STAT3 in the dorsal raphe gates behavioural reactivity and regulates gene networks associated with psychopathology

The study addresses how STAT3 signalling within the midbrain serotonergic system, particularly the dorsal raphe (DR), contributes to behavioural traits relevant to psychopathology. Against the backdrop of high global burden of major depressive disorder, bipolar disorder, and schizophrenia, and incomplete understanding of their pathophysiology, the authors hypothesised that STAT3 in serotonergic neurons regulates negative emotional behaviours and responses to aversive stimuli. Motivated by evidence that STAT3 is activated by diverse immunogenic and non-immunogenic signals, influences synaptic plasticity and epigenetic regulation, and may interact with serotonergic neurotransmission, the purpose was to test whether disrupting STAT3 in serotonergic neurons alters behaviour, neuronal activity, and gene expression programs linked to mental illness.

The introduction synthesizes prior work indicating: (i) STAT3 is a key mediator of cytokine-driven immune processes implicated in psychopathology (immune hypothesis of psychiatric disease); (ii) STAT3 activation can also reflect actions of growth factors, hormones, and endocannabinoids, all relevant to neural function; (iii) STAT3 modulates synaptic plasticity and epigenetic control of gene expression, potentially impacting neural circuits independently of immune activation; (iv) emerging links exist between STAT3 and the serotonergic system, central to psychiatric disease aetiology. Prior reports, including those by the authors, suggest STAT3 signalling as a regulator of negative emotional behaviour and responses to aversive conditions, motivating a focused investigation of serotonergic STAT3 in behaviour and underlying neural mechanisms.

- Animals: Conditional STAT3 knockout mice with serotonergic cell-specific deletion (SertCre/+; Stat3fl/fl; KO) and littermate controls (SertCre/+; Stat3+/+). For viral experiments, Stat3fl/fl mice received AAV-Cre or AAV-GFP in the DR. Both sexes, 8–16 weeks old, single-housed, standard 12/12 light/dark, 22±1°C, food and water ad libitum. Blinded experimenters; sample sizes per Supplementary Table 1; ethical approval per EU Directive 2010/63/EU. - In vivo electrophysiology: Extracellular recordings of DR 5-HT neurons using Axoclamp-2B amplifier, Digidata-1440, pClamp-10/Clampfit for acquisition and analysis, following established protocols. - Behavioural testing: • Sucrose Preference Test (SPT): hedonic behaviour measured as % sucrose preference. • Novelty-Suppressed Feeding (NSF): latency to first bite (s) in a brightly lit arena after food deprivation. • Forced Swim Test (FST): % immobility using automated tracking (VideoTrack v3). • Open Field Test (OFT): locomotor activity (total distance travelled) via beam breaks (Activity Monitor v5). • Light-Dark Box (LDB): anxiety-like behaviour (time in light zone, %). • Elevated Plus Maze (EPM): anxiety-like behaviour (time in open arms, %; Videotrack). • Rotarod (RR): motor coordination; average latency to fall over 3 trials. • Amphetamine Sensitisation: locomotor sensitisation assessed as % change in distance vs day 1 following i.p. d-amphetamine (2 mg/kg free base) over repeated sessions in the open field. • Conditioned Place Preference (CPP): biased paradigm; d-amphetamine (5 mg/kg free base) vs saline pairings over 6 training days; side preference (%) tested. - Drugs: d-amphetamine sulphate in saline, free base conversion factor 1.36 applied. - Immunohistology: Perfusion with 4% PFA; 30 µm DR sections (Bregma −4.0 to −5.2 mm). Primary antibodies for 5-HT, STAT3, Iba1, GFAP, NeuN; appropriate fluorescent secondaries. Confocal imaging (Nikon A1) with 20x/60x/100x objectives. Quantification of STAT3 immunoreactivity within defined ROIs and within serotonergic neurons, microglia, and astrocytes via ImageJ using thresholded masks; DR-wide STAT3 intensity quantified post-viral manipulation in a standardized rectangular ROI (~700×800 µm²). - Tissue collection: Rapid brain extraction; DR microdissection from four 300 µm coronal sections (Bregma −4.0 to −5.2 mm) into RNALater. - RNA isolation and sequencing: miRNEasy Mini Kit; RIN > 7.0. Library prep and sequencing per prior work; alignment with STAR; differential expression with DESeq2; multiple testing correction by Benjamini-Hochberg at FDR 5%. Dataset deposited in GEO (GSE146101). - qRT-PCR: cDNA from 450 ng DR RNA (DyNamo kit); STAT3 expression relative to β-actin by ΔΔCt; primers in Supplementary Table 2. - Viral DR knockdown: Stereotaxic bilateral DR injections of AAV5-Cre-eGFP or AAV5-GFP (0.5 µl per site over 8 min) at coordinates adjusted by individual scaling: (a) x=0.0, y=−4.5, z=−2.9; (b) x=0.0, y=−4.5, z=−2.7. Post-behaviour histological verification of GFP spread and STAT3 reduction. - Statistics: Outliers pre-excluded if outside mean ±2 SD. Two-tailed Student’s t-tests (Welch correction as needed) for two-group comparisons; repeated-measures ANOVA for longitudinal designs; analyses in GraphPad Prism 7.0.

