFC-BLA neurons regulates genes involved in synaptogenesis. A Schematic representing the triple viral approach used for the vTRAP experiment. B Tile scans showing the viral spread in the dmPFC and BLA as well as dmPFC neurons presenting cells infected by AAV5-FLEX-EGFPL10a (green), cells infected by rAAV2 retro Cre-mCherry (red), and cells showing co-infection of AAV5-FLEX-EGFPL10a and rAAV2 retro Cre-mCherry (yellow). C Principal component analysis showing separation of Prdm2 KD samples and scrambled control into distinct clusters. D Volcano Plot illustrating the most significantly altered genes following Prdm2 KD. E Hierarchical clustering dendrogram grouping together interconnected, highly co-expressed genes. Colormaps beneath the dendrogram corresponds to modules of co-expressed genes. Top colormap: initial identified modules. Bottom colormap: modules after merging modules with similar expression profiles. F Differential expression analysis for each co-expression module, comparing Prdm2 KD with scrambled control. Red horizontal dashed line denotes a significance level of FDR-corrected p value of 0.05. G Boxplot comparing the gene expression profile of module “MEblue” between conditions. KD: Prdm2 KD, SCR: Scramble control. Statistical test: Two-sided unpaired t-test. H Gene ontology enrichment for genes in the module “MEblue”. I Gene network analysis performed using IPA. Prdm2 KD increases expression of genes that code for the cadherin, neurexin/neuroligin and ephrin/ephrin receptors family as well as for proteins that belongs to the SNARE complex. J Differential expression and significance level for selected synaptogenesis-related genes. Vertical dashed line in the bar plot denotes a significance level of FDR-corrected p value of 0.05. BLA basolateral amygdala, dmPFC dorsomedial prefrontal cortex.
Prdm2 knock-down (KD) in the dmPFC increases glutamate release probability in the BLA
A Representative image showing the viral expression (AAV9.HI.shR.ratPrdm2.CMV.ZsGreen.SV40) in dmPFC terminals targeting the BLA. B Representative sEPSCs traces recorded from BLA putative principal neurons (PNs) in Scrambled or Prdm2 KD rats. Scale bars: 50 pA × 500 ms. Cumulative distributions and bar graphs showing the frequency (C) and amplitude (D) of sEPSCs recorded from putative BLA PNs in Scrambled or Prdm2 KD rats. E Schematic representation of the recording and stimulating electrodes sites for evoked (e)EPSCs recordings. F Representative eEPSCs traces evoked by paired electrical stimulations recorded from putative BLA PNs in Scrambled or Prdm2 KD rats. Scale bars: 50 pA × 25 ms. G Bar graphs showing the mean values of PPR recorded from putative BLA PNs in Scrambled or Prdm2 KD rats. Bar graphs showing the mean values of 1/CV² (H) and VMR (I) of eEPSCs recorded from Scrambled or Prdm2 KD rats (N = 5–6/group). *p < 0.05. J Schematic representing the fiber photometry approach (top) and a representative image (bottom) showing the implanted optical fiber aimed at the BLA and the expression of two viruses, AAV9.HI.shR.ratPrdm2.CMV.ZsGreen.SV40 in dmPFC terminals targeting the BLA as well as AAV9.CaMKII.GCaMP6s.WPRE.SV40 in BLA glutamatergic neurons. K Traces showing normalized calcium-dependent GCaMP fluorescent signal (mean ± SEM) in BLA neurons of Scrambled or Prdm2 KD rats, averaged over the first 2 tones from the fear expression test. Duration of the tone is indicated by the shaded gray box. Bar graphs showing larger peak normalized GCaMP signal in response to tone onset (L) and increased Area Under the Curve (AUC) of the normalized GCaMP signal during the 5 s preceding the tone and the first 5 s of the tone presentation (M) in Prdm2 KD rats compared to Scrambled control rats. BLA basolateral amygdala, CeA central amygdala, dmPFC dorsomedial prefrontal cortex, LA lateral amygdala, VMR variance to mean ratio, sEPSCs spontaneous excitatory postsynaptic currents.
Excessive fear is a hallmark of anxiety disorders, a major cause of disease burden worldwide. Substantial evidence supports a role of prefrontal cortex-amygdala circuits in the regulation of fear and anxiety, but the molecular mechanisms that regulate their activity remain poorly understood. Here, we show that downregulation of the histone methyltransferase PRDM2 in the dorsomedial prefrontal cortex enhances fear expression by modulating fear memory consolidation. We further show that Prdm2 knock-down (KD) in neurons that project from the dorsomedial prefrontal cortex to the basolateral amygdala (dmPFC-BLA) promotes increased fear expression. Prdm2 KD in the dmPFC-BLA circuit also resulted in increased expression of genes involved in synaptogenesis, suggesting that Prdm2 KD modulates consolidation of conditioned fear by modifying synaptic strength at dmPFC-BLA projection targets. Consistent with an enhanced synaptic efficacy, we found that dmPFC Prdm2 KD increased glutamatergic release probability in the BLA and increased the activity of BLA neurons in response to fear-associated cues. Together, our findings provide a new molecular mechanism for excessive fear responses, wherein PRDM2 modulates the dmPFC -BLA circuit through specific transcriptomic changes.
Freezing is a conserved defensive behaviour that constitutes a major stress-coping mechanism. Decades of research have demonstrated a role of the amygdala, periaqueductal grey and hypothalamus as core actuators of the control of fear responses, including freezing. However, the role that other modulatory sites provide to this hardwired scaffold is not known. Here, we show that freezing elicited by exposure to electrical foot shocks activates laterodorsal tegmentum (LDTg) GABAergic neurons projecting to the VTA, without altering the excitability of cholinergic and glutamatergic LDTg neurons. Selective chemogenetic silencing of this inhibitory projection, but not other LDTg neuronal subtypes, dampens freezing responses but does not prevent the formation of conditioned fear memories. Conversely, optogenetic-activation of LDTg GABA terminals within the VTA drives freezing responses and elicits bradycardia, a common hallmark of freezing. Notably, this aversive information is subsequently conveyed from the VTA to the amygdala via a discrete GABAergic pathway. Hence, we unveiled a circuit mechanism linking LDTg-VTA-amygdala regions, which holds potential translational relevance for pathological freezing states such as post-traumatic stress disorders, panic attacks and social phobias.
Spironolactone decreased binge-like alcohol drinking in mice
A Spironolactone dose-dependently reduced alcohol intake (g/kg of body weight) in mice drinking a sweetened alcohol solution [20% alcohol (v/v), 3% glucose (w/v), and 0.1% saccharin (w/v)], and female mice drank significantly more alcohol than male mice. Males: n = 8; Females: n = 7. B Spironolactone dose-dependently reduced alcohol intake (g/kg of body weight) in mice drinking an unsweetened alcohol solution [20% (v/v)]. Males: n = 8; Females: n = 7. C Spironolactone had no effect on the intake (mL/kg of body weight) of a sweet solution without alcohol [0.3% glucose (w/v) and 0.01% saccharin (w/v)] in mice. Males: n = 8; Females: n = 7. Separate cohorts of mice were used for each drinking solution. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, vs. vehicle; #p < 0.05, male vs. female. DID drinking-in⁻the-dark.
Spironolactone did not affect motor coordination or spontaneous locomotion in mice
A Spironolactone treatment had no effect on motor coordination in mice that received a saline injection and were tested 30 min and 90 min later, on the rotarod. Males: n = 6; Females: n = 5. B Systemic administration of alcohol (1.5 g/kg) significantly impaired motor coordination in mice. Males: n = 6; Females: n = 5. Spironolactone treatment had no effect on alcohol-induced ataxia on the rotarod test at any time point. ####p < 0.0001, vs. Baseline. C Spironolactone had no effect on blood alcohol levels 30 min and 90 min after systemic administration of alcohol (1.5 g/kg). ####p < 0.0001, 30 min vs. 90 min. Males: n = 6; Females: n = 5. D Spironolactone had no effect on spontaneous locomotion in the circular corridor. Males: n = 8; Females: n = 6.
Spironolactone decreased operant alcohol self-administration in alcohol-dependent (DEP) and nondependent (NON) rats
A Spironolactone administration decreased alcohol self-administration in nondependent and alcohol-dependent male rats tested under a fixed-ratio 1 schedule of reinforcement. ****p < 0.0001, vs. vehicle. ####p < 0.0001, vs. NON. Nondependent: n = 12; Dependent: n = 12. B Alcohol-induced ataxia was higher in nondependent than dependent male rats; spironolactone did not affect alcohol-induced ataxia in either group. **p < 0.01, difference between dependent and nondependent male rats. Nondependent: n = 15; Dependent: n = 9. C Spironolactone had no effect on blood alcohol levels 30, 60, 120, and 180 min after systemic administration of alcohol (1.5 g/kg) in male rats. Nondependent: n = 15; dependent: n = 9. D Spironolactone administration decreased alcohol self-administration in nondependent and alcohol-dependent female rats tested under a fixed-ratio 1 schedule of reinforcement. *p < 0.05, ****p < 0.0001, vs. vehicle. ####p < 0.0001, vs. NON. Nondependent: n = 7; Dependent: n = 8.
Difference-in-difference estimates and 95% confidence intervals of self-reported changes in Alcohol Use Disorders Identification Test-Consumption-C (AUDIT-C) scores associated with spironolactone exposure, overall, by baseline AUDIT-C score, and by average daily dose of spironolactone
Difference-in-differences = reported AUDIT-C decrease among spironolactone-exposed individuals minus reported AUDIT-C decrease among propensity score-matched unexposed controls during the study period. *p < 0.05, ****p < 0.0001, NS not significant.
Evidence suggests that spironolactone, a nonselective mineralocorticoid receptor (MR) antagonist, modulates alcohol seeking and consumption. Therefore, spironolactone may represent a novel pharmacotherapy for alcohol use disorder (AUD). In this study, we tested the effects of spironolactone in a mouse model of alcohol drinking (drinking-in-the-dark) and in a rat model of alcohol dependence (vapor exposure). We also investigated the association between spironolactone receipt for at least 60 continuous days and change in self-reported alcohol consumption, using the Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), in a pharmacoepidemiologic cohort study in the largest integrated healthcare system in the US. Spironolactone dose-dependently reduced the intake of sweetened or unsweetened alcohol solutions in male and female mice. No effects of spironolactone were observed on drinking of a sweet solution without alcohol, food or water intake, motor coordination, alcohol-induced ataxia, or blood alcohol levels. Spironolactone dose-dependently reduced operant alcohol self-administration in dependent and nondependent male and female rats. In humans, a greater reduction in alcohol consumption was observed among those who received spironolactone, compared to propensity score-matched individuals who did not receive spironolactone. The largest effects were among those who reported hazardous/heavy episodic alcohol consumption at baseline (AUDIT-C ≥ 8) and those exposed to ≥ 50 mg/day of spironolactone. These convergent findings across rodent and human studies demonstrate that spironolactone reduces alcohol use and support the hypothesis that this medication may be further studied as a novel pharmacotherapy for AUD.
The precise development of the neocortex is a prerequisite for higher cognitive and associative functions. Despite numerous advances that have been made in understanding neuronal differentiation and cortex development, our knowledge regarding the impact of specific genes associated with neurodevelopmental disorders on these processes is still limited. Here, we show that Taok2, which is encoded in humans within the autism spectrum disorder (ASD) susceptibility locus 16p11.2, is essential for neuronal migration. Overexpression of de novo mutations or rare variants from ASD patients disrupts neuronal migration in an isoform-specific manner. The mutated TAOK2α variants but not the TAOK2β variants impaired neuronal migration. Moreover, the TAOK2α isoform colocalizes with microtubules. Consequently, neurons lacking Taok2 have unstable microtubules with reduced levels of acetylated tubulin and phosphorylated JNK1. Mice lacking Taok2 develop gross cortical and cortex layering abnormalities. Moreover, acute Taok2 downregulation or Taok2 knockout delayed the migration of upper-layer cortical neurons in mice, and the expression of a constitutively active form of JNK1 rescued these neuronal migration defects. Finally, we report that the brains of the Taok2 KO and 16p11.2 del Het mouse models show striking anatomical similarities and that the heterozygous 16p11.2 microdeletion mouse model displayed reduced levels of phosphorylated JNK1 and neuronal migration deficits, which were ameliorated upon the introduction of TAOK2α in cortical neurons and in the developing cortex of those mice. These results delineate the critical role of TAOK2 in cortical development and its contribution to neurodevelopmental disorders, including ASD.
Childhood apraxia of speech (CAS), the prototypic severe childhood speech disorder, is characterized by motor programming and planning deficits. Genetic factors make substantive contributions to CAS aetiology, with a monogenic pathogenic variant identified in a third of cases, implicating around 20 single genes to date. Here we aimed to identify molecular causation in 70 unrelated probands ascertained with CAS. We performed trio genome sequencing. Our bioinformatic analysis examined single nucleotide, indel, copy number, structural and short tandem repeat variants. We prioritised appropriate variants arising de novo or inherited that were expected to be damaging based on in silico predictions. We identified high confidence variants in 18/70 (26%) probands, almost doubling the current number of candidate genes for CAS. Three of the 18 variants affected SETBP1, SETD1A and DDX3X, thus confirming their roles in CAS, while the remaining 15 occurred in genes not previously associated with this disorder. Fifteen variants arose de novo and three were inherited. We provide further novel insights into the biology of child speech disorder, highlighting the roles of chromatin organization and gene regulation in CAS, and confirm that genes involved in CAS are co-expressed during brain development. Our findings confirm a diagnostic yield comparable to, or even higher, than other neurodevelopmental disorders with substantial de novo variant burden. Data also support the increasingly recognised overlaps between genes conferring risk for a range of neurodevelopmental disorders. Understanding the aetiological basis of CAS is critical to end the diagnostic odyssey and ensure affected individuals are poised for precision medicine trials.
RtTg glutamatergic neurons exhibit elevated activities during FPS
a Schematic for the establishment of FPS paradigm in mice. b Startle amplitudes of non-conditioned or fear-conditioned mice in the presence or absence of the light (CS) (n = 10 per group; t = 0.1155, P = 0.9917 (non-conditioned); t = 7.4760, P < 0.0001 (conditioned)). c The quantity distribution of mice with different %FPS values. Representative images (d) and quantitative analysis (e) showing c-fos expression in various brain regions of control, startle and FPS mice (n = 6 per group; for control vs startle, t = 0.3036, P = 0.9992 (BLA); t = 0.8096, P = 0.9355 (LHb); t = 6.903, P < 0.0001 (PVT); t = 0.4843, P = 0.9931 (DRN); t = 16.69, P < 0.0001 (RtTg); for startle vs FPS, t = 3.504, P = 0.0049 (BLA); t = 3.404, P = 0.0066 (LHb); t = 6.220, P < 0.0001 (PVT); t = 6.258, P < 0.0001 (DRN); t = 8.151, P < 0.0001 (RtTg)). Scale bar, 200 μm. BLA basolateral amygdaloid nucleus, LHb lateral habenular nucleus, PVT paraventricular thalamic nucleus, DRN dorsal raphe nucleus. f Quantification of c-fos⁺ cell number in consecutive RtTg sections of control, startle and FPS mice (n = 6 per group). g, h Representative images (g) and percentage (h) of RtTg c-fos⁺ cells co-labelled with glutamate during startle and FPS respectively (n = 8 per group; t = 1.628, P = 0.2163 (c-fos⁺Glu⁺/c-fos⁺); t = 6.804, P < 0.0001 (c-fos⁺Glu⁺/Glu⁺)). Scale bar, 50 μm. i Schematic of fiber photometry setup for monitoring calcium signals of RtTg glutamatergic neurons during FPS. Sample traces (j) and quantifications (k, n = 25 bouts from 5 mice) of calcium signal changes of RtTg glutamatergic neurons during startle and FPS. Thick line indicates mean and the area shaded in lighter color indicates s.e.m. l Schematic experimental procedure for assessing the effect of chemogenetic inhibition of RtTg glutamatergic neurons on the FPS behavior. m Representative image showing the expression of hM4Di-mCherry in the RtTg. Scale bar, 200 μm. n Quantitative %FPS values of hM4Di⁺ group (n = 10 mice; t = 6.101, P < 0.0001) and mCherry⁺ group (n = 10 mice; t = 0.9397, P = 0.3598) of mice receiving CNO or saline administration. All data are presented as the mean ± s.e.m. Significance was assessed between groups by two-way ANOVA with Sidak post hoc in b, e, h, and two-sided unpaired t-tests in n. ** P < 0.01; *** P < 0.001; ns not significant.
