Understanding complex dynamics of behavioral, neurochemical and transcriptomic changes induced by prolonged chronic unpredictable stress in zebrafish

Abstract

Stress-related neuropsychiatric disorders are widespread, debilitating and often treatment-resistant illnesses that represent an urgent unmet biomedical problem. Animal models of these disorders are widely used to study stress pathogenesis. A more recent and historically less utilized model organism, the zebrafish (Danio rerio), is a valuable tool in stress neuroscience research. Utilizing the 5-week chronic unpredictable stress (CUS) model, here we examined brain transcriptomic profiles and complex dynamic behavioral stress responses, as well as neurochemical alterations in adult zebrafish and their correction by chronic antidepressant, fluoxetine, treatment. Overall, CUS induced complex neurochemical and behavioral alterations in zebrafish, including stable anxiety-like behaviors and serotonin metabolism deficits. Chronic fluoxetine (0.1 mg/L for 11 days) rescued most of the observed behavioral and neurochemical responses. Finally, whole-genome brain transcriptomic analyses revealed altered expression of various CNS genes (partially rescued by chronic fluoxetine), including inflammation-, ubiquitin- and arrestin-related genes. Collectively, this supports zebrafish as a valuable translational tool to study stress-related pathogenesis, whose anxiety and serotonergic deficits parallel rodent and clinical studies, and genomic analyses implicate neuroinflammation, structural neuronal remodeling and arrestin/ubiquitin pathways in both stress pathogenesis and its potential therapy.

Figure 1

A brief diagram outlining the study experimental design, including the chronic unpredictable stress (CUS) protocol and behavioral testing (see Table 1 for details of CUS stressors applied in the present study). NTT the novel tank test, LDT the light–dark test, SH shoaling test, ZTI the zebrafish tail immobilization test, HPLC high-performance liquid chromatography, RNASeq RNA sequencing.

Figure 2

Weekly dynamics of behavioral alterations induced by chronic unpredictable stress (CUS) exposure and fluoxetine treatment in adult zebrafish tested in the novel tank test (time spent in top and distance traveled), the light–dark test (time spent in light), shoaling test (average inter-fish distance and distance to water surface) and the zebrafish tail immobilization test (ZTI, time spent active). Data is represented as mean ± S.E.M. (n = 20 in weeks 1–3 and n = 15 in weeks 4–5), *p < 0.05 control vs. stress, post-hoc Tukey’s test for significant Wald Chi-squared test (ANOVA Type II) for GZLM1 for group (control and stress), week (1–5) and their interaction as predictors, ^#p < 0.05 stress vs. control group and ^$p < 0.05 fluoxetine vs. stress group, post-hoc Tukey’s test for significant Wald Chi-squared test ANOVA (Type II) for GZLM2 for group (control, stress and fluoxetine) at week 5 as predictor. Graphs were constructed using the ggplot2 R package, also see Tables 2 and 3 and Supplementary Tables S1–S5 for statistical details.

Figure 3

Weekly dynamics of neurochemical alterations induced by chronic unpredictable stress (CUS) exposure and fluoxetine treatment, assessed by HPLC in the whole-brain samples of adult zebrafish (n = 10). Data are represented as mean ± S.E.M. *p < 0.05 control vs. stress, post-hoc Tukey’s test for significant Wald Chi-squared test ANOVA (Type II) for GZLM1 for group (control and stress), week (1–5) and their interaction as predictors, ^#p < 0.05 stress vs. control group and ^$p < 0.05 fluoxetine vs. stress group and ^&p < 0.05 fluoxetine vs. control, post-hoc Tukey’s test for significant Wald Chi-squared test ANOVA (Type II) for GZLM2 using group (control, stress and fluoxetine) at week 5 as predictor. Graphs were constructed using the ggplot2 R package, also see Tables 2 and 3 and Supplementary Tables S1–S5 for statistical details.

Figure 4

The network of protein–protein interactions (PPI) constructed for differentially expressed genes (found in all analyses) using the STRING online database (see “Methods” section and Supplementary Figs. S1–S3 for details). Genes represented as numbers refer to “ENSDARG000000*”, where * denotes the last 5 digits of the Ensembl gene names (ID). The network was visualized using the CytoScape application^,. SvC stress vs. control, FvC fluoxetine vs. stress, SFvC differentially expressed in both SvC and FvC (in same l2fc direction), FvS fluoxetine vs. stress, SvCF differentially expressed in SvC and FvS (in opposite directions, color refers to the direction of FvS expression change), Additional—20 proteins with the highest interaction score in STRING for suggested PPI networks. Letters denote several clusters of genes, including (A) arrestins and G protein-coupled receptors (GPCRs) related genes, (B) ubiquitin-related genes and their inflammatory modulators, (C) inflammation-related transcription factors, (D) cytoskeletal and motility related proteins, (E) vitellogenins.

Figure 5

Gene co-expression (GE) network constructed for differentially expressed genes (found in all analyses) using the GeneMANIA^– online database (see the “Methods” section, and Supplementary Figs. S1–S3 for details of treatment and network construction). The network was graphically presented using the CytoScape application^,. SvC stress vs. control, FvC fluoxetine vs. stress, SFvC differentially expressed in both SvC and FvC (in same l2fc direction), FvS fluoxetine vs. stress, SvCF differentially expressed in both SvC and FvS (in the opposite direction, color refers to the direction of FvS expression change), Additional—20 genes with the highest co-expression score in GeneMANIA for suggested PPI networks. (A) fluoxetine vs. stress-related genes that are commonly co-expressed.

Figure 6

Qualitative real-time polymerase chain reaction results of the last (fifth) week of CUS showing differential expression of selected four reference genes in adult zebrafish brain samples with significant expression differences detected previously by the RNA-seq. Data is analyzed and represented using the Pfaffl method. *p < 0.05, **p < 0.01, ***p < 0.001 vs. controls or vs. the group connected by a horizontal line, post-hoc Dunn’s test for pairwise comparisons for significant Kruskal–Wallis data (see Supplementary Table S10 for details). Graphs were constructed using the ggplot2 R package.