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

Abstract

The signal transducer and activator of transcription 3 (STAT3) signalling pathway is activated through phosphorylation by Janus kinases in response to a diverse set of immunogenic and non-immunogenic triggers. Several distinct lines of evidence propose an intricate involvement of STAT3 in neural function relevant to behaviour in health and disease. However, in part due to the pleiotropic effects resulting from its DNA binding activity and the consequent regulation of expression of a variety of genes with context-dependent cellular consequences, the precise nature of STAT3 involvement in the neural mechanisms underlying psychopathology remains incompletely understood. Here, we focused on the midbrain serotonergic system, a central hub for the regulation of emotions, to examine the relevance of STAT3 signalling for emotional behaviour in mice by selectively knocking down raphe STAT3 expression using germline genetic (STAT3 KO) and viral-mediated approaches. Mice lacking serotonergic STAT3 presented with reduced negative behavioural reactivity and a blunted response to the sensitising effects of amphetamine, alongside alterations in midbrain neuronal firing activity of serotonergic neurons and transcriptional control of gene networks relevant for neuropsychiatric disorders. Viral knockdown of dorsal raphe (DR) STAT3 phenocopied the behavioural alterations of STAT3 KO mice, excluding a developmentally determined effect and suggesting that disruption of STAT3 signalling in the DR of adult mice is sufficient for the manifestation of behavioural traits relevant to psychopathology. Collectively, these results suggest DR STAT3 as a molecular gate for the control of behavioural reactivity, constituting a mechanistic link between the upstream activators of STAT3, serotonergic neurotransmission and psychopathology.

Fig. 1. Loss of serotonergic STAT3 alters 5-HT neuronal excitability in vivo.

A STAT3 immunoreactivity was evaluated within 5-HT-positive cells (ROI: white outlines shown in STAT3 channel) in images of the DR area (representative 4 × 5 stitched 60X images; scale bar: 100 µm), illustrating a visible reduction of STAT3 signal within the specified ROI (i.e., serotonergic cells) in KO animals. White dashed boxes mark the detailed area depicted in (B). B Corresponding single panel images of 5-HT and STAT3 immunoreactivity and a merged image in control and KO mice (60X, scale bar: 50 µm). C Quantification of STAT3 immunoreactivity within the ROI confirmed the reduction of STAT3 expression in KO serotonergic neurons of the DR. D, E STAT3 KO mice displayed significantly increased serotonergic firing frequencies compared to controls. 5-HT: 5-hydroxytryptamine; A.U.: arbitrary units; DR: dorsal raphe; KO: knockout; ROI: region of interest; STAT3: signal transducer and activator of transcription 3. All data are presented as mean ± SD. *p < 0.05.

Fig. 2. Loss of serotonergic STAT3 leads to differential behavioural reactivity in stressful behavioural paradigms.

Motor function was not impacted by knockout of STAT3 in serotonergic cells, as (A) locomotor activity in the OFT and (B) motor coordination in the RR were similar between groups. C No differences between genotypes in sucrose preference were found in the SPT. D Average latency to feed in an adverse environment was reduced in KO animals relative to controls in the NSF. No effect of serotonergic STAT3 knockout on (E) body mass, (F) body mass lost during the food restriction period preceding the NSF, or (G) food consumption immediately after the NSF test. H KO animals showed markedly reduced passive immobility behaviour in the FST in comparison to controls. Avoidance of the adverse compartments in (I) LDB and (J) EPM were unchanged by serotonergic STAT3 knockout. EPM: elevated plus maze; FST: forced swim test; KO: knockout; LDB: light-dark box; NSF: novelty-suppressed feeding; OFT: open field test; RR: Rotarod; SPT: sucrose preference test; STAT3: signal transducer and activator of transcription 3. All data are presented as mean ± SD. *p < 0.05; ***p < 0.001.

Fig. 3. Transcriptional networks altered by serotonergic STAT3 deletion overlap with gene sets and pathways functionally implicated in psychopathology.