Screening of candidate RtTg upstream regulatory nucleus involved in the regulation of FPS
a Schematic for retrograde tracing experiment starting from the RtTg. Representative images showing RtTg injection site (b) as well as retrogradely labeled EGFP⁺ cells in different brain nuclei (c). Scale bar, 300 μm. LCN lateral cerebellar nucleus, CN cochlear nucleus, MHb medial habenular nucleus, PrH nucleus prepositus hypoglossus, Cg1 cingulate cortex, area 1, Cg cingulate cortex, area 2, M1 primary motor cortex; M2, secondary motor cortex, AuC auditory cortex, S1 primary somatosensory cortex. d Quantification of retrogradely labeled EGFP⁺ cells of different brain nuclei (n = 8 mice). e Schematic for chemogenetic activation of RtTg-projecting DRN neurons. f, g Quantitative startle amplitudes of hM3Dq⁺ group and mCherry⁺ group of mice receiving CNO (n = 8 mice) or saline (n = 8 mice) administration into the DRN (f, t = 4.171, P = 0.0004 (85 dB); t = 4.009, P = 0.0007 (90 dB); t = 4.757, P < 0.0001 (95 dB); gt = 0.6783, P = 0.8760 (85 dB); t = 0.0019, P = 0.9999 (90 dB); t = 0.3334, P = 0.7405 (95 dB)). h Representative image showing RtTg-projecting DRN EGFP⁺ neurons co-labelled with 5-HT (white arrowheads). Scale bar, 50 μm. i Pie chart indicating the percentage of RtTg-projecting DRN EGFP⁺ neurons co-labelled with or without 5-HT. All data are presented as the mean ± s.e.m. Significance was assessed between groups by two-way ANOVA with Sidak post hoc. ** P < 0.01; *** P < 0.001; ns not significant.
DRN 5-HTergic neurons specifically project to RtTgP GABAergic neurons that form inhibitory microcircuit with RtTgC glutamatergic neurons
a Schematic for the anterograde transsynaptic tracing from DRN to RtTgP. b Representative images showing DRN injection site (middle) and tdT⁺ signals specifically distributed in the RtTgP. Scale bar, 200 μm. c Left: schematic for the anterograde tracing from DRN 5-HTergic neurons to RtTgP GABAergic neurons by injecting AAV-DIO-EGFP-T2A-Synaptophysin:mCherry into the DRN of Pet1-Cre:GAD1-EGFP mice. Representative image (middle) and quantitative analysis (right) showing co-existence of mCherry⁺ presynaptic terminals of DRN 5-HTergic neurons with GAD⁺ neurons in the RtTgP (white arrowheads). Scale bar, 50 μm. d Schematic for the retrograde mono-transsynaptic tracing from RtTgP to DRN by injecting RV-EnvA-ΔG-DsRed and helper AAVs into the RtTgP of Vgat-Cre mice. e Representative images showing the starter cells (blue arrowheads) in the RtTgP injection site. DsRed⁺ DRN neurons traced from RtTgP were co-labelled with 5-HT (white arrowheads). Scale bar, 200 μm (Starter cell) and 50 μm (Merged). f Pie chart indicating the percentage of RtTgP-projecting DRN RV⁺ neurons co-labelled with or without 5-HT. g Left: schematic for optogenetic activation of RtTgP neuronal terminals and simultaneous whole-cell patch clamp recording of RtTg neurons in the RtTg brain slice of Vgat-Cre mice receiving injection of the AAV-DIO-ChR2-EGFP. Right: representative image showing RtTgP EGFP⁺ presynaptic terminals co-existed with RtTgC neurons immunopositive for glutamate (white arrowheads). Scale bar, 30 μm. h Sample traces (left) and quantitative analysis (right) for IPSCs of RtTg neurons evoked by photostimulation (blue bar, 1 ms) before or after bath application of bicuculline (Bic, 20 μM) (n = 6 cells from 3 mice, P = 0.0009). TTX and 4-AP were added into aCSF. i Left: schematic for chemogenetic inhibition of GABAergic neurons in the RtTgP of Vgat-Cre mice. j Startle amplitudes of hM3Dq⁺ group of mice receiving CNO or saline administration (n = 8 per group; t = 2.542, P = 0.0412 (85 dB); t = 4.516, P = 0.0001 (90 dB); t = 5.030, P < 0.0001 (95 dB)). All data are presented as the mean ± s.e.m. Significance was assessed between groups by two-sided paired t-tests in h and two-way ANOVA with Sidak post hoc in j. * P < 0.05; ** P < 0.01; *** P < 0.001; ns not significant.
Functional verification of a DRN-RtTgP-RtTgC disinhibitory circuit
a Schematic for monitoring calcium signals of RtTgP-projecting DRN 5-HTergic neurons in freely moving Pet1-Cre mice receiving injection of a Retro-AAV-DIO-GCaMP6m into the RtTgP. b Representative traces of the DRN calcium signal transients aligned to the light (CS) onset. Heat map (c, n = 3 bouts from individual mouse) and quantification of calcium signal changes (d) in the RtTgP-projecting DRN 5-HTergic neurons of FPS mice (n = 15 bouts from 5 mice) and non-conditioned mice (n = 15 bouts from 5 mice) during 3-s light (CS). Thick line indicate mean and the area shaded in lighter color indicates s.e.m. e Schematic for the optogenetic activation of the terminals of DRN 5-HTergic neurons and simultaneous cell-attached recording of RtTgP EGFP⁺ neurons in the RtTg brain slice of Pet1-Cre::GAD1-EGFP mice receiving an injection of AAV-DIO-ChR2-mCherry into the DRN. f, g Sample traces (f) and quantitative analysis (g) for the spontaneous firing frequency of RtTgP EGFP⁺ neurons before or after optogenetic activation of 5-HTergic DRN-RtTgP projections (n = 9 cells from 3 mice; P < 0.0001 (g, left); P < 0.0001 (g, right)). h Schematic for chemogenetic activation of the terminals of DRN 5-HTergic neurons and simultaneous whole-cell patch clamp recording of RtTg EGFP⁻ neurons in the RtTg brain slice of mice receiving an injection of AAV-DIO-hM3Dq-mCherry into the DRN. Sample traces (i) and quantitative analysis (j) for the GABA sIPSCs in the RtTgC neurons pre and post chemogenetic activation of 5-HTergic DRN-RtTgP projections (n = 8 cells from 4 mice; P = 0.0297 (j, left); P = 0.2102 (j, right)). k Left: schematic experimental procedure for assessing the effect of chemogenetic activation of DRN-RtTgP projections on startle level in Pet1-Cre mice receiving injection of an AAV-DIO-hM3Dq-mCherry or AAV-DIO-mCherry into the DRN. Right: representative image showing the expression of hM3Dq-mCherry in the DRN. Scale bar, 200 μm. l Startle amplitudes of hM3Dq⁺ group (n = 8; t = 2.545, P = 0.0434 (85 dB); t = 2.713, P = 0.0286 (90 dB); t = 3.558, P = 0.0028 (95 dB)) and mCherry⁺ control group (n = 8; t = 0.3421, P = 0.9812 (85 dB); t = 0.2478, P = 0.9926 (90 dB); t = 0.3230, P = 0.9840 (95 dB)) of mice receiving CNO (0.2 μg/μl) or saline administration into the RtTgP. m Left: schematic experimental procedure for assessing the effect of chemogenetic inhibition of the DRN-RtTgP projections on FPS behavior in Pet1-Cre mice receiving injection of an AAV-DIO-hM4Di-mCherry or AAV-DIO-mCherry into the DRN. Right: representative image showing the expression of hM4Di-mCherry in the DRN. Scale bar, 200 μm. n Quantitative FPS values (%) of hM4Di⁺ group (n = 10; P = 0.0005) and control mCherry⁺ group (n = 10; P = 0.9214) of mice receiving CNO (0.2 μg/μl) or saline administration. All data are presented as the mean ± s.e.m. Significance was assessed between groups by two-sided paired t-tests in g, j, two-way ANOVA with Sidak post hoc in l, and two-sided unpaired t-tests in n. * P < 0.05; ** P < 0.01; *** P < 0.001; ns not significant.
Screening of RtTgP 5-HT receptor subtypes responsible for FPS
a Schematic experimental procedure for evaluating the effect of intra-RtTgP injection of various 5-HT1R agonists on startle level. Quantitative startle amplitudes of mice receiving intra-RtTgP injection of the 5-HT1R agonist 5-CT (b, n = 7 per group; t = 2.521, P = 0.0481 (85 dB); t = 3.241, P = 0.0077 (90 dB); t = 3.795, P = 0.0016 (95 dB)), the 5-HT1BR agonists CP93129 (c, n = 8 per group; t = 2.840, P = 0.0206 (85 dB); t = 2.957, P = 0.0152 (90 dB); t = 4.698, P < 0.0001 (95 dB)) or the vehicle. d Representative image (left) and pie chart (right) indicating the percentage of GAD-EGFP⁺ RtTgP neurons co⁻labelled with or without 5-HT1BRs. Scale bar, 50 μm. Sample traces (e) and quantitative analysis (f) for the spontaneous firing frequency of RtTgP EGFP⁺ neurons during optogenetic activation of 5-HTergic DRN-RtTgP projections with or without bath application of 5-HT1BR antagonists GR55562 (20 μM, n = 9 cells from 4 mice; for baseline vs BL, P < 0.0001; for BL vs BL + GR55562, P < 0.0001; for baseline vs BL + GR55562, P = 0.5770). g Schematic experimental procedure for evaluating the effect of intra-RtTgP injection of 5-HT1BR antagonists GR55562 (5 μg/μl) on the FPS behavior. h Quantitative FPS value of mice receiving intra-RtTgP injection of the 5-HT1BR antagonist GR55562 or the vehicle (n = 8 per group; P = 0.0280). i Schematic of Cre-dependent AAV based 5-HT1BR silencing. j Relative mRNA level of 5-HT1BRs in RtTgP of Vgat-Cre mice injected with 5-HT1BR (n = 9) shRNA or control shRNA (n = 10; P < 0.0001). k Representative Western blot (left) and quantitative analysis (right) of 5-HT1BR expression in the RtTgP of Vgat-Cre mice injected with 5-HT1BR shRNA or control shRNA (n = 5 per group; P < 0.0001). l Quantitative FPS value of Vgat-Cre mice receiving bilateral intra-RtTgP injection of 5-HT1BR shRNA (n = 9) or control shRNA (n = 10; P < 0.0001). m Schematic experimental procedure for evaluating the effect of i.p. injection of fluoxetine on startle behavior. n Quantitative ASR amplitudes of mice receiving i.p. injection of fluoxetine (10 mg/kg) or the vehicle (n = 10 per group; t = 1.136, P = 0.5966 (85 dB); t = 0.6066, P = 0.9068 (90 dB); t = 0.3116, P = 0.9856 (95 dB)). o Schematic experimental procedure for evaluating the rescue role of GR55562 on %FPS change caused by i.p. injection of fluoxetine. p Quantitative FPS value of mice receiving i.p. injection of GR55562/fluoxetine/fluoxetine + GR55562 or the vehicle (n = 10 per group; vehicle vs GR55562, t = 6.101, P < 0.0001; vehicle vs fluoxetine, t = 2.182, P = 0.0426; fluoxetine vs fluoxetine + GR55562, t = 4.590, P = 0.0002). All data are presented as the mean ± s.e.m. Significance was assessed between groups by two-way ANOVA with Sidak post hoc in b, c, n, two-sided paired t-tests in f, and two-sided unpaired t-tests in h, j–l, p. * P < 0.05; ** P < 0.01; *** P < 0.001; ns not significant.
Exaggerated startle has been recognized as a core hyperarousal symptom of multiple fear-related anxiety disorders, such as post-traumatic stress disorder (PTSD) and panic disorder. However, the mechanisms driving this symptom are poorly understood. Here we reveal a neural projection from dorsal raphe nucleus (DRN) to a startle-controlling center reticulotegmental nucleus (RtTg) that mediates enhanced startle response under fear condition. Within RtTg, we identify an inhibitory microcircuit comprising GABAergic neurons in pericentral RtTg (RtTgP) and glutamatergic neurons in central RtTg (RtTgC). Inhibition of this RtTgP-RtTgC microcircuit leads to elevated startle amplitudes. Furthermore, we demonstrate that the conditioned fear-activated DRN 5-HTergic neurons send inhibitory projections to RtTgP GABAergic neurons, which in turn upregulate neuronal activities of RtTgC glutamatergic neurons. Chemogenetic activation of the DRN-RtTgP projections mimics the increased startle response under fear emotions. Moreover, conditional deletion of 5-HT1B receptor from RtTgP GABAergic neurons largely reverses the exaggeration of startle during conditioned fear. Thus, our study establishes the disinhibitory DRN-RtTgP-RtTgC circuit as a critical mechanism underlying exaggerated startle under fear emotions, and provides 5-HT1B receptor as a potential therapeutic target for treating hyperarousal symptom in fear-associated psychiatric disorders.
Virtually all neuropsychiatric disorders display sex differences in prevalence, age of onset, and/or clinical symptomology. Although altered dopamine (DA) signaling is a feature of many of these disorders, sex-dependent mechanisms uniquely responsive to DA that drive sex-dependent behaviors remain unelucidated. Previously, we established that anomalous DA efflux (ADE) is a prominent feature of the DA transporter (DAT) variant Val559, a coding substitution identified in two male-biased disorders: attention-deficit/hyperactivity disorder and autism spectrum disorder. In vivo, Val559 ADE induces activation of nigrostriatal D2-type DA autoreceptors (D2ARs) that magnifies inappropriate, nonvesicular DA release by elevating phosphorylation and surface trafficking of ADE-prone DAT proteins. Here we demonstrate that DAT Val559 mice exhibit sex-dependent alterations in psychostimulant responses, social behavior, and cognitive performance. In a search for underlying mechanisms, we discovered that the ability of ADE to elicit D2AR regulation of DAT is both sex and circuit-dependent, with dorsal striatum D2AR/DAT coupling evident only in males, whereas D2AR/DAT coupling in the ventral striatum is exclusive to females. Moreover, systemic administration of the D2R antagonist sulpiride, which precludes ADE-driven DAT trafficking, can normalize DAT Val559 behavioral changes unique to each sex and without effects on the opposite sex or wildtype mice. Our studies support the sex- and circuit dependent capacity of D2ARs to regulate DAT as a critical determinant of the sex-biased effects of perturbed DA signaling in neurobehavioral disorders. Moreover, our work provides a cogent example of how a shared biological insult drives alternative physiological and behavioral trajectories as opposed to resilience.
Structural and functional locus coeruleus (LC) connectivity
Illustration of structural (a, c; SIFT2 filtered tractography) and functional (b, 35 min resting-state fMRI) LC connectivity in one example participant. The segmented brain regions represent the individual subjects’ brain anatomy of the areas that are connected with the LC. The opacity and color of those regions illustrate the strength of connectivity where blue colored regions show highest connectivity.
Group statistics and relationship between functional and structural locus coeruleus (LC) connectivity
Regions with high structural connectivity (a, DTI) are also strongly functionally (a, fc) connected to the LC (FDR p < 0.05), most evident in the subcortical thalamic, ventral diencephalic, cerebellar regions and the presubiculum (regions counter-clockwise ordered by strongest structural and functional connections, see also Supplementary Table 3 for Glasser abbreviations). In (b), colors and opacity illustrate group statistics of strength of functional (left column, group level FDR p < 0.05, h-values) and structural connectivity (DTI, right column, group level FDR p < 0.05, F values) to the whole brain, with blue colored regions representing strongest connectivity.
Structural and functional locus coeruleus (LC) whole-brain connectivity showed a linear relationship (Pearson’s r = 0.687, p = 0.001)
Big circles represent mean connectivity values to each brain region (right and left hemisphere) whilst small symbols illustrate connectivity on the individual subject level (e.g., two small red triangles represent LC connectivity to the right and left thalamus for one subject). This association is driven by regions with both high structural and functional connectivity, which reveals that brain areas with high structural connectivity to the LC are also tightly functionally linked to the LC in the resting state, e.g., the thalamus or ventral diencephalon.
Relationship between locus coeruleus (LC) structural connectivity and psychological traits
Most brain areas (a, b) that are structurally connected to the locus coeruleus (LC) showed a negative correlation to anxiety (STAI, a) and positive correlation to alertness (b) scores. All areas depicted in the further-back anatomy illustrations in (a, b) significantly correlated to anxiety (a) and alertness (b) with respect to LC structural connectivity, whereas the foreground illustrations single out the correlations with a very high r-coefficient only (r > 0.7, see Supplementary Table 3 for Glasser abbreviations). c Shows LC whole brain structural connectivity (raw z-values, see also Supplementary Fig. 2 for all subjects) of three example subjects: subject ‘8’, whose behavioral score was high in anxiety/low in alertness and who exhibits overall low structural LC connectivity, subject ‘5’ with medium values, and subject ‘7’ with low anxiety/high alertness effects and overall dense LC structural connectivity. c Illustrates, how structural connectivity of single subjects may provide a first impression of psychological traits.