- Selective deletion of STAT3 in serotonergic neurons (SertCre/+; Stat3fl/fl) significantly reduced STAT3 immunoreactivity within DR 5-HT neurons and altered in vivo DR serotonergic neuronal firing activity. - Behaviourally, mice lacking serotonergic STAT3 showed reduced negative behavioural reactivity across assays probing anxiety- and depression-like dimensions, and exhibited normal motor coordination and baseline locomotion. - STAT3 KO mice displayed a blunted locomotor sensitisation response to repeated d-amphetamine exposure; reward learning assessed by conditioned place preference was evaluated using a biased paradigm. - Transcriptomic profiling of DR tissue revealed STAT3-dependent regulation of gene networks enriched for pathways and genes associated with neuropsychiatric disorders. - Adult-onset, localized STAT3 knockdown in the DR via AAV-Cre phenocopied the behavioural alterations seen in the germline serotonergic STAT3 KO, indicating that the behavioural phenotype is not strictly developmentally determined and that DR STAT3 in adults is sufficient to gate behavioural reactivity.

The findings support the hypothesis that STAT3 signalling within the dorsal raphe serotonergic system functions as a molecular gate controlling behavioural reactivity to aversive conditions and psychostimulant sensitisation. By demonstrating altered DR 5-HT neuron firing and broad STAT3-dependent transcriptional changes in networks implicated in psychiatric illness, the study bridges upstream STAT3-activating factors (cytokines, growth factors, hormones, endocannabinoids) with serotonergic circuit function and behavioural outcomes. The convergence of germline serotonergic cell-specific KO data with adult DR-targeted viral knockdown strengthens the causal role of DR STAT3 in modulating anxiety- and depression-related behaviours and dampening amphetamine sensitisation. These results advance mechanistic understanding of how immune and neuromodulatory signals can reshape serotonergic output and behavioural phenotypes relevant to MDD, bipolar disorder, and schizophrenia.

This work identifies STAT3 in dorsal raphe serotonergic neurons as a key regulator of behavioural reactivity and a transcriptional hub connecting upstream modulators to gene networks linked to psychopathology. Both constitutive serotonergic STAT3 deletion and adult DR-specific knockdown reduce negative emotional behaviours, blunt amphetamine sensitisation, alter DR neuron firing, and reprogram DR transcriptomes. These insights highlight DR STAT3 signalling as a potential target for interventions aimed at modulating stress- and reward-related behavioural dimensions across psychiatric diagnoses. Future research should delineate the specific upstream ligands and receptors driving DR STAT3 activation in vivo, resolve cell-type and circuit-level specificity within and beyond serotonergic neurons, test causal roles of identified downstream gene networks, and evaluate translational relevance and therapeutic manipulability of DR STAT3 signalling in disease models.

Limitations were not explicitly detailed in the provided text. Potential considerations include: reliance on mouse behavioural assays that model facets of human psychiatric dimensions; focus primarily on the dorsal raphe and serotonergic populations without exhaustive cell-type resolution; possible off-target or developmental effects of Cre driver lines, although adult viral knockdown mitigates this concern; and limited generalisability across strains, ages, and environmental conditions. Detailed effect sizes and full statistical results are in supplementary materials.