A Results of RNA-Seq in DR tissue of STAT3 KO and control animals revealed 194 differentially expressed genes (DEG, red dots: q < 0.05). B qRT-PCR targeting recombined exons (18, 19, 20) of STAT3 confirms reduced expression of functional STAT3 mRNA vs. β-actin in the DR of the STAT3 KO group. C Of the DEG, 111 transcripts (DEG×STAT3) are reported to be direct targets of STAT3 in published ChIP-Seq experiments according to ChIP-Atlas. D Several gene sets relating to different aspects of synaptic transmission as well as nervous system development (GO: biological processes, white bars) and genes belonging to the synaptic vesicle KEGG pathway (black bars) were disproportionately represented among DEG×STAT3. E Venn analysis using lists of MDD-, SCZ- and BP- associated genes in combination with the DEG set showed 52 genes differentially expressed in KO mice which were previously associated with SMI (DEG×SMI). F Significantly enriched gene sets related predominantly to biological processes (white bars) and KEGG pathways (black bars) involved in neuronal excitability and cell-to-cell communication. G Daily d-amphetamine administration induced locomotor sensitisation in control mice, but this effect was significantly blunted in KO. H Levels of reward-associated learning in the CPP were not impacted by STAT3 loss. BP: bipolar disorder; CPP: conditioned place preference; DEG: differentially expressed genes; GO: gene ontology; KEGG: Kyoto Encyclopaedia of Genes and Genomes; KO: knockout; MDD: major depressive disorder; SCZ: schizophrenia; SMI: severe mental illnesses; STAT3: signal transducer and activator of transcription 3. All data are presented as mean ± SD. Red line in (D) and (F) denotes -log(adjusted p = 0.05). *p < 0.05; **p < 0.01.

Fig. 4. Viral-mediated knockdown of STAT3 in DR recapitulates behaviours relevant to psychopathology observed in serotonergic STAT3 KO mouse.

A Adult Stat3^fl/fl mice were injected with AAV-Cre or AAV-GFP in the DR. A representative image (left panel; 10X, scale bar: 100 µm; area represented by box in right panel) and a heat map (right panel) showing the overlap of transfected areas in the cohort of AAV-Cre-injected mice are shown, with the darkest area representing the area that was targeted in all animals. B Timeline of experiments and procedures for the acute viral knockdown of STAT3. C STAT3 immunoreactivity in the DR was used to represent the efficiency of the viral knockdown strategy. STAT3 fluorescence signal intensity was quantified within the ROI (white rectangle) in the DR of AAV-Cre- vs. AAV-GFP-injected mice to (representative 4 × 5 stitched 60X images; scale bar: 100 µm). White dashed boxes mark the detailed area represented in (D), where single panel images (60X, scale bar: 50 µm) are shown. White arrows indicate examples of cells which are AAV-Cre or AAV-GFP transfected and have significant STAT3 immunoreactivity. These cells appear less numerous in the AAV-Cre group. E Quantification of overall STAT3 fluorescence intensity in the ROI confirmed Cre-mediated knockdown of STAT3 protein expression in the DR of the AAV-Cre group compared to AAV-GFP controls. F Transfection efficiency (DAPI + /GFP + coexpression vs. total DAPI + in ROI) and neurotropism (NeuN + /GFP + coexpression vs. total NeuN+ in ROI) were quantified in the DR of AAV-Cre injected mice. Behavioural evaluation of AAV-Cre mice did not reveal any differences in (G) locomotor activity in the open field or (H) sucrose preference in the SPT. I AAV-Cre mice showed significantly reduced latency to feed during the NSF while (J) their body mass, (K) the extent of weight loss due to food restriction, and (l) the amount of food consumed post-NSF was not impacted by DR STAT3 knockdown. M AAV-Cre mice displayed significantly reduced immobility in the FST when compared to the AAV-GFP group. N In contrast to AAV-GFP mice, AAV-Cre mice showed virtually no locomotor sensitisation to d-amphetamine. A.U.: arbitrary units; DR: dorsal raphe; FST: forced swim test; NSF: novelty-suppressed feeding; OFT: open field test; ROI: region of interest; SPT: sucrose preference test; STAT3: signal transducer and activator of transcription 3. All data are presented as mean ± SD. *p < 0.05.