The locus coeruleus (LC) in the brainstem as the main regulator of brain noradrenaline gains increasing attention because of its involvement in neurologic and psychiatric diseases and its relevance in general to brain function. In this study, we created a structural connectome of the LC nerve fibers based on in vivo MRI tractography to gain an understanding into LC connectivity and its impact on LC-related psychological measures. We combined our structural results with ultra-high field resting-state functional MRI to learn about the relationship between in vivo LC structural and functional connections. Importantly, we reveal that LC brain fibers are strongly associated with psychological measures of anxiety and alertness indicating that LC-noradrenergic connectivity may have an important role on brain function. Lastly, since we analyzed all our data in subject-specific space, we point out the potential of structural LC connectivity to reveal individual characteristics of LC-noradrenergic function on the single-subject level.
Results from the static connectivity: Positive association of right amygdala seed-based connectivity with PSS10 scores
A positive association between PSS10 scores and right amygdala seed-based connectivity was observed within 17 clusters. ARepresentation of the most significant clusters obtained. Each voxel is colored by its statistical significance, and the right amygdala seed region is colored in blue. B3D representation of all clusters obtained, as well as its connection with right amygdala. Each cluster is represented by a sphere colored by the strength of the connectivity (stronger connection in yellow). Spheres sizes were defined as an approximation of clusters sizes, using a pre-defined range interval. Seed-Based connectivity analysis were made using FSL, with individual seed connectivity maps as the variable of interest, PSS10 scores as the independent term, and age and sex as covariates. The statistical significance was considered for p values < 0.05, after threshold-free cluster enhancement (TFCE) and family-wise error rate (FWE-R) correction.
Lower perceived stress relates to more counterbalanced activity between amygdala-hippocampus and the rest of the brain
A The functional phase-locking pattern represented by the cluster centroid Vc obtained for k = 16, c = 4. Elements in Vc(n) are sorted in descending order and colored (from orange to dark blue) according to their relative phase shift. The arrows on the left indicate the seed region (red) and the significant clusters (blue) from the seed-based analysis. On the right, a 3D rendering of the brain regions color-coded according to Vc(n). B The probability of occurrence of this pattern is plotted against PSS10 scores for all 252 subjects, as well as the trendline of the negative association found. B1–B2 For two representative subjects, the BOLD signals averaged across subsystems with the same color in (A). The gray patches in the background highlight the time points when this pattern was detected, revealing clearly more occurrence for the subject with lower perceived stress. Notably, even when this pattern is not detected, there is clearly less synchronicity between the fMRI signals in subject 2.
The significant link between stress and psychiatric disorders has prompted research on stress’s impact on the brain. Interestingly, previous studies on healthy subjects have demonstrated an association between perceived stress and amygdala volume, although the mechanisms by which perceived stress can affect brain function remain unknown. To better understand what this association entails at a functional level, herein, we explore the association of perceived stress, measured by the PSS10 questionnaire, with disseminated functional connectivity between brain areas. Using resting-state fMRI from 252 healthy subjects spanning a broad age range, we performed both a seed-based amygdala connectivity analysis (static connectivity, with spatial resolution but no temporal definition) and a whole-brain data-driven approach to detect altered patterns of phase interactions between brain areas (dynamic connectivity with spatiotemporal information). Results show that increased perceived stress is directly associated with increased amygdala connectivity with frontal cortical regions, which is driven by a reduced occurrence of an activity pattern where the signals in the amygdala and the hippocampus evolve in opposite directions with respect to the rest of the brain. Overall, these results not only reinforce the pathological effect of in-phase synchronicity between subcortical and cortical brain areas but also demonstrate the protective effect of counterbalanced (i.e., phase-shifted) activity between brain subsystems, which are otherwise missed with correlation-based functional connectivity analysis.
Two residues important for normal neurological function interact during NMDA receptor gating
a Structural model of a core GluN1/GluN2A receptor lacking NTD and CTD (N1∆N∆C, tan; N2A∆N∆C, cyan). b Within the GluN2A subunit, the interaction between P552 and F652 (top) is activity-dependent as indicated by smaller center-of-mass distance (COM) (bottom left), and stronger Van der Waals contact energy (VdW) (bottom right), in open vs. closed conformations. *p < 0.05 Kolmogorov–Smirnoff test. c Substitutions at both P552 and F652 change the VdW contact energy between these residues in structural models of open receptors, consistent with the disruption of a gating-favorable interaction. *p < 0.05 Kolmogorov–Smirnoff test. d Side chains of both P552 and F652 contribute to the gating kinetics of full-length NMDA receptors; substitutions at these sites (P/A and F/V) changed the pattern of current recorded from individual receptors, the distribution of closed (black) and open (red) intervals, and the gating rate constants calculated with the indicted state models. Macroscopic current responses to pulses (5 s) of glutamate (1 mM) predicted by each model (red) are shown superimposed with experimentally recorded whole-cell currents (green and yellow). *p < 0.05 two-tailed t-test. e, left Diagram of the thermodynamic cycle used to calculate the coupling energy between P552 and F652 using the rates illustrated in d. right Energy landscapes calculated for the gating reactions of individual receptors illustrate increased barriers to activation in receptors with mutations at disease-associated residues.
NMDA receptor variants associated with neurological dysfunction display a broad range of gating perturbations
a Structural models of open NMDA receptors variants illustrate the relative positions of two disease-associated residues. b Relative to receptors with wild-type residues, the modeled open states of receptors with disease-associated mutations have distinct Van der Waals contact energies (VdW) between residues 552 and 652 of GluN2A (*p < 0.05; Kolmogorov–Smirnov test). c, top Currents recorded from individual full-length GluN1/GluN2AP552R receptors (n = 16); (middle) Dwell-time histograms of closed (left) and open (right) interval durations with superimposed distributions (lines) predicted by the model illustrated below; (bottom) Energy landscapes calculated from the kinetic models derived for the indicated receptors. d Distributions of gating parameters estimated for the indicated full length receptor types: open probabilities (Po), mean open (MOT) and mean closed (MCT) durations estimated for entire records or for bursts of activity. (*p < 0.05; Student’s t test).
NMDA receptor variants associated with neurological dysfunction display changes in conductance and permeability
aTop, Whole-cell current trace recorded in response to Glu application (1 mM) illustrates change in steady-state current amplitude during a ramp in the membrane potential. Bottom, Macroscopic current-voltage relationships measured from recordings as in a. b Ca²⁺ permeability properties inferred from macroscopic current recordings as in a. c Top, Unitary current traces recorded from cell-attached patches with +100 mV applied potential and external Ca²⁺ as indicated. Bottom, Unitary current-voltage relationships for the indicated receptors and summary of results. (*p < 0.05; Student’s t test relative to WT).
Disease-associated variants display complex functional changes
a, left Whole-cell currents evoked with several concentrations of glutamate in saturating glycine (0.1 mM); (right) Glutamate dose-dependence of the macroscopic steady-state current amplitude. b, left The rise and decay phases of the macroscopic current (black) recorded from the GluN1/GluN2AP552R variant superimposed with fits to exponential functions (red). (Right) Glutamate dose-dependence of the rise time (circles) and fit to linear function (line) used to estimate the apparent glutamate binding rate. c, left Reaction mechanisms derived from fitting each model simultaneously to all single-channel recordings in each data set. (Right) Macroscopic current responses simulated with the respective kinetic models (left) and corresponding experimentally recorded whole-cell currents (right). d Simulated responses to a synaptic-like glutamate exposure (1 ms, 1 mM) predict drastic changes in peak current levels (left), time course (middle), and charge transfer. e Simulated responses to extrasynaptic-like glutamate exposure (5 s, 2 μM) predict complex changes in steady-state current levels (left), kinetics (middle), and charge transfer (right). fTop, Simulated responses to repetitive exposure to synaptic-like pulses predict complex changes in frequency-dependent facilitation. Bottom, Cumulative charge transfer over 60 s of repetitive stimulation over varying physiologic frequencies. gTop, Simulation response to standard theta-burst stimulation. Middle, Expanded view of the normalized response to a single train of stimuli. Bottom, Expanded view of the normalized response to the first and last epoch of the first train of stimuli.
Proposed role of P552/F652 interaction in the gating reaction of NMDA receptors
1 Residues on the D2-M1 linker of GluN2A subunits (blue), such as L550 and P552, interact directly with residues located on the gate-forming M3 helix of the same subunit (P552/F652) or of the adjacent GluN1 subunit (yellow) (L550/I642). 2 Agonist-triggered contraction of the LBD domain induces outward movement of the D2-M1 linker and causes (3) stabilization of the open M3 position.
NMDA receptors have essential roles in the physiology of central excitatory synapses and their dysfunction causes severe neuropsychiatric symptoms. Recently, a series of genetic variants have been identified in patients, however, functional information about these variants is sparse and their role in pathogenesis insufficiently known. Here we investigate the mechanism by which two GluN2A variants may be pathogenic. We use molecular dynamics simulation and single-molecule electrophysiology to examine the contribution of GluN2A subunit-residues, P552 and F652, and their pathogenic substitutions, P552R and F652V, affect receptor functions. We found that P552 and F652 interact during the receptors’ normal activity cycle; the interaction stabilizes receptors in open conformations and is required for a normal electrical response. Engineering shorter side-chains at these positions (P552A and/or F652V) caused a loss of interaction energy and produced receptors with severe gating, conductance, and permeability deficits. In contrast, the P552R side chain resulted in stronger interaction and produced a distinct, yet still drastically abnormal electrical response. These results identify the dynamic contact between P552 and F652 as a critical step in the NMDA receptor activation, and show that both increased and reduced communication through this interaction cause dysfunction. Results show that subtle differences in NMDA receptor primary structure can generate complex phenotypic alterations whose binary classification is too simplistic to serve as a therapeutic guide.
Features of SCZ-specific CRDs
A Difference in medians per peak activity between SCZ cases and controls as a function of the strength of association given in -log10 p-values; purple dots denote peaks that are differentially active between SCZ cases and controls at FDR 5% (3450 peaks) with light purple and dark purple indicating lower and higher median activity, respectively, in SCZ cases compared to controls. B Comparison of differences in per CRD peak activity correlation estimates between SCZ cases and controls for SCZ-specific CRDs (3078 CRDs). C Example region of a correlation structure between 250 peaks on chromosome 5 in SCZ cases and controls, revealing a well-delimited SCZ-specific CRD that is composed of five regulatory elements. The higher the correlation between peaks, the darker the colour blue. D Proportion of SCZ-specific CRD peaks that overlap with active regulatory regions identified in fetal samples (Fisher’s exact test p = 0.001, OR = 1.52) compared to those captured in adults (Fisher’s exact test p = 3.04 × 10⁻⁵⁰, OR = 0.57).
Association between CRDs and genes
A Expression of differentially expressed genes (DEGs) is correlated with the activity of differentially active CRDs (DACs) significantly more often than expected by chance (Fischer’s exact test p = 3.43 × 10⁻⁵, OR = 1.65); coloured dots denote DEGs genome-wide identified at FDR 5%: purple dots mark DEGs, blue dots mark DEGs whose expression correlates with the activity of a DAC in the same direction, red dots mark DEGs whose expression correlates with the activity of a DAC in the opposite direction. B Distribution of gene-to-CRD distances for genes localizing outside the associated CRD boundary (545 gene-CRD associations). C Distribution of the relative position of gene TSS to the boundary of an associated CRD (652 gene-CRD associations).
SCZ-specific QTL effects
Genotype-dependent effect on (A) CRD activity and (B) gene expression identified only in SCZ cases.
Regulatory mechanisms for eCRDQTL-CRD-gene triplets in SCZ cases and controls
Comparison of the direction of effect from eCRD-QTL onto gene expression and CRD activity for tested triplets between SCZ cases and controls: A triplet count for models showing the same regulatory mechanism in SCZ cases and controls, and B triplet count for models showing a change in the regulatory mechanism between SCZ cases and controls (term “different” indicates either reactive or independent model; light blue bar indicates triplets, for which the causal model (i.e., mediation via CRD for QTL effect) was not identified in SCZ cases nor in controls). C Distinct regulatory mechanism of the genetic regulation on gene expression for SCZ cases and controls for a triplet consisting of an eCRD-QTL chr6:169646282:A:T, gene THBS2 and a CRD composed of 18 REs on chr6:169,541,739–169,999,929; the probabilities based on the BN analysis for each tested model is given above schematics; shading of the colour for the gene and for the CRD indicates strength in expression and activity, respectively, relative to the other disease status group (SCZ cases vs controls). D, E Distribution of CRD activity and THBS2 gene expression for SCZ cases and controls.
Schizophrenia is a polygenic psychiatric disorder with limited understanding about the mechanistic changes in gene expression regulation. To elucidate on this, we integrate interindividual variability of regulatory activity (ChIP-sequencing for H3K27ac histone mark) with gene expression and genotype data captured from the prefrontal cortex of 272 cases and controls. By measuring interindividual correlation among proximal chromatin peaks, we show that regulatory element activity is structured into 10,936 and 10,376 cis-regulatory domains in cases and controls, respectively. The schizophrenia-specific cis-regulatory domains are enriched for fetal-specific (p = 0.0014, OR = 1.52) and depleted of adult-specific regulatory activity (p = 3.04 × 10⁻⁵⁰, OR = 0.57) and are enriched for SCZ heritability (p = 0.001). By studying the interplay among genetic variants, gene expression, and cis-regulatory domains, we ascertain that changes in coordinated regulatory activity tag alterations in gene expression levels (p = 3.43 × 10⁻⁵, OR = 1.65), unveil case-specific QTL effects, and identify regulatory machinery changes for genes affecting synaptic function and dendritic spine morphology in schizophrenia. Altogether, we show that accounting for coordinated regulatory activity provides a novel mechanistic approach to reduce the search space for unveiling genetically perturbed regulation of gene expression in schizophrenia.
Effect of SAFit2 on hippocampal neurite outgrowth
Hippocampal neurons were isolated from E18 mice and exposed 48 h to SAFit2 or DMSO before fixation and beta II tubulin immunohistochemistry (A). SAFit2 increased neurite length, insets show ×2 magnifications (B). Quantification revealed a dose-dependent increase in neurite length (C) and nodes (D). Comparison to BDNF (40 ng/mL) confirmed neurotrophic effect of SAFit2 (500 nM) (E). Quantification revealed that SAFit2 induced a larger increase in neurite length and nodes (F, G). Results correspond to three independent cultures with triplicates in each (mean + SEM, n = 9 per group). DIV, day in vitro; ihc, immunohistochemistry; BDNF, brain-derived neurotrophic factor. *p < 0.05, **p < 0.01, ***p < 0.001 vs DMSO, #p < 0.05, ###p < 0.001 vs BDNF by one-way ANOVA with Tukey post hoc test.
Effect of SAFit2 on hippocampal NPC differentiation
Hippocampal NPCs were isolated from P28 male rats, grown for four days as neurospheres, then dissociated and kept for 7 days under differentiation conditions while exposed to 1, 10, or 100 nM SAFit2 or DMSO (A). SAFit2 increased NPC differentiation into beta III tubulin positive cells and the neurite length (B). Quantification revealed that 100 nM significantly increased the amount of beta III tubulin positive cells (C). Quantification revealed that SAFit2 induced a dose-dependent increase in neurite length (D) and an increase in the proportion of neurons with secondary and tertiary processes (E, F). SAFit2 induced no change in the number of neurons with primary branches (G) but increased those with secondary and tertiary branches (H, I). Results correspond to three independent cultures with triplicates in each (mean + SEM, n = 9 per group). NPC neuroprecursor cells, DIV day in vitro, P postnatal day. *p < 0.05, **p < 0.01, ***p < 0.001 vs DMSO, by one-way ANOVA with Tukey post hoc test.
Effect of SAFit2 on hippocampal NPC proliferation
Hippocampal NPC were isolated from P28 male rats, grown for four days as neurospheres, then dissociated and kept in a proliferative environment with 0.02% BrdU for 4 hs while exposed to 1, 10 or 100 nM SAFit2 or DMSO (A). Representative images of BrdU staining (B). Quantification of nuclear BrdU-positive NPCs revealed that 100 nM SAFit2 significantly increased NPC proliferation (C). Results correspond to three independent cultures with triplicates in each (mean + SEM, n = 9 per group). NPC neuroprecursor cells, DIV day in vitro, P postnatal day. *p < 0.05 vs DMSO, by one-way ANOVA with Tukey post hoc test.
Time dependent action of SAFit2 in the forced swim test (FST)
SAFit2 (20 mg/kg) or vehicle was administered i.p. 16 or 1 h prior to FST. Blood was collected before the behavioral testing and 30 and 90 min after (A). SAFit2 decreased the time spent immobile in FST when administered 16 h before the test but not 1 h (B). mean + SEM, n = 7–8 per group, *p < 0.05 vs Vehicle by Student t test.
Behavioral and biochemical effect of chronic SAFit2 administration on chronic psychosocial stress
Schematic representation of experimental design (A). Briefly, SAFit2 (20 mg/kg) or vehicle was administered i.p. twice-a-day for five weeks while animals were subjected to social defeat and overcrowding. A separate group was administered fluoxetine i.p. (10 mg/kg once a day) while exposed to the stress. Blood was collected at days 17 and 35 of experiment and behavioral testing took place during the last two weeks of experiment. Administration of SAFit2 increased the latency to the first social defeat (B). Both SAFit2 and fluoxetine prevented the decrease in social interaction caused by chronic psychosocial stress (C). Stress induced a decrease in the distance traveled in the central area of the open field that was prevented by the administration of SAFit2 during the second 5 min (D). SAFit2 and fluoxetine decreased the latency difference in the NIH but neither stress nor drug treatment affected transitions in the LDB (E). Stress-induced decrease in preference for female urine was only prevented by the administration of Fluoxetine (F). Neither stress nor drug treatment affected immobility time in FST (F). At experimental day 17, there was no effect of chronic SAFit2 administration on chronic stress-induced increase in plasma corticosterone levels (G). At experimental day 35, there was no effect of chronic stress or chronic drug administration on acute stress-induced corticosterone levels (H). Quantification of immature DCX + new neurons in dorsal, ventral and whole hippocampus show a discrete effect of SAFit2. SIT, social interaction test; OF open field, LDB light-dark box, NIH novelty induced hypophagia. FUST female urine sniffing test, FST forced swim test. mean + SEM, n = 6–10 per group. % p < 0.05 by two-way ANOVA followed by Tukey post hoc test, & p < 0.05 by one-way ANOVA followed by Tukey post hoc test. *p < 0.05, **p < 0.01 ***p < 0.01 vs no stress–vehicle, and #p < 0.05, ###p < 0.001 vs stress-vehicle and @<0.01 vs no stress-SAFit2 by Kruskal-Wallis followed by Dunn’s comparison.
Stress-related psychiatric disorders such as depression are among the leading causes of morbidity and mortality. Considering that many individuals fail to respond to currently available antidepressant drugs, there is a need for antidepressants with novel mechanisms. Polymorphisms in the gene encoding FK506-binding protein 51 (FKBP51), a co-chaperone of the glucocorticoid receptor, have been linked to susceptibility to stress-related psychiatric disorders. Whether this protein can be targeted for their treatment remains largely unexplored. The aim of this work was to investigate whether inhibition of FKBP51 with SAFit2, a novel selective inhibitor, promotes hippocampal neuron outgrowth and neurogenesis in vitro and stress resilience in vivo in a mouse model of chronic psychosocial stress. Primary hippocampal neuronal cultures or hippocampal neural progenitor cells (NPCs) were treated with SAFit2 and neuronal differentiation and cell proliferation were analyzed. Male C57BL/6 mice were administered SAFit2 while concurrently undergoing a chronic stress paradigm comprising of intermittent social defeat and overcrowding, and anxiety and depressive -related behaviors were evaluated. SAFit2 increased neurite outgrowth and number of branch points to a greater extent than brain derived neurotrophic factor (BDNF) in primary hippocampal neuronal cultures. SAFit2 increased hippocampal NPC neurogenesis and increased neurite complexity and length of these differentiated neurons. In vivo, chronic SAFit2 administration prevented stress-induced social avoidance, decreased anxiety in the novelty-induced hypophagia test, and prevented stress-induced anxiety in the open field but did not alter adult hippocampal neurogenesis in stressed animals. These data warrant further exploration of inhibition of FKBP51 as a strategy to treat stress-related disorders.
Molecular and behavioral characterization of acutely stressed mice
A. A general schematic of the conducted experiments. For most experiments, mice were subjected to a single bout of acute stress and tested 24 h later. Some mice were given metyrapone 1 h prior to stress and then tested 48 h later. B Animals subjected to forced swim stress display anxiety-like behavior, as measured in the LDB (two-tailed student t-test p = 0.0015) and the OFT (two-tailed student t-test p = 0.0171). C The induction of anxiety-like behavior in males is also observed in response to RS paradigm (two-tailed student t-test, p = 0.0028). D Blocking CORT synthesis through administering metyrapone (IP; 40 mg/kg; 1 h pre-stress) inhibits stress-induced anxiety-like behavior (significant interaction of stress × drug; two-way ANOVA F (1, 44) = 7.051, p = 0.0110; Tukey’s post hoc analysis of control versus stress within vehicle p = 0.0444, within metyrapone p = 0.7281). E Acute swim stress induces significant induction of TNF protein in the vHPC measured 24 h post-stress (p < 0.0001). F Swim stress induces serum CORT levels independent of TNF signaling (ANOVA F (1, 12) = 63.79, p < 0.0001; Tukey’s post hoc analysis of control versus stress within WT animals p = 0.0031, within TNF−/− animals p = 0.0001). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± SEM. TNF tumor necrosis factor, CORT corticosterone, LDB light-dark box. OFT open field test. Sample sizes are indicated on the figures.
Microglial TNF signaling is important for the induction of stress-induced anxiety-like behavior
A. Mice that lack global TNF production do not show induction in anxiety-like behavior. (two-way ANOVA genotype x stress interaction F(1, 55) = 6.536, p = 0.0134; Tukey’s post hoc analysis of control versus stress within WT p = 0.0324, TNF−/−p = 0.8689). B Stress induces anxiety-like behavior in GFAP-Cre positive animals compared to their unstressed GFAP-Cre positive counterparts, similar to the phenotype in WT animals (24 h; two-way ANOVA main effect of stress F(1, 31) = 25.53, p < 0.0001; Tukey’s post hoc analysis: Cre negative control versus stress p = 0.0127, Cre positive control versus stress p = 0.0028). C Stressed CX3CR1-Cre positive animals lack a stress-induced increase in anxiety-like behavior compared to their unstressed counterparts. Control CX3CR1-Cre negative animals do have a stressed-induced increase in anxiety-like behaviour, which is comparable to WT (24 h; two-way ANOVA main effect of stress F (1, 35), p = 0.0227; main effect of genotype F (1, 35) = 1.222, p = 0.2765; interaction of stress x genotype F (1, 35) = 3.679, p = 0.0633). Tukey’s post hoc reveals that for Cre negative animals, control versus stress p = 0.0384 while for Cre positive, control versus stress P = 0.9836. D Microglial-TNF-lacking animals lack stress-induction of TNF in the vHPC compared to their controls (two-tailed student t-test, p = 0.5355). E Sample images demonstrating the results of RNAscope in situ hybridization combined with IBA1 immunohistochemistry staining in the vCA1-SR, using a bacterial probe and TNF mRNA probes in unstressed controls and stressed animals. The results are quantified in F, G. F Stress induces TNF mRNA in the SR of the vHPC (4 h post-stress; one-way ANOVA, main effect F(2,22), p = 0.0096; Tukey’s post hoc analysis of control versus stress p = 0.0288, stress versus nonspecific signal p = 0.0271, control versus nonspecific signal p = 0.9182, Sample size n (number of animals N) for assay control = 5 (3), for unstressed controls = 8 (4), for stressed animals = 12 (6)). G Quantification of the overlap in in situ hybridization signal and microglial cells in all three conditions. (4 h post-stress; one-way ANOVA main effect F(2, 197) = 7.144, p = 0.001; Tukey’s post-hoc analysis of control versus stress p = 0.0029, control versus nonspecific signal p = 0.9108, stress versus nonspecific signal p = 0.0082, number of detected microglia n’ (number of sections n–number of animals N) for assay control = 37 (3-2), for unstressed controls = 87 (6-3), for stressed animals = 76 (5-3)). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± SEM. TNF tumor necrosis factor; vHPC ventral hippocampus. Sample sizes are indicated on the figures.
AMPAR-mediated currents at the Schaffer collateral synapses in vCA1 are potentiated in WT animals and not in TNF−/− mice
A. Acute swim stress increases the AMPA/NMDA current ratio at Schaffer collateral synapses in the vCA1 at 2 and 24 h post-stress, as seen in sample traces and group data (Kruskal Wallis H(2) = 8.068, p = 0.0177; Control: n = 17, N = 11; 2 h post-stress: n = 13, N = 8; 24 h post-stress: n = 9, N = 5). B Stress potentiates the AMPA receptor (AMPAR) input-output relationship in the ventral Schaffer collaterals (24 h post-stress; two-way ANOVA of the main effect of stress F (1,40) = 11.439, p = 0.0016; control: n = 5, N = 4, stress: n = 7, N = 3). C There is no significant stress-induced potentiation of the NMDA receptor (NMDAR) mediated input-output curve in the ventral Schaffer collaterals (24 h post-stress; two-way ANOVA of the main effect of stress F (1,52) = 1 .0999, p = 0.2991; control: n = 7, N = 4, stress: n = 8, N = 4). D TNF lacking animals (TNF−/−) do not exhibit potentiation in the AMPAR currents in the ventral hippocampus in response to stress-exposure (24 h post-stress; two-way ANOVA of the main effect of stress F (1, 44) = 6.315, p = 0.0157; control: n = 6, N = 3, stress: n = 7, N = 4). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± SEM. Sample sizes are indicated on the bar figure or in the caption.
TNF antagonism post-stress reverses stress-induced plasticity and behavior
A. A schematic of the experiments performed in B, C. B Systemic (IP) administration of saline prior to stress does not interfere with stress-induced AMPAR current potentiation at vCA1-SC synapses (two-way ANOVA of the main effect of stress F (1, 58) = 14.5093, p = 0.0003; control: n = 7, N = 5, stress: n = 10, N = 6). C. Blocking TNF signaling through systemic (IP) administration of dominant negative TNF (DNTNF; 20 mg/kg) prior to stress blocks the observed potentiation (two-way ANOVA of the main effect of stress F (1, 44) = 0.4727, p = 0.4954; control: n = 7, N = 3, stress: n = 6, N = 4). D A schematic of the experiments performed in E–I. E Administering saline IP at 5 h post-stress does not block AMPAR mediated, vCA1-SC synaptic potentiation observed at 48 h post-stress (two-way ANOVA of the main effect of stress F (1, 84) = 6.5693, p = 0.0122; control: n = 11, N = 7, stress: n = 12, N = 8). F Systemic (IP) administration of DN-TNF 5 h after stress blocks the expression of stress-induced synaptic potentiation (two-way ANOVA of the main effect of stress F(1, 64) = 14.78, p = 0.0003; control: n = 10, N = 5, stress: n = 8, N = 5). G. Blocking TNF signaling via administering DN-TNF (IP; 20 mg/kg; 5 h post-stress) prevents anxiety-like behavior 48 h post-swim stress (two-way ANOVA of the main effect of stress F (1, 41) = 4.825, p = 0.0338; main effect of drug treatment F (1, 41) = 1.428, p = 0.2390; interaction of stress x drug treatment F (1, 41) = 2.470, p = 0.1237; Tukey’s post hoc analyses of control saline versus FST saline p = 0.0494, control DN-TNF versus FST DN-TNF p = 0.9714). H TNF is similarly necessary for stress-induction of anxiety-like behavior following restraint stress (two-way ANOVA of the interaction of stress x drug treatment F (1, 46) = 4.692, p = 0.0355; Tukey’s post hoc analyses of control saline versus RS saline p = 0.0017, control DN-TNF versus RS DN-TNF p = 0.7908). I Blocking TNF in the vHPC through local administration of DN-TNF (IC; 4.8 mg/kg; 5 h post-RS) prevents the development of anxiety-like behavior 48 h in response to stress (two-way ANOVA of the interaction of stress x drug treatment F (1, 24) = 20.58, p = 0.0001; Tukey’s post hoc analyses of control saline versus RS saline p = 0.0078, of control DN-TNF versus RS DN-TNF p = 0.0419). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± SEM. Sample sizes are indicated on the bar figure or in the caption.
Acute stress triggers plasticity of forebrain synapses as well as behavioral changes. Here we reveal that Tumor Necrosis Factor α (TNF) is a required downstream mediator of the stress response in mice, necessary for stress-induced synaptic potentiation in the ventral hippocampus and for an increase in anxiety-like behaviour. Acute stress is sufficient to activate microglia, triggering the long-term release of TNF. Critically, on-going TNF signaling specifically in the ventral hippocampus is necessary to sustain both the stress-induced synaptic and behavioral changes, as these could be reversed hours after induction by antagonizing TNF signaling. This demonstrates that TNF maintains the synaptic and behavioral stress response in vivo, making TNF a potential novel therapeutic target for stress disorders.
Diagnostic criteria of a depressive episode in the DSM-5 and ICD-11.
Depressive disorders are the most burdensome psychiatric disorders worldwide. Although huge efforts have been made to advance treatment, outcomes remain unsatisfactory. Many factors contribute to this gridlock including suboptimal animal models. Especially limited study comparability and replicability due to imprecise terminology concerning depressive-like states are major problems. To overcome these issues, new approaches are needed. Here, we introduce a taxonomical concept for modelling depression in laboratory mice, which we call depression-like syndrome (DLS). It hinges on growing evidence suggesting that mice possess advanced socioemotional abilities and can display non-random symptom patterns indicative of an evolutionary conserved disorder-like phenotype. The DLS approach uses a combined heuristic method based on clinical depression criteria and the Research Domain Criteria to provide a biobehavioural reference syndrome for preclinical rodent models of depression. The DLS criteria are based on available, species-specific evidence and are as follows: (I) minimum duration of phenotype, (II) significant sociofunctional impairment, (III) core biological features, (IV) necessary depressive-like symptoms. To assess DLS presence and severity, we have designed an algorithm to ensure statistical and biological relevance of findings. The algorithm uses a minimum combined threshold for statistical significance and effect size (p value ≤ 0.05 plus moderate effect size) for each DLS criterion. Taken together, the DLS is a novel, biologically founded, and species-specific minimum threshold approach. Its long-term objective is to gradually develop into an inter-model validation standard and microframework to improve phenotyping methodology in translational research.
Shank2/3 dKO mice show severe autistic-like behaviours
A Representative immunoblot showing the loss of Shank2A/E and Shank3a/c/d/e isoforms in cortical homogenates of dKO mice. B dKO did not bury any marble in comparison to WT mice; WT = 13, dKO = 10. C Impaired nest building in dKO mice; WT n = 16, KO n = 16. D Representative heatmaps of the three-chamber sociability trial (left) and dKO mice showed social deficits (right); WT = 11, dKO = 11. E Representative heatmaps of the three-chamber social novelty trial (left) and dKO mice displayed social memory deficits (right); WT = 11, dKO = 11. F Frequency spectrogram of typical ultrasonic vocalizations (USVs) emitted by WT and dKO male mice during female urine presentation. G, H dKO male mice emitted reduced USVs in the presence of female urine (G), as well as during direct interaction with a female (H); WT = 8, dKO = 8. I, J dKO mice showed social interaction deficits (I) and increased aggressive acts (J); WT = 7, dKO = 8. K dKO mice detected odour cues; WT = 8, dKO = 5. L, M dKO mice displayed skin lesions (L) due to increased self-grooming (M); WT n = 14, dKO n = 11. N–Q A significant number of dKO mice showed increased time spent jumping (N, O) and upright scrabbling (P, Q); WT n = 12, dKO n = 11 (N, O); WT n = 14, dKO n = 11 (P, Q). See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.
Shank2/3 loss in the nucleus accumbens leads to repetitive behaviours
A Schematic of bilateral injections of AAV9-CMV-GFP or AAV9-CMV-GFP-Cre into nucleus accumbens (ACB) of dKOfx/fx. B Representative images showing GFP (left) and GFP-Cre (right) expression in the ACB. C, D Immunohistochemistry staining of the ACB using the Shank2 (C) and Shank3 (D) markers; GFP n = 4, GFP-Cre n = 5. E Cre-expressing mice displayed repetitive behaviours measured as time spent grooming; GFP n = 5, GFP-Cre n = 6. F Nest building behaviour; GFP n = 5, GFP-Cre n = 6. G Representative heatmaps of the three-chamber sociability trial. H–J GFP- and Cre-expressing mice spent more time exploring the S1 instead of the empty cage; GFP n = 5, GFP-Cre n = 6. K Representative heatmaps of the three-chamber social novelty trial. L–N GFP- and Cre-expressing mice spent more time together with the S2 instead of the S1 (L, M), but Cre-expressing mice displayed reduced preference for the novel stimulus in comparison to control animals (N); GFP n = 5, GFP-Cre n = 6. Scale bars: 500 μm in (B) and 5 μm in (C, D). See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.
Cortical screening
A Schematic of bilateral injections of AAV9-CMV-GFP-Cre into 10 cortical coordinates (coordinate a-j). B–K Representative images showing GFP-Cre expression (left) and volumetric measurements of targeted single brain areas (right). ACA Anterior cingulate area; MOs, secondary motor area; SSp Primary somatosensory area, MOp Primary motor area, OLF Olfactory areas, VIS Visual areas, PTLp Posterior parietal association areas, RSP Retrosplenial area. L–O Time spent self-grooming (L), nesting score (M), preference index for sociability (N) and preference index for the novel stimulus (O) of dKOfx/fx mice injected with AAV9-CMV-GFP-Cre in the different cortical coordinates. Nucleus accumbens (ACB) was used as a reference brain area for the screening; ACB (GFP) n = 5, ACB (GFP-Cre) n = 6, screening (GFP-Cre) n = 3 per each coordinate. Scale bar: 1 mm. See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.
Lack of Shank2/3 in the retrosplenial area leads to social memory deficits and excitatory synapses loss, which cannot be rescued by acute DREADD-mediated neuronal activation
A Representative images showing GFP (left) and GFP-Cre (right) expression in the retrosplenial area (RSP). B GFP- and Cre-expressing mice preferred to investigate S1 instead of the empty cage; GFP n = 5, GFP-Cre n = 4. C Cre-expressing mice had no preference between the S1 and the S2; GFP n = 5, GFP-Cre n = 4. D Immunohistochemistry (IHC) and quantification of excitatory synapses in the RSP using the Homer1b/c and VGlut1 markers; GFP n = 3, GFP-Cre n = 3. E Immunohistochemistry (IHC) and quantification of neurons positive for c-Fos after three-chamber test; GFP n = 3, GFP-Cre n = 3. F Representative images showing GFP (left) and GFP-Cre/Gq (right) expression in the RSP. G GFP- and Cre-expressing mice treated with either vehicle (veh) or CNO are sociable; GFP + veh n = 4, GFP-Cre + veh n = 5, GFP + CNO[1x] n = 3, GFP-Cre + CNO[1x] n = 4, GFP + CNO[5x] n = 5, GFP-Cre + CNO[5x] n = 5. H Cre-expressing mice treated with CNO still showed social memory deficits; GFP + veh n = 4, GFP-Cre + veh n = 5, GFP + CNO[1x] n = 3, GFP-Cre + CNO[1x] n = 4, GFP + CNO[5x] n = 5, GFP-Cre + CNO[5x] n = 5. I IHC showing that 93% of Cre-positive neurons also expressed Gq; GFP-Cre + CNO n = 3. J IHC showing that CNO administration increased c-Fos levels in the RSP; GFP-Cre + Veh[5x] n = 3, GFP-Cre + CNO[5x] n = 3. K IHC showing that repeated increase of neuronal firing did not rescue the loss of excitatory synapses in the RSP of Cre-expressing mice; GFP-Cre + Veh[5x] n = 3, GFP-Cre + CNO[5x] n = 3. Scale bars: 500 μm in (A, F), 5 μm in (D–K), 20 μm in (E, I, and J). See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.
Single brain regions causally linked to ASD-related behaviours
In this schematic overview, identified subregions of the brain are highlighted and associated behavioural alterations seen in ASD models are listed [63–81].
Members of the Shank protein family are master scaffolds of the postsynaptic architecture and mutations within the SHANK genes are causally associated with autism spectrum disorders (ASDs). We generated a Shank2-Shank3 double knockout mouse that is showing severe autism related core symptoms, as well as a broad spectrum of comorbidities. We exploited this animal model to identify cortical brain areas linked to specific autistic traits by locally deleting Shank2 and Shank3 simultaneously. Our screening of 10 cortical subregions revealed that a Shank2/3 deletion within the retrosplenial area severely impairs social memory, a core symptom of ASD. Notably, DREADD-mediated neuronal activation could rescue the social impairment triggered by Shank2/3 depletion. Data indicate that the retrosplenial area has to be added to the list of defined brain regions that contribute to the spectrum of behavioural alterations seen in ASDs.
Shared genetic basis between schizophrenia and eight brain volumetric phenotypes
a The MiXeR-estimated heritability, polygenicity, and discoverability for each phenotype. The SNP heritability is a sum of effect across all trait-influencing variants. The polygenicity of each trait is represented by the number of trait-influencing variants to explain 90% heritability. The discoverability of each trait is defined as effect size per trait-influencing variant. A trait-influencing variant is defined as a common variant associated with the trait of interest after controlling for linkage disequilibrium (LD). b The MiXeR-modeled the number of shared and trait-specific trait-influencing variants in thousands. The colors reflect the shared (gray) and trait-specific (in color) trait-influencing variants. The standard deviations are shown in bracket. c The LD Score-estimated genetic correlation and MiXeR-modeled proportion of shared variants with concordant effect direction on both traits. Only a significant false discovery rate-corrected P value (FDR) of genetic correlation is labeled within the figure. The error bar of genetic correlation presents the standard error. In the proportion of concordant shared variants, the error bar reflects standard deviation. All details are provided in Supplementary Table 1. SCZ schizophrenia, ICV intracranial volume.
Individual shared associations between schizophrenia and brain volumetric phenotypes and relevant gene expression patterns
a The Manhattan plot of shared associations between schizophrenia and brain volumetric phenotypes. The x-axis stands for the chromosomal number and position and the y-axis represents -log10 transformed conjunctional false discovery rate (FDR) values with a dotted horizontal line reflecting significance. Each dot represents a SNP and the border indicates a lead SNP. b The SNP sign test for shared lead SNPs. NSNP counts the number of lead SNPs available in the independent datasets and NCON calculates the number of lead SNPs showing consistent effect between discovery and independent cohorts. The x-axis reflects the proportion of consistency. The solid points indicate significance of SNP sign test after FDR correction with the P value shown in FDR. c Co-expression patterns among shared genes. Shared genes between schizophrenia and four brain volumetric phenotypes, including accumbens, hippocampus, pallidum, putamen, and ICV, are enriched in five co-expression modules reported by PsychoENCODE human brain development study. Those five co-expression modules were labeled as modules (MEs) 2, 3, 5, 7, and 9 to be consistent with the PsychoENCODE study. d The five sets of co-expressed shared genes showed distinct temporal expression trajectories. The cluster red, pink, azure, blue, and black represented the shared genes enriched in MEs 2, 3, 5, 7, and 9 respectively. The whole-brain expression level (y-axis) represents the log2-median transformed mean expression value across brain tissues. The whole-brain expression trajectory was shown by a fitted non-linear LOESS regression line with the 95% confidence interval presented by shaded areas. The age (x-axis) was presented using post-conception days, which were further divided into five stages—fetal development (8 PCW < = age < 28 PCW), infancy (40 PCW < = age < 1 PY), childhood (1 PY < = age < 12 PY), adolescence (12 PY < = age < 20 PY), and adulthood (20 PY < = age < = 40 PY). Details of those five stages and nine anatomic tissues are provided in Supplementary Method. ICV intracranial volume.
The flowchart of gene-based functional analysis
For all functional enrichment analyses in the present study, we excluded shared genes located in complex linkage disequilibrium (LD) regions, including major histocompatibility complex (MHC), Chromosome 8p23.1 deletion (8p23.1), microtubule-associated protein tau (MAPT), and apolipoprotein E (APOE) regions. The gene-based association analysis by PrediXcan was illustrated using the pair of schizophrenia and thalamus as an example. A green rectangle represents a process, with a decision indicated by a diamond shape. The input and output are reflected in purple parallelograms. The data from PsychoENCODE, GTEx, and the Adolescent Brain Cognitive Development (ABCD) cohort are shown in database symbols in light blue. More details are provided in Methods. MSigDB: Molecular Signatures Database; eQTLs: expression quantitative trait loci.
Two candidate genes for schizophrenia and thalamus volume in late childhood
a The expression-trait associations between two candidate genes (ARL17A and CRHR1) and traits of interest. The x-axis indicates data resources and corresponding tissue, as well as the traits of interest labeled below. The expression-trait associations were genetically imputed from PrediXcan and S-PrediXcan, and differential gene expression profiles were based on CommonMind Consortium (CMC) and BrainSeq Phase 2 studies. The asterisk implies significance. DLPFC: dorsolateral prefrontal cortex. b Two candidate genes were mapped by a shared locus (chr17:43859929–44865603) between schizophrenia and thalamus. The violin plot represents the original GWAS Z scores of candidate SNPs within this shared locus. c The brain cis-expression quantitative trait locus (cis-eQTL) between the lead SNP and each candidate gene. The significant cis-eQTLs are shown in colors with the indication of the direction of effect. The significant brain cis-eQTLs were annotated from GTEx portal (v8). d The lifespan temporal expression trajectory of two candidate genes in nine brain tissues. The brain tissue expression (y-axis) was log2-median transformed. The expression trajectory for each brain tissue was revealed by a fitted non-linear LOESS regression line with the 95% confidence interval (shaded areas). The time span was shown using five stages, including fetal development (8 PCW < = age < 28 PCW), infancy (40 PCW < = age < 1 PY), childhood (1 PY < = age < 12 PY), adolescence (12 PY < = age < 20 PY), and adulthood (20 PY < = age < = 40 PY). ICV: intracranial volume.
Patients with schizophrenia have consistently shown brain volumetric abnormalities, implicating both etiological and pathological processes. However, the genetic relationship between schizophrenia and brain volumetric abnormalities remains poorly understood. Here, we applied novel statistical genetic approaches (MiXeR and conjunctional false discovery rate analysis) to investigate genetic overlap with mixed effect directions using independent genome-wide association studies of schizophrenia (n = 130,644) and brain volumetric phenotypes, including subcortical brain and intracranial volumes (n = 33,735). We found brain volumetric phenotypes share substantial genetic variants (74–96%) with schizophrenia, and observed 107 distinct shared loci with sign consistency in independent samples. Genes mapped by shared loci revealed (1) significant enrichment in neurodevelopmental biological processes, (2) three co-expression clusters with peak expression at the prenatal stage, and (3) genetically imputed thalamic expression of CRHR1 and ARL17A was associated with the thalamic volume as early as in childhood. Together, our findings provide evidence of shared genetic architecture between schizophrenia and brain volumetric phenotypes and suggest that altered early neurodevelopmental processes and brain development in childhood may be involved in schizophrenia development.
Despite the substantial heritability of antisocial behavior (ASB), specific genetic variants robustly associated with the trait have not been identified. The present study by the Broad Antisocial Behavior Consortium (BroadABC) meta-analyzed data from 28 discovery samples (N = 85,359) and five independent replication samples (N = 8,058) with genotypic data and broad measures of ASB. We identified the first significant genetic associations with broad ASB, involving common intronic variants in the forkhead box protein P2 (FOXP2) gene (lead SNP rs12536335, P = 6.32 x 10-10). Furthermore, we observed intronic variation in Foxp2 and one of its targets (Cntnap2) distinguishing a mouse model of pathological aggression (BALB/cJ strain) from controls (BALB/cByJ strain). Polygenic-risk-score (PRS) analyses in independent samples revealed that the genetic risk for ASB was associated with several antisocial outcomes across the lifespan, including diagnosis of conduct disorder, official criminal convictions, and trajectories of antisocial development. We found substantial genetic correlations of ASB with mental health (depression rg=0.63, insomnia rg = 0.47), physical health (overweight rg = 0.19, waist-to-hip ratio rg = 0.32), smoking (rg=0.54), cognitive ability (intelligence rg= -0.40), educational attainment (years of schooling rg = -0.46) and reproductive traits (age at first birth rg=-0.58, father’s age at death rg= -0.54). Our findings provide a starting point towards identifying critical biosocial risk mechanisms for the development of ASB.
ATAC-seq data analysis in the amygdala of acute ethanol exposed rats
A Heatmap showing differential ATAC peaks (FDR < 0.2; 345 peaks) and chromatin accessibility in the amygdala after acute ethanol exposure. Red represents increases in peaks and blue represents decreases in peaks. Individual peaks are represented in rows and individual animals from control and ethanol groups (n = 6 animal per treatment) are shown in columns. B Distribution of ATAC peak fold changes indicating that out of the 164 peaks at FDR < 0.05, 148 peaks were associated with ‘open’ chromatin regions in the amygdala of acute ethanol-treated compared to 16 peaks in the control rats. C ATAC-seq data analysis in the amygdala of acute ethanol exposed rats showing distribution of ATAC peaks across genomic features. D Footprinting images for the top 3 (out of 41) motifs derived from FIMO analysis of 572 vertebrate motifs from the JASPAR database. The motifs satisfied the following criteria: q < 0.01 and >50% enrichment (log2 ratio >0.585). The blue lines represent controls and the red lines represent ethanol-treated samples. [(NR3C1- Glucocorticoid Receptor; Nuclear Receptor Subfamily 3 Group C Member 1), (AR- Androgen Receptor), NR3C2 (Mineralocorticoid Receptor; Nuclear Receptor Subfamily 3 Group C Member 2)].
RNA-seq data analysis in the amygdala of acute ethanol-exposed rats
A HeatMap of RNA-seq differentially expressed transcripts at FDR < 0.2.Hierarchical clustering and heatmap of differentially expressed mRNAs (FDR < 0.2) from acute ethanol and normal saline (control) treated rats. Red represents increases in overall expression and blue represents decreases in expression. Individual mRNAs are represented in rows and individual experiments (n = 3 in each treatment; RNA from two rats are pooled for each treatment) are shown in columns. Gene names highlighted in blue were used for the validations. B Validations of mRNA levels of selected candidates from RNA-seq data in the amygdala of acute ethanol exposed rats. Expression levels of mRNAs were significantly different in the amygdala of acute ethanol-exposed rats compared to saline-treated control rats, as measured by qPCR. Data represents the mean ± SEM and individual values are shown on bar diagrams with circle dots for control and ethanol groups (n = 6–14; Two-tailed Student’s t test; *p < 0.05; **p < 0.01; ***p < 0.001).
Histone acetylation (H3K9/14ac; H3K27ac) and DNA methylation (5-methylcytosine, 5mC) changes reveal relaxed chromatin architecture at the ATAC-seq peak regions near the transcription start site (TSS) of Hif3a and Slc10a6 genes
A Schematic showing the location of ATAC-seq peaks and location of primers for qPCR validation for Hif3a and Slc10a6. B The “open peak” regions at the transcriptional control regions of Hif3a and Slc10a6 were evaluated by chromatin immunoprecipitation (ChIP) assay representing fold changes of H3K27ac and H3K9/14ac occupancy at selected regions of Hif3a and Slc10a6 in ethanol-treated and control rats. C Changes in DNA methylation were also evaluated at the same loci of Hif3a and Slc10a6 in amygdala of ethanol-treated and control rats and represented as fold changes in DNA methylation (5mC levels). Values are represented as the mean ± SEM and individual values are shown on bar diagrams with circle dots for control and ethanol groups (n = 7–8; two-tailed Student’s t test *p < 0.05; **p < 0.01).
Hif3a siRNA infusion into the central nucleus of amygdala (CeA) blocks ethanol-induced anxiolysis and reduces mRNA levels of Hif3a, but not Slc10a6. Hif3a mRNA is decreased during withdrawal after chronic ethanol exposure
A Schematic representation of the experimental design for the siRNA infusion and behavioral measurement. B Bar diagram showing anxiety-like behaviors following Hif3a siRNA infusion into the CeA with or without acute ethanol treatment (1 g/kg, intraperitoneal) in rats. Hif3a siRNA infusion into CeA (24 h prior) prevented anxiolytic effects of acute ethanol in rats. Values are represented as mean ± SEM (n = 10–11; two-way analysis of variance (Percent open arm entries: treatment effect, F1,37 = 72.397, p < 0.001, group effect F1,37 = 71.445, p < 0.001, treatment × group interaction, F1,37 = 3.078, p = 0.088; Percent time spent in open arms: treatment effect, F1,37 = 97.836, p < 0.001, group effect, F1,37 = 90.159, p < 0.001, treatment × group interaction, F1,37 = 10.175, p < 0.01) followed by post-hoc Tukey’s test: ***p < 0.001). C The mRNA levels of Hif3a and Slc10a6 were also measured in the amygdala of rats infused with control siRNA or Hif3a siRNA into CeA then treated with either saline or ethanol. Values are represented as mean ± SEM [n = 9–10; Two-way analysis of variance (Hif3a: treatment effect, F1,35 = 23.54, p < 0.001; group effect, F1,35 = 19.366, p < 0.001; Slc10a6: group effect, F1,33 = 11.003, p < 0.01) followed by post-hoc Tukey’s test: *p < 0.05; ***p < 0.001]. D Bar diagram showing changes in Hif3a mRNA levels in the amygdala of control, chronic ethanol-treated and ethanol-withdrawn (24 h) rats. Values are represented as mean ± SEM (n = 8; Kruskal–Wallis one-way analysis of variance on ranks (H2 = 7.3, p < 0.05) followed by Tukey’s test, *p < 0.05). Individual values are shown on bar diagrams with circle dots for various groups.
This model depicts the ability of acute ethanol to rapidly alter the epigenome in the amygdala and produce transcriptomic changes
These genome-wide chromatin accessibility and transcriptomic signatures in the amygdala are associated with anti-anxiety effects of ethanol in rats. One such candidate gene is hypoxia-induced gene transcription factor, Hif3a, which is epigenetically induced by acute ethanol, and anxiolytic effects associated with acute ethanol are prevented by inhibiting Hif3a expression in the central nucleus of amygdala of rats. The rapid and dynamic epigenomic modifications from a low dose of ethanol clearly suggest that these molecular processes may prime the amygdala to the emotional negative consequences that are most commonly associated with and promote alcohol use disorder.
Positive effects of alcohol drinking such as anxiolysis and euphoria appear to be a crucial factor in the initiation and maintenance of alcohol use disorder (AUD). However, the mechanisms that lead from chromatin reorganization to transcriptomic changes after acute ethanol exposure remain unknown. Here, we used Assay for Transposase-Accessible Chromatin followed by high throughput sequencing (ATAC-seq) and RNA-seq to investigate epigenomic and transcriptomic changes that underlie anxiolytic effects of acute ethanol using an animal model. Analysis of ATAC-seq data revealed an overall open or permissive chromatin state that was associated with transcriptomic changes in the amygdala after acute ethanol exposure. We identified a candidate gene, Hif3a (Hypoxia-inducible factor 3, alpha subunit), that had ‘open’ chromatin regions (ATAC-seq peaks), associated with significantly increased active epigenetic histone acetylation marks and decreased DNA methylation at these regions. The mRNA levels of Hif3a were increased by acute ethanol exposure, but decreased in the amygdala during withdrawal after chronic ethanol exposure. Knockdown of Hif3a expression in the central nucleus of amygdala attenuated acute ethanol-induced increases in Hif3a mRNA levels and blocked anxiolysis in rats. These data indicate that chromatin accessibility and transcriptomic signatures in the amygdala after acute ethanol exposure underlie anxiolysis and possibly prime the chromatin for the development of AUD.
Alcohol-use-disorders are chronic relapsing illnesses, often co-morbid with anxiety. We have previously shown using the “drinking-in-the-dark” model in mice that the stimulation of the serotonin receptor 1A (5-HT1A) reduces ethanol binge-drinking behaviour and withdrawal-induced anxiety. The 5-HT1A receptor is located either on Raphe neurons as autoreceptors, or on target neurons as heteroreceptors. By combining a pharmacological approach with biased agonists targeting the 5-HT1A auto- or heteroreceptor and a chemogenetic approach (DREADDs), here we identified that ethanol-binge drinking behaviour is dependent on 5-HT1A autoreceptors and 5-HT neuronal function, with a transition from DRN-dependent regulation of short-term (6 weeks) ethanol intake, to MRN-dependent regulation after longer ethanol exposure (12 weeks). We further identified a serotonergic microcircuit (5-HT MRN->DG) originating from the MRN and projecting to the dentate gyrus (DG) of the hippocampus, that is specifically affected by, and modulates long-term ethanol consumption. The present study indicates that targeting Raphe nuclei 5-HT1A autoreceptors with agonists might represent an innovative pharmacotherapeutic strategy to combat alcohol abuse.
Prefrontal cortex (PFC) is a site of information convergence important for behaviors relevant to psychiatric disorders. Despite the importance of inhibitory GABAergic parvalbumin-expressing (PV+) interneurons to PFC circuit function and decades of interest in N-methyl-D-aspartate receptors (NMDARs) in these neurons, examples of defined circuit functions that depend on PV+ interneuron NMDARs have been elusive. Indeed, it remains controversial whether all PV+ interneurons contain functional NMDARs in adult PFC, which has major consequences for hypotheses of the pathogenesis of psychiatric disorders. Using a combination of fluorescent in situ hybridization, pathway-specific optogenetics, cell-type-specific gene ablation, and electrophysiological recordings from PV+ interneurons, here we resolve this controversy. We found that nearly 100% of PV+ interneurons in adult medial PFC (mPFC) express transcripts encoding GluN1 and GluN2B, and they have functional NMDARs. By optogenetically stimulating corticocortical and thalamocortical inputs to mPFC, we show that synaptic NMDAR contribution to PV+ interneuron EPSCs is pathway-specific, which likely explains earlier reports of PV+ interneurons without synaptic NMDAR currents. Lastly, we report a major contribution of NMDARs in PV+ interneurons to thalamus-mediated feedforward inhibition in adult mPFC circuits, suggesting molecular and circuit-based mechanisms for cognitive impairment under conditions of reduced NMDAR function. These findings represent an important conceptual advance that has major implications for hypotheses of the pathogenesis of psychiatric disorders.
Cardinal features of ADHD
Three behavioral paradigms were used in this study to assess ADHD-like phenotypes in Gpr88−/− mice. The five-Choice Serial Reaction Time Task (5-CSRTT) evaluates hyperactivity, motor impulsivity and inattention, which characterize clinical features of ADHD. The Go/No-Go task and Attentional Set Shifting Task (ASST) specifically address impulsivity and inattention, respectively. Human testing paradigms, which best parallel animal testing and are used in this study (Table 1), are indicated in italics, and include the Diagnostic Interview for Children (DISC), the Continuous Performance Test (CPT) and the Wisconsin Card Sorting Test (WCST).
Gpr88 mutant mice show ADHD-like behavior in the 5-CSRTT and higher inattention in the ASST
A. Gpr88−/− mice show lower performance in all aspects of the 5-CSRTT. This test is performed in a touchscreen apparatus (Supplementary Fig. S1). Mice must maintain attention to five spatial locations on a screen, and withhold from responding until a signal (flashlight) is presented randomly at one of the locations. The animal then needs to touch the screen where light was presented to obtain a food reward, a task requiring spatial and temporal attention, and effective inhibitory controls to avoid premature responding. The % accuracy was calculated as the number of correct trials divided by total number of trials x 100 (correct and incorrect), and the % omission was calculated as the number of missed trials divided by number of presented trials ×100. Lower accuracy and higher omissions were found across the four stimulus durations, and the number of premature responses to stimulus presentation was higher. Animal activity (infrared beams) was higher at the screen and on the reward side of the chamber. Latency to collect the reward was increased or unchanged. n = 6–8. Stars show genotype effects. *p < 0.05; **p < 0.01; ***p < 0.001. B Gpr88−/− mice show lower performance in the ASST. In this test series, mice need to associate an odor and/or a medium with a reward in seven steps of increasing difficulty (Supplementary Fig. S3). Steps proceed in the following order: simple discrimination (SD), compound discrimination (CD), reversal (R1), intra-dimensional shift IDS1, IDS2, reversal (R2) and extra-dimensional shift (EDS). For each step, mice are required to perform eight consecutive correct responses before moving onto the next step. Gpr88−/− mice needed more trials to achieve EDS, made more wrong trials in CD and EDS, and showed higher latencies to correct choice in SD and EDS. Data are represented as mean (±SEM) per session. n = 6–8. Stars show genotype effects *p < 0.05.
Gpr88 mutant mice show higher motor impulsivity in the Go/No-Go task
Mice are initially trained to nose poke for food reward, signaled by a light cue in operant boxes (autoshaping Supplementary Fig. S4). Ability to inhibit the already initiated response was then measured in two successive experimental series. A Go phase. Intertrial (no light) and pretrial (house light) periods are introduced before the cue light is illuminated, to test whether animals are able to withhold responding. Gpr88−/− mice and their controls showed similar learning patterns, shown by same numbers of active nosepokes and earned rewards during the first and last session. The % premature responses (number of premature responses divided by number of pre-trials ×100) was increased in Gpr88−/− mice in sessions 4–11, and between first and last session. B No-Go phase. Animal’s ability to inhibit inappropriate behavior was further tested. A light cue only (Go, omission error) or a paired light/sound cue (no-Go, commission error) are randomly presented and rewarded. The % premature responses was increased for Gpr88−/− mice in sessions 1, 7, 8, 9, 10 and 11, and over all sessions. The % commission errors (number of commission errors divided by number of No-Go trials, ×100) was increased, with a significant effect in the last session compared to first session, and over the last 5 sessions. The impulsivity index (percentage of correct go trials minus percentage of correct No-Go trials) was increased, with significant difference during the last session comparing to first session and over the last 5 sessions. Data are represented as mean (±SEM) per session. n = 12–15. Gray stars show session effects; colored stars show genotype effects. *p < 0.05; **p < 0.01; ***p < 0.001.
A2A-GPR88 and D1-GPR88 show higher waiting and stopping impulsivity, respectively, in the Go/No-Go task
The experiment was performed as in Fig. 3. A2AR- and D1R-Gpr88 mice and their respective controls (A2A-Ctl and D1R-Ctl) were initially trained to nose poke for food reward and all animals reached the criterion at the end of the AutoShaping phase (autoshaping Supplementary Fig. S4). Next, mice underwent the Go phase, followed by the No-Go phase. A Go phase. Intertrial (no light) and pretrial (house light) periods are introduced before the cue light is illuminated, to test whether animals are able to withhold responding. The % premature responses (number of premature responses divided by number of pre-trials ×100) was increased in A2AR-Gpr88 but not D1R-Gpr88 mice in sessions 3–5, 11, 16–17 and 21, over all the sessions, and between first and last session. B–D No-Go phase. B The % premature responses was again increased A2AR-Gpr88 mice, with higher significance compared to the Go phase. D1R-Gpr88 mice showed higher premature responses in 3 session (14–16) only. C The % commission errors (number of commission errors divided by number of No-Go trials, ×100). D The impulsivity index (percentage of correct go trials minus percentage of correct No-Go trials) were increased in DR1-Gpr88, but not D1R-Gpr88, mice over the last 5 sessions and when comparing first and last session. Data are represented as mean (±SEM) per session. n = 5–11. Gray stars show session effects; colored stars show genotype effects. *p < 0.05; **p < 0.01; ***p < 0.001.
Association of GPR88 tag SNPs with ADHD, clinical/behavioral traits, response to treatment and cognition.
The neural orphan G protein coupled receptor GPR88 is predominant in the striatum and cortex of both rodents and humans, and considered a potential target for brain disorders. Previous studies have shown multiple behavioral phenotypes in Gpr88 knockout mice, and human genetic studies have reported association with psychosis. Here we tested the possibility that GPR88 contributes to Attention Deficit Hyperactivity Disorder (ADHD). In the mouse, we tested Gpr88 knockout mice in three behavioral paradigms, best translatable between rodents and humans, and found higher motor impulsivity and reduced attention together with the reported hyperactivity. Atomoxetine, a typical ADHD drug, reduced impulsivity in mutant mice. Conditional Gpr88 knockout mice in either D1R-type or D2R-type medium spiny neurons revealed distinct implications of the two receptor populations in waiting and stopping impulsivity. Thus, animal data demonstrate that deficient GPR88 activity causally promotes ADHD-like behaviors, and identify circuit mechanisms underlying GPR88-regulated impulsivity. In humans, we performed a family-based genetic study including 567 nuclear families with DSM-IV diagnosis of ADHD. There was a minor association for SNP rs2036212 with diagnosis, treatment response and cognition. A stronger association was found for SNP rs2809817 upon patient stratification, suggesting that the T allele is a risk factor when prenatal stress is involved. Human data therefore identify GPR88 variants associated with the disease, and highlight a potential role of life trajectories to modulate GPR88 function. Overall, animal and human data concur to suggest that GPR88 signaling should be considered a key factor for diagnostic and treatment of ADHD. Mutant mouse behavior and human genetic data concur to suggest that the orphan receptor GPR88 contributes to Attention-Deficit/Hyperactivity Disorder.
T1w MPRAGE image and R1, MPF and R2* parametric maps of an example HC participant
Subcortical regions of interest (ROIs) are overlaid on the T1w MPRAGE image. The slices for each view are chosen to have most of the ROIs visible. The bright regions at the caudal borders of R1 and MPF maps are artifactual and arise from low effective flip angle at the edges of the 3D imaging slab as the B1⁺ correction does not account for the slab profile of the 3D excitation. This artifact does not overlap with the ROIs in the study.
HC and PSD group differences in quantitative MRI measures
Sample distributions of R1 (A), synthetic R2* (B), MPF (C), and volume (D) across bilateral regions of interest are shown by group. Boxplots indicate the first and third quartiles of the sample distribution while the white dots represent the median. Asterisks indicate significant group differences after multiple comparison correction (corrected p < 0.05).
HC and PSD diagnostic subgroup differences in quantitative MRI measures
Sample distributions of R1 (A), synthetic R2* (B), and volume (C) across bilateral regions of interest are shown by group. Boxplots indicate the first and third quartiles of the sample distribution while the white dots represent the median. Asterisks indicate significant group differences after multiple comparison correction (corrected p < 0.05). The dashed line in panel C indicates trend level group difference with uncorrected p < 0.01.
Partial plots of the relationship between synthetic R2* in left thalamus and the SIS, SANS, and SAPS scores
Linear fitting lines are shown for significant correlations. Covariates include age, sex, and structure volume for all plots, and an additional categorical variable indicating group membership (HC vs. PSD) for the correlation with SIS.
Iron deficits have been reported as a risk factor for psychotic spectrum disorders (PSD). However, examinations of brain iron in PSD remain limited. The current study employed quantitative MRI to examine iron content in several iron-rich subcortical structures in 49 young adult individuals with PSD (15 schizophrenia, 17 schizoaffective disorder, and 17 bipolar disorder with psychotic features) compared with 35 age-matched healthy controls (HC). A parametric approach based on a two-pool magnetization transfer model was applied to estimate longitudinal relaxation rate (R1), which reflects both iron and myelin, and macromolecular proton fraction (MPF), which is specific to myelin. To describe iron content, a synthetic effective transverse relaxation rate (R2*) was modeled using a linear fitting of R1 and MPF. PSD patients compared to HC showed significantly reduced R1 and synthetic R2* across examined regions including the pallidum, ventral diencephalon, thalamus, and putamen areas. This finding was primarily driven by decreases in the subgroup with schizophrenia, followed by schizoaffective disorder. No significant group differences were noted for MPF between PSD and HC while for regional volume, significant reductions in patients were only observed in bilateral caudate, suggesting that R1 and synthetic R2* reductions in schizophrenia and schizoaffective patients likely reflect iron deficits that either occur independently or precede structural and myelin changes. Subcortical R1 and synthetic R2* were also found to be inversely related to positive symptoms within the PSD group and to schizotypal traits across the whole sample. These findings that decreased iron in subcortical regions are associated with PSD risk and symptomatology suggest that brain iron deficiencies may play a role in PSD pathology and warrant further study.
Overview of the three stages of the analysis
The color indicates the homogeneity of the samples (dark blue=most homogeneous in terms of STB assessment instruments and type of psychiatric disorders, light blue=most heterogeneous in terms of STB assessment instruments and type of psychiatric disorders). C-SSRS Columbia Suicide Severity Rating Scale; HC healthy controls; CC clinical controls.
Boxplot showing the mean surface area of the frontal pole in young people without a lifetime history of any suicide attempt (in red), and those with a lifetime history of an actual suicide attempt (in blue)
Lifetime history of an actual suicide attempt was assessed using the C-SSRS.
Identifying brain alterations associated with suicidal thoughts and behaviors (STBs) in young people is critical to understanding their development and improving early intervention and prevention. The ENIGMA Suicidal Thoughts and Behaviours (ENIGMA-STB) consortium analyzed neuroimaging data harmonized across sites to examine brain morphology associated with STBs in youth. We performed analyses in three separate stages, in samples ranging from most to least homogeneous in terms of suicide assessment instrument and mental disorder. First, in a sample of 577 young people with mood disorders, in which STBs were assessed with the Columbia Suicide Severity Rating Scale (C-SSRS). Second, in a sample of young people with mood disorders, in which STB were assessed using different instruments, MRI metrics were compared among healthy controls without STBs (HC; N = 519), clinical controls with a mood disorder but without STBs (CC; N = 246) and young people with current suicidal ideation (N = 223). In separate analyses, MRI metrics were compared among HCs (N = 253), CCs (N = 217), and suicide attempters (N = 64). Third, in a larger transdiagnostic sample with various assessment instruments (HC = 606; CC = 419; Ideation = 289; HC = 253; CC = 432; Attempt=91). In the homogeneous C-SSRS sample, surface area of the frontal pole was lower in young people with mood disorders and a history of actual suicide attempts (N = 163) than those without a lifetime suicide attempt (N = 323; FDR-p = 0.035, Cohen’s d = 0.34). No associations with suicidal ideation were found. When examining more heterogeneous samples, we did not observe significant associations. Lower frontal pole surface area may represent a vulnerability for a (non-interrupted and non-aborted) suicide attempt; however, more research is needed to understand the nature of its relationship to suicide risk.
Quantification of serotonin transporter (SERT) binding potential (BPP)
a Average SERT BPP from 79 participants included in this study are displayed in transversal planes overlaid on a MR template for placebo (left) and citalopram (right) scans. High baseline binding was observed in regions rich in SERT such as thalamus and striatum. Time activity curves (b) for the thalamus and metabolite corrected plasma activity (c) ±SE for PET/MR scans with citalopram and placebo challenge are plotted for groups defined by ABCB1rs2235015 genotype.
The effect of genotype, sex and citalopram plasma concentrtion on serotonin transporter occupancy
a Approximately one hour after infusion of 8 mg citalopram, lower serotonin transporter occupancy was observed in ABCB1rs2235015 minor allele carriers (A + AC) compared to major allele homozygotes (C) and female compared to male participants. b SERT occupancy is plotted against the area under the curve (AUC) of citalopram in plasma. Regression lines are plotted for participants grouped according to ABCB1rs2235015 genotype. Grey ribbons indicate 95% confidence intervals.
Modeling SERT binding based on clinical variables and ABCB1 genotype
a The ratio of occupied to unoccupied (O/U) SERT after citalopram infusion was predicted for different combinations of sex, ABCB1rs2235015 genotype, age and weight based on a linear regression model fitted to the study data. O/U SERT is directly proportional to the product of drug affinity and concentration at target sites in equilibrium and is reported as a percentage of the sample’s average to aid interpretation of effects. O/U SERT was predicted to be −14.48 ± 5.38% lower in rs2235015 minor allele carriers, +19.10 ± 6.95% higher in women, −4.83 ± 2.70% lower per 10 kg bodyweight, and −2.68 ± 3.07% lower per 10 years of age. b Predictions of O/U SERT were transformed into SERT occupancy after citalopram 8 mg infusion based on equation 1 after multiplication with the sample’s mean O/U SERT.
Strategies to personalize psychopharmacological treatment promise to improve efficacy and tolerability. We measured serotonin transporter occupancy immediately after infusion of the widely prescribed P-glycoprotein substrate citalopram and assessed to what extent variants of the ABCB1 gene affect drug target engagement in the brain in vivo. A total of 79 participants (39 female) including 31 patients with major depression and 48 healthy volunteers underwent two PET/MRI scans with the tracer [¹¹C]DASB and placebo-controlled infusion of citalopram (8 mg) in a cross-over design. We tested the effect of six ABCB1 single nucleotide polymorphisms and found lower SERT occupancy in ABCB1 rs2235015 minor allele carriers (n = 26, MAF = 0.18) compared to major allele homozygotes (t73 = 2.73, pFWE < 0.05) as well as in men compared to women (t73 = 3.33, pFWE < 0.05). These effects were robust to correction for citalopram plasma concentration, age and diagnosis. From occupancy we derived the ratio of occupied to unoccupied SERT, because in theory this measure is equal to the product of drug affinity and concentration at target sites. A model combining genotype with basic clinical variables, predicted that, at the same dosage, occupied to unoccupied SERT ratio was −14.48 ± 5.38% lower in rs2235015 minor allele carriers, +19.10 ± 6.95% higher in women, −4.83 ± 2.70% lower per 10 kg bodyweight, and −2.68 ± 3.07% lower per 10 years of age. Our results support the exploration of clinical algorithms with adjustment of initial citalopram dosing and highlight the potential of imaging-genetics for precision pharmacotherapy in psychiatry.
Tractographic planning
Inline coronal (left panel) and sagittal (righ panel) tractographic planning of deep brain stimulation quadripolar electrode in the supero lateral branch of medial forebrain bundle during its course through the anterior limb of the internal capsule in a patient with refractory obsessive-compulsive disorder.
Clinical symptom scores
Average scores on the Y-BOCS (Yale-Brown Obsessive Compulsive Scale), HAM-D (Hamilton Depression Rating Scale), and HAM-A (Hamilton Anxiety Scale) in patients with tractography-based deep brain stimulation (DBS) (N = 20) and anatomical landmark-based DBS (N = 20).
Deep brain stimulation (DBS) of the ventral anterior limb of the internal capsule (vALIC) is effective for refractory obsessive-compulsive disorder (OCD). Retrospective evaluation showed that stimulation closer to the supero-lateral branch of the medial forebrain bundle (slMFB), within the vALIC, was associated with better response to DBS. The present study is the first to compare outcomes of DBS targeted at the vALIC using anatomical landmarks and DBS with connectomic tractography-based targeting of the slMFB. We included 20 OCD-patients with anatomical landmark-based DBS of the vALIC that were propensity score matched to 20 patients with tractography-based targeting of electrodes in the slMFB. After one year, we compared severity of OCD, anxiety and depression symptoms, response rates, time to response, number of parameter adjustments, average current, medication usage and stimulation-related adverse effects. There was no difference in Y-BOCS decrease between patients with anatomical landmark-based and tractography-based DBS. Nine (45%) patients with anatomical landmark-based DBS and 13 (65%) patients with tractography-based DBS were responders (BF10 = 1.24). The course of depression and anxiety symptoms, time to response, number of stimulation adjustments or medication usage did not differ between groups. Patients with tractography-based DBS experienced fewer stimulation-related adverse effects than patients with anatomical landmark-based DBS (38 vs 58 transient and 1 vs. 17 lasting adverse effects; BF10 = 14.968). OCD symptoms in patients with anatomical landmark-based DBS of the vALIC and tractography-based DBS of the slMFB decrease equally, but patients with tractography-based DBS experience less adverse effects.
Groups of individuals defined by APOE genotype and local ancestry inference (n = 309)
While groups A1, A4, B1, and B3 contain individuals with a single local ancestry at APOE locus, groups A2, A3, and B2 contain individuals with dual ancestry at the APOE locus (mixed). Group A has APOE4 carriers (APOE4+) and group B is composed of noncarriers (APOE4−). Local ancestries referred as EUR (European) and non-EUR (Non-European).
Association between African global ancestry and cognitive outcomes stratified by neuropathology and APOE4 status
Linear regression models of African ancestry and Clinical Dementia Rating Sum of Boxes (CDR-SOB) considering an interaction term between African ancestry and neuritic plaques evaluated by the CERAD score (A, C, E), or neurofibrillary tangle burden evaluated by the Braak & Braak stage (B, D, F). A neuritic plaque burden in all individuals [Black diamond: None or sparse (n = 303); Gray triangle: Moderate (n = 49); Black circle: Frequent (n = 48)]; (B) neurofibrillary tangle burden in all individuals [Black diamond: 0-II (n = 263); Gray triangle: III-IV (n = 99); Black circle: V-VI (n = 38)]; (C) neuritic plaques in APOE4− individuals [Black diamond: None or sparse plaques (n = 239); Gray triangle: Moderate plaques (n = 31); Black circle: Frequent plaques (n = 22)]; (D) neurofibrillary tangles in APOE4− individuals [Black diamond: Braak 0-II (n = 206); Gray triangle: Braak III-IV (n = 64); Black circle: Braak V-VI (n = 22)]; (E) neuritic plaques in APOE4+ individuals [Black diamond: None or sparse plaques (n = 64)]; Gray triangle: Moderate plaques (n = 18); Black circle: Frequent plaques (n = 26); and (F) neurofibrillary tangles in APOE4 + individuals [Black diamond: Braak 0-II (n = 57); Gray triangle: Braak III-IV (n = 35); Black circle: Braak V-VI (n = 16)]. P values for the interaction terms included in linear regression models adjusted for age, sex, education, and APOE4 status (A, B). Neuritic plaques were evaluated by the Consortium to Establish a Registry for Alzheimer’s disease (CERAD) score, and neurofibrillary tangles were evaluated by the Braak staining system.
Dementia is more prevalent in Blacks than in Whites, likely due to a combination of environmental and biological factors. Paradoxically, clinical studies suggest an attenuation of APOE ε4 risk of dementia in African ancestry (AFR), but a dearth of neuropathological data preclude the interpretation of the biological factors underlying these findings, including the association between APOE ε4 risk and Alzheimer’s disease (AD) pathology, the most frequent cause of dementia. We investigated the interaction between African ancestry, AD-related neuropathology, APOE genotype, and functional cognition in a postmortem sample of 400 individuals with a range of AD pathology severity and lack of comorbid neuropathology from a cohort of community-dwelling, admixed Brazilians. Increasing proportions of African ancestry (AFR) correlated with a lower burden of neuritic plaques (NP). However, for individuals with a severe burden of NP and neurofibrillary tangles (NFT), AFR proportion was associated with worse Clinical Dementia Rating sum of boxes (CDR-SOB). Among APOE ε4 carriers, the association between AFR proportion and CDR-SOB disappeared. APOE local ancestry inference of a subset of 309 individuals revealed that, in APOE ε4 noncarriers, non-European APOE background correlated with lower NP burden and, also, worse cognitive outcomes than European APOE when adjusting by NP burden. Finally, APOE ε4 was associated with worse AD neuropathological burden only in a European APOE background. APOE genotype and its association with AD neuropathology and clinical pattern are highly influenced by ancestry, with AFR associated with lower NP burden and attenuated APOE ε4 risk compared to European ancestry.
PRISMA flowchart
Number of studies identified, screened for eligibility, and included in final analyses, with tallied reasons for exclusion.
Moderators of the effect of ketamine vs. placebo on standardized % improvement in MADRS scores
In all figures, larger scores on the y-axis = greater improvement from baseline, expressed in standard deviation units relative to the overall sample mean. A moderation by study’s eligibility threshold for the number of previous failed, adequate antidepressant medication trials that were required for study enrollment (post-rapid timepoint); B moderation by use of a crossover design (rapid timepoint); C moderation by study performance in the US (post-rapid timepoint). Regression prediction lines based on models predicting MADRS % improvement from baseline (standardized across the full dataset) at post-infusion (rapid or post-rapid) timepoint with a random effect for study. All individual patient-level datapoints are depicted by red triangles (ketamine-treated patients) or black circles (placebo-treated patients). Statistics overlaid on each figure depict the simple effects of the moderator variable within ketamine-treated patients alone and within placebo-treated patients alone.
Description of included studies.
Depression is disabling and highly prevalent. Intravenous (IV) ketamine displays rapid-onset antidepressant properties, but little is known regarding which patients are most likely to benefit, limiting personalized prescriptions. We identified randomized controlled trials of IV ketamine that recruited individuals with a relevant psychiatric diagnosis (e.g., unipolar or bipolar depression; post-traumatic stress disorder), included one or more control arms, did not provide any other study-administered treatment in conjunction with ketamine (although clinically prescribed concurrent treatments were allowable), and assessed outcome using either the Montgomery-Åsberg Depression Rating Scale or the Hamilton Rating Scale for Depression (HRSD-17). Individual patient-level data for at least one outcome was obtained from 17 of 25 eligible trials [pooled n = 809]. Rates of participant-level data availability across 33 moderators that were solicited from these 17 studies ranged from 10.8% to 100% (median = 55.6%). After data harmonization, moderators available in at least 40% of the dataset were tested sequentially, as well as with a data-driven, combined moderator approach. Robust main effects of ketamine on acute [~24-hours; β*(95% CI) = 0.58 (0.44, 0.72); p < 0.0001] and post-acute [~7 days; β*(95% CI) = 0.38 (0.23, 0.54); p < 0.0001] depression severity were observed. Two study-level moderators emerged as significant: ketamine effects (relative to placebo) were larger in studies that required a higher degree of previous treatment resistance to federal regulatory agency-approved antidepressant medications (≥2 failed trials) for study entry; and in studies that used a crossover design. A comprehensive data-driven search for combined moderators identified statistically significant, but modest and clinically uninformative, effects (effect size r ≤ 0.29, a small-medium effect). Ketamine robustly reduces depressive symptoms in a heterogeneous range of patients, with benefit relative to placebo even greater in patients more resistant to prior medications. In this largest effort to date to apply precision medicine approaches to ketamine treatment, no clinical or demographic patient-level features were detected that could be used to guide ketamine treatment decisions. Review Registration: PROSPERO Identifier: CRD42021235630 Molecular Psychiatry; https://doi.
Details of the genome-wide top hit rs12930712
a Manhattan plot and (b) Regional association plot. The red line represents the genome-wide (5 × 10⁻⁶) threshold in Manhattan plot. The lead variant of GWAS (rs12930712) is indicated by a square symbol in regional plot. Variants in linkage disequilibrium with rs12930712 are highlighted in yellow (r² > 0.6 in the East Asian populations of 1KG Phase3).
Epigenome-wide association results and enrichment analysis of top-100 CpG sites regarding Mets in schizophrenia cohorts
a Manhattan plot of top differentially methylated positions (DMPs) in Mets versus Non-Mets. The red lines designate the false discovery rate (FDR) 0.05 threshold. b CpG probe-level methylation of cg16740586 and cg27243685 in ABCG1. c GO enrichment summary of genes located in the top-100 DMPs. GO categories are grouped according to semantic similarity. Mets metabolic syndrome. Non-Mets Not metabolic syndrome.
Differentially methylated regions (DMRs) associated with Mets in the schizophrenia cohort
a coMET plots of top DMRs associated with Mets. Genomic annotations to gene, CpG islands and chromatin regulation are from UCSC CpG Island, UCSC DNase Cluster, and the Broad UCSC ChromatinHMM tracks. b box plot for DMR1 (chr21:43641146–43642366) and DMR2 (chr21:43655256–43655919). c Triglyceride (TG) and total cholesterol (TC) contents in HepG2 cells treated with OLA or vehicle at 24 h after CRISPR-based methylation modification. n = 3 per group. OLA olanzapine, hypo hypomethylation, hyper hypermethylation.
RNAi screen identifies genes associated with OLA-induced fat deposit in C. elegans
a schematic diagram of OLA-treated C. elegans with quantitative observation of fat content in liquid culture. N2 worms were synchronized, and bacteria was cultured. Synchronized L1 animals and diluted bacteria were added to 96 well plate after 20 h and grew to L4 for 43 h at 20 °C. OLA and FUDR was added to the plate, resulting in a final volume of 50 μl. After that, worms were cultured for 4 days. b Nile red signal increases after OLA (100 μM) treatment compared with vehicle (DMSO). Nile red mean intensity was measured for >30 animals from at least two biological replicates. c Increased or decreased relative fat levels were evaluated by Nile red staining for RNAi of the 17 genes emerging from the RNAi screen in OLA treatment relative to vehicle. Low fat sbp-1 indicated RNAi screen work fine. d Quantification of candidate gene expression in the vehicle and OLA treatment group. mRNA levels of target genes were normalized to act-1 expression, and data are presented as fold change to vehicle. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s two-tailed, unpaired t test (c), Mann–Whitney U test (d).
Gene expression level in models of APs-induced Mets and pharmacological inhibition of PTPN11 rescues OLA-induced Mets in vitro
a Heatmap of gene expression in Mets and Non-Mets patients. The pink rectangle indicates higher expression level. b Relative expression levels in vehicle and OLA group in HepG2 cells. c, d Relative expression of candidate genes in the livers of mice at wide type and OB/OB group at 8 weeks of OLA treatment. n = 5 animals per group. e Quantification for PTPN11 in HepG2 cells treated with PTPN11 OE transfection by qRT-PCR. f, g Line and histogram plot showing the levels of glucose response and elevated hepatic lipid accumulation. h Compared with the PTPN11 OE group, the activity of glucose response was significantly increased in pretreatment with SHP099, a selective inhibitor of PTPN11. Error bars, means ± S.D., n = 3. P < 0.05 by One-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are shown as mean ± SEM.
Antipsychotic-induced metabolic syndrome (APs-induced Mets) is the most common adverse drug reaction, which affects more than 60% of the psychiatric patients. Although the etiology of APs-induced Mets has been extensively investigated, there is a lack of integrated analysis of the genetic and epigenetic factors. In this study, we performed genome-wide, whole-exome sequencing (WES) and epigenome-wide association studies in schizophrenia (SCZ) patients with or without APs-induced Mets to find the underlying mechanisms, followed by in vitro and in vivo functional validations. By population-based omics analysis, we revealed that rare functional variants across in the leptin and peroxisome proliferator-activated receptors (PPARs) gene sets were imbalanced with rare functional variants across the APs-induced Mets and Non-Mets cohort. Besides, we discovered that APs-induced Mets are hypermethylated in ABCG1 (chr21:43642166–43642366, adjusted P < 0.05) than Non-Mets, and hypermethylation of this area was associated with higher TC (total cholesterol) and TG (triglycerides) levels in HepG2 cells. Candidate genes from omics studies were furtherly screened in C. elegans and 17 gene have been verified to associated with olanzapine (OLA) induced fat deposit. Among them, several genes were expressed differentially in Mets cohort and APs-induced in vitro/in vivo models compared to controls, demonstrating the validity of omics study. Overexpression one of the most significant gene, PTPN11, exhibited compromised glucose responses and insulin resistance. Pharmacologic inhibition of PTPN11 protected HepG2 cell from APs-induced insulin resistance. These findings provide important insights into our understanding of the mechanism of the APs-induced Mets.
Flowchart of the study procedure
Before the walk participants filled out questionnaires and underwent the fMRI scanning procedure, which included the Fearful Faces Task and the Montreal Imaging Stress Task. Subsequently, each participant was randomly assigned to a 60-min walk, in either a natural or urban environment. After the walk, the participants underwent the fMRI scanning procedure again and filled out the questionnaires.
Location of the nature and urban walk
a GPS data of two participants during the walk in the natural environment (Berlin, Grunewald) and the urban environment (Berlin, Schloßstraße) displayed on the OpenStreetMap ( b Sample picture of the walk in the natural environment. c Sample picture of the walk in the urban environment.
Bilateral amygdala activity during the Fearful Faces Task before and after the walk in the urban and in the natural environment
a a Bilateral amygdala activity while watching fearful faces (Fear condition) decreased after the walk in the natural environment. b Bilateral amygdala activity while watching neutral faces (Neutral condition) decreased after the walk in the natural environment. c Region of interest, the bilateral amygdala as defined in Automated Anatomic Labelling Atlas 2. Bottom: Stimuli in the Fearful Faces Task showing fearful facial expression, within the Fear condition (left) and neutral facial expression within the Neutral condition (right). Note: BOLD stands for Blood-Oxygen Level-Dependent; Significant differences are indicated with asterisks (*P < 0.05; **P < 0.01); error bars represent one standard error of the mean.
a GPS data of two participants during the walk in the natural environment (Berlin, Grunewald) and the urban environment (Berlin, Schloßstraße) displayed on the OpenStreetMap ( b Sample picture of the walk in the natural environment. c Sample picture of the walk in the urban environment.
Before the walk participants filled out questionnaires and underwent the fMRI scanning procedure, which included the Fearful Faces Task and the Montreal Imaging Stress Task. Subsequently, each participant was randomly assigned to a 60-min walk, in either a natural or urban environment. After the walk, the participants underwent the fMRI scanning procedure again and filled out the questionnaires.
Since living in cities is associated with an increased risk for mental disorders such as anxiety disorders, depression, and schizophrenia, it is essential to understand how exposure to urban and natural environments affects mental health and the brain. It has been shown that the amygdala is more activated during a stress task in urban compared to rural dwellers. However, no study so far has examined the causal effects of natural and urban environments on stress-related brain mechanisms. To address this question, we conducted an intervention study to investigate changes in stress-related brain regions as an effect of a one-hour walk in an urban (busy street) vs. natural environment (forest). Brain activation was measured in 63 healthy participants, before and after the walk, using a fearful faces task and a social stress task. Our findings reveal that amygdala activation decreases after the walk in nature, whereas it remains stable after the walk in an urban environment. These results suggest that going for a walk in nature can have salutogenic effects on stress-related brain regions, and consequently, it may act as a preventive measure against mental strain and potentially disease. Given rapidly increasing urbanization, the present results may influence urban planning to create more accessible green areas and to adapt urban environments in a way that will be beneficial for citizens’ mental health.
Previous studies have underscored the importance of breastfeeding and parental care on offspring development and behavior. However, their contribution as dynamic variables in animal models of early life stress are often overlooked. In the present study, we investigated how lipopolysaccharide (LPS)-induced maternal immune activation (MIA) on postnatal day (P)10 affects maternal care, milk, and offspring development. MIA was associated with elevated milk corticosterone concentrations on P10, which recovered by P11. In contrast, both milk triglyceride and percent creamatocrit values demonstrated a prolonged decrease following inflammatory challenge. Adolescent MIA offspring were heavier, which is often suggestive of poor early life nutrition. While MIA did not decrease maternal care quality, there was a significant compensatory increase in maternal licking and grooming the day following inflammatory challenge. However, this did not protect against disrupted neonatal huddling or later-life alterations in sensorimotor gating, conditioned fear, mechanical allodynia, or reductions in hippocampal parvalbumin expression in MIA offspring. MIA-associated changes in brain and behavior were likely driven by differences in milk nutritional values and not by direct exposure to LPS or inflammatory molecules as neither LPS binding protein nor interleukin-6 milk levels differed between groups. These findings reflected comparable microbiome and transcriptomic patterns at the genome-wide level. Animal models of early life stress can impact both parents and their offspring. One mechanism that can mediate the effects of such stressors is changes to maternal lactation quality which our data show can confer multifaceted and compounding effects on offspring physiology and behavior.
Depression and anxiety are major global health burdens. Although SSRIs targeting the serotonergic system are prescribed over 200 million times annually, they have variable therapeutic efficacy and side effects, and mechanisms of action remain incompletely understood. Here, we comprehensively characterise the molecular landscape of gene regulatory changes associated with fluoxetine, a widely-used SSRI. We performed multimodal analysis of SSRI response in 27 mammalian brain regions using 310 bulk RNA-seq and H3K27ac ChIP-seq datasets, followed by in-depth characterisation of two hippocampal regions using single-cell RNA-seq (20 datasets). Remarkably, fluoxetine induced profound region-specific shifts in gene expression and chromatin state, including in the nucleus accumbens shell, locus coeruleus and septal areas, as well as in more well-studied regions such as the raphe and hippocampal dentate gyrus. Expression changes were strongly enriched at GWAS loci for depression and antidepressant drug response, stressing the relevance to human phenotypes. We observed differential expression at dozens of signalling receptors and pathways, many of which are previously unknown. Single-cell analysis revealed stark differences in fluoxetine response between the dorsal and ventral hippocampal dentate gyri, particularly in oligodendrocytes, mossy cells and inhibitory neurons. Across diverse brain regions, integrative omics analysis consistently suggested increased energy metabolism via oxidative phosphorylation and mitochondrial changes, which we corroborated in vitro; this may thus constitute a shared mechanism of action of fluoxetine. Similarly, we observed pervasive chromatin remodelling signatures across the brain. Our study reveals unexpected regional and cell type-specific heterogeneity in SSRI action, highlights under-studied brain regions that may play a major role in antidepressant response, and provides a rich resource of candidate cell types, genes, gene regulatory elements and pathways for mechanistic analysis and identifying new therapeutic targets for depression and anxiety.
Chronic neuropathic pain leads to cognitive impairment and neuronal atrophy which are attenuated by the analgesic drug, gabapentin
A Schematic representation of the experimental design of 16-week-long SNI and Gabapentin (Gab) treatment for 11 weeks. B Von Frey test revealed that SNI induced allodynia as indicated by decreased hindlimb withdrawal threshold which was attenuated by Gabapentin. C Gabapentin-evoked pain relief conditioned SNI animals in the Conditioned place preference test as indicated by increased Δ time of SNI animals spent in Gab-paired chamber. SNI induced memory deficits as indicated by reduced discrimination index in NOR test (D) and reduced spontaneous alterations in Y-maze test (E); note that Gab-driven analgesia reverted these cognitive deficits. F No alteration in total distance traveled in Open field test among all groups indicating no locomotion differences [one-way ANOVA, 8–9 animals/group for all behavioral tests except CPP test (student’s t-test, 7–8 animals/group)]. Golgi-based reconstruction of CA1 neurons (G) revealed that SNI animals exhibited reduced apical, but not basal, dendritic length (H, I) as well as reduced dendritic arborization in the hippocampus (assessed by Sholl analysis); J when compared to controls and gabapentin-treated animals (5 animals/group; 27–29 neurons/group). All data are expressed as group mean ± SEM (*p < 0.05, ***<0.001).
Prolonged neuropathic pain triggers Tau accumulation and inhibition of autophagy in the hippocampus
A Levels of IF staining of total Tau were increased in the hippocampus of SNI animals, while Gabapentin-treated SNI animals show total Tau IF staining similar to sham mice. B–F Representative immunoblots and quantification of hippocampal protein levels of total Tau (C) 4R-Tau (D), Rab35 (E) and autophagy-related protein, p62 (F). With the exception of the small GTPase, Rab35, SNI triggered an accumulation of these proteins. Note that Gabapentin treatment reverted these changes induced by SNI. G, H IF staining of different autophagic markers showed that SNI caused reduced levels of LC3 and LAMP1 followed by accumulated levels of p62 indicating autophagic inhibition; Gabapentin treatment in SNI animals reverted these changes (for IF analysis, five animals/group, for WB 6–7 animals/group, one-way ANOVA). All data are expressed as group mean ± SEM (*p < 0.05, **p < 0.01, ***<0.001, **** <0.0001).
Rab35 overexpression blocked the SNI-evoked Tau accumulation and related memory deficits
A Experimental design showing the three animal groups used: sham, SNI, and SNI + Rab35. B SNI and SNI + Rab35 animals exhibited reduced hindlimb withdrawal threshold compared to sham animals indicating that allodynia was established in both SNI groups. C SNI animals showed decreased % freezing in Contextual fear conditioning test compared to sham animals while Rab35 exogenous expression in SNI animals (SNI + Rab35) reverted this SNI-driven reduction. D Discrimination index of Novel object recognition test was reduced in SNI, but not SNI-Rab35, animals when compared to sham animals. E, F No difference of locomotion was found among all groups as indicated by similar levels of total distance traveled in the Open field arena (E); body weight was also unaltered (F). G–J Rab35 exogenous expression blocked the SNI-driven accumulation of total Tau and pSer396/404 Tau in the hippocampus (5–6 animals/group, one-way ANOVA). All data are expressed as group mean ± SEM (*p < 0.05, **p < 0.01, **** <0.0001).
Tau ablation blocked memory loss and hippocampal atrophy induced by chronic neuropathic pain
A Experimental design showing the use of sham and SNI operation in animals lacking Tau (Tau-KO) and their wild-type (WT) littermates. B SNI decreased the withdrawal threshold of both WT and Tau-KO animals in the von Frey test. C WT SNI animals showed decreased freezing time when compared to WT sham in the Contextual fear conditioning test indicating memory impairment. However, Tau-KO SNI animals were not different from Tau-KO sham suggesting absence of SNI-driven memory deficits. Discrimination index of NOR test (D) and number of entries in the new arm of Y-maze (E) were decreased in WT, but not Tau-KO, SNI animals indicating that SNI does not induce memory decline in Tau-KO animals (7–10 animals/group). F SNI decreased dendritic length in the hippocampus (CA1 area) of WT, but not Tau-KO, animals. G Sholl analysis revealed SNI-driven reduced intersections in WTs indicative of reduced dendritic arborization (two-way ANOVA, five animals/group; 33–35 neurons/group). H–J Representative immunoblots and quantification of hippocampal protein levels of total Tau as well as pSer202-, pThr231- and pSer396/404-Tau showing increased total Tau (I) and phosphorylation levels of Thr231 and pSer396/404 Tau epitopes (J) in the hippocampus of WT SNI animals. K Representative blots and quantification of sarkosyl-insoluble and soluble Tau levels in the hippocampus of P301L-Tau transgenic animals. Note that SNI increased the levels of both sarkosyl-insoluble and soluble Tau indicating increased Tau aggregation and accumulation (5–6 animals/group). L von Frey test in P301L-Tau animals showing that SNI severely decreased withdrawn threshold indicating mechanical allodynia. M SNI-driven reduced freezing time of P301L-Tau animals in contextual fear conditioning test (Student’s t-test, 10 animals/group). N Hypothetical model showing that SNI causes accumulation of phospho- and insoluble-Tau species through reduction of Rab35 and autophagy leading to neuronal atrophy and cognitive deficits. All data are expressed as group mean ± SEM (*p < 0.05, **p < 0.01, ***<0.001, **** <0.0001).
Persistent pain has been recently suggested as a risk factor for dementia. Indeed, chronic pain is frequently accompanied by maladaptive brain plasticity and cognitive deficits whose molecular underpinnings are poorly understood. Despite the emerging role of Tau as a key regulator of neuronal plasticity and pathology in diverse brain disorders, the role of Tau has never been studied in the context of chronic pain. Using a peripheral (sciatic) neuropathy to model chronic pain in mice—spared nerve injury (SNI) for 4 months—in wildtype as well as P301L-Tau transgenic mice, we hereby demonstrate that SNI triggers AD-related neuropathology characterized by Tau hyperphosphorylation, accumulation, and aggregation in hippocampus followed by neuronal atrophy and memory deficits. Molecular analysis suggests that SNI inhibits autophagy and reduces levels of the Rab35, a regulator of Tau degradation while overexpression of Rab35 or treatment with the analgesic drug gabapentin reverted the above molecular changes leading to neurostructural and memory recovery. Interestingly, genetic ablation of Tau blocks the establishment of SNI-induced hippocampal morphofunctional deficits supporting the mediating role of Tau in SNI-evoked hippocampal pathology and memory impairment. These findings reveal that exposure to chronic pain triggers Tau-related neuropathology and may be relevant for understanding how chronic pain precipitates memory loss leading to dementia.
The brain remains a key reservoir of latent HIV infection, and people living with HIV (PLWH) face a high risk for cognitive impairment and psychiatric disorders. Although the burden of HIV infection and co-morbidities is greatest in the Global South, a large proportion of HIV mental health research is carried out in the Global North. Large, well-funded observational cohort studies exploring HIV-associated psychopathology generally involve participant groups from WEIRD (Western, educated, industrialised, rich and democratic) settings. The socioeconomic status and institutional access afforded to these participant groups on average does not reflect those of the majority of beneficiaries of HIV mental health research. This misalignment may lead to limitations in generalising findings and developing effective interventions to improve the mental health of PLWH. Here, I offer recommendations to actively cultivate authentic diversity and inclusion in the field, with four focus points: (1) for funding bodies, to actively invest in neuroscientists in the Global South for investigations of HIV-related psychopathology; (2) for scientific publishers, to fund professional support services for researchers in the Global South; (3) for academic institutions, to facilitate meaningful, equitable collaborations with researchers in the Global South and incentivise studies with diverse participant groups; and (4) for individual neuroscientists, to actively cite and converse with colleagues in the Global South, tackle personal biases in those conversations, and avoid overgeneralising findings from primarily WEIRD participant groups.
As part of his lifelong effort to develop optimal nosologic categories for the non-affective delusional syndromes, in the 1913 8th edition of his textbook, Kraepelin proposed a new diagnosis of paraphrenia presenting with extensive bizarre delusions and auditory hallucinations but no prominent negative symptoms or personality deterioration. He tentatively suggested it was distinct from dementia praecox (DP). His proposal was met with controversy. In an attempt to resolve this matter, Wilhelm Mayer, working with Kraepelin in Munich, published in 1921 the result of a follow-up study of the 78 cases of paraphrenia on the basis of which Kraepelin had developed his new diagnosis. In the 74 cases with adequate follow-up, Mayer’s final diagnoses were 43% DP, 38% paraphrenia, and 18% other. He also presented limited family data, suggesting co-aggregation of DP and paraphrenia. On the basis of these results, Mayer argued that paraphrenia was likely better considered to represent a form of DP and not an independent disorder. His opinion was accepted by nearly all subsequent authors. Mayer’s work appeared nearly a half-century before the proposal of Robin and Guze for the validation of psychiatric disorders by follow-up and family studies. The idea of deciding psychiatric questions on empirical grounds—rather than on the prestige of debating parties—is not a recent discovery but can be traced to the roots of our current diagnostic system in the work of Emil Kraepelin and his associates.
Top-cited authors
James L Kennedy
  • Centre for Addiction and Mental Health
Markus M Nöthen
  • University of Bonn
Peter Mcguffin
  • King's College London
Julio Licinio
  • State University of New York Upstate Medical University
Marcella Rietschel
  • Central Institute of Mental Health