Microarray analyses reveal novel targets of exercise-induced stress resistance in the dorsal raphe nucleus

Serotonin (5-HT) is implicated in the development of stress-related mood disorders in humans. Physical activity reduces the risk of developing stress-related mood disorders, such as depression and anxiety. In rats, 6 weeks of wheel running protects against stress-induced behaviors thought to resemble symptoms of human anxiety and depression. The mechanisms by which exercise confers protection against stress-induced behaviors, however, remain unknown. One way by which exercise could generate stress resistance is by producing plastic changes in gene expression in the dorsal raphe nucleus (DRN). The DRN has a high concentration of 5-HT neurons and is implicated in stress-related mood disorders. The goal of the current experiment was to identify changes in the expression of genes that could be novel targets of exercise-induced stress resistance in the DRN. Adult, male F344 rats were allowed voluntary access to running wheels for 6 weeks; exposed to inescapable stress or no stress; and sacrificed immediately and 2 hours after stressor termination. Laser capture microdissection selectively sampled the DRN. mRNA expression was measured using the whole genome Affymetrix microarray. Comprehensive data analyses of gene expression included differential gene expression, log fold change (LFC) contrast analyses with False Discovery Rate correction, KEGG and Wiki Web Gestalt pathway enrichment analyses, and Weighted Gene Correlational Network Analysis (WGCNA). Our results suggest that physically active rats exposed to stress modulate expression of twice the number of genes, and display a more rapid and strongly coordinated response, than sedentary rats. Bioinformatics analyses revealed several potential targets of stress resistance including genes that are related to immune processes, tryptophan metabolism, and circadian/diurnal rhythms

Acute stressor exposure modifies plasma exosome-associated heat shock protein 72 (Hsp72) and microRNA (miR-142-5p and miR-203).

Exosomes, biologically active nanoparticles (40-100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but there are no studies examining in vivo exosomal changes evoked by the acute stress response. The present study reveals that exposing male Fisher 344 rats to an acute stressor modulates the protein and miRNA profile of circulating plasma exosomes, specifically increasing surface heat shock protein 72 (Hsp72) and decreasing miR-142-5p and -203. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-evoked modulation of immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Together, these findings demonstrate that activation of the acute stress response modifies the proteomic and miRNA profile of exosomes released into the circulation

Exposure to an acute stressor modifies the proteomic profile of circulating plasma exosomes.

<p>A. Male Fisher 344 rats exposed to inescapable tail shocks (Stress) had elevated plasma concentrations of Hsp72 compared to non-stressed rats (Control). Following exosome enrichment with ExoQuick precipitation, B. concentrations of Hsp72 were the highest in the exosome enriched fractions from stressed rats. C. Correction with CD63 particles confirmed that exposure to stress increases Hsp72 concentrations in the exosome enriched fraction compared to control conditions. D. Lysing the exosome enriched fraction increased Hsp72 concentrations for both the stress and control groups, but the impact of stressor exposure was restricted to the not lysed group. E. Following pre-treatment with Thromboplastin-D, plasma was divided and either treated with magnetic Dynabeads coated in CD63 antibodies (Anti-CD63) to immunoprecipitate CD63 particles from the plasma or left undisturbed (No Treatment) prior to ExoQuick precipitation. Anti-CD63 treatment attenuates CD63 concentrations in the plasma exosome enriched fractions from stressed rats compared no treatment conditions. F. Anti-CD63 treatment also attenuates Hsp72 in the plasma exosome enriched fractions from stressed rats compared to no treatment conditions. Results are expressed in means ± SE; 6–8 rats/condition. *indicates significant difference when compared to control rats (p<0.05). #indicates significant difference when compared to exosome depleted fraction (p<0.05). Two-way ANOVA with Fisher PLSD post hoc test was used.</p

Six weeks of voluntary wheel running reduces the deficit in instrumental escape learning produced by 5-HT_2C receptor activation in the dorsal striatum.

<p>Fifteen minutes prior to behavioral testing, sedentary and physically active (Run) rats received intra-DS microinjections of the 5-HT_2C receptor agonist CP-809101 (0.3 mM, 2.0 mM or 6.0 mM). (<b>A</b>) Cannula tip placement within the DS. Sedentary rats are denoted with black triangles, physically active rats are denoted with gray triangles, and off-site placements are denoted with an <b>X</b>. Brain illustrations adapted from Paxinos and Watson (published in the Rat Brain in Stereotaxic Coordinates, 4^th ed., Copyright Elsevier (1998)). Numbers left of illustrations refer to distance from Bregma (mm). (<b>B</b>) Freezing behavior over the duration of the post-FR-1 freezing observation period presented in 2 minute blocks (pre-shock scores are not different and therefore overlap). (<b>C</b>) The mean percent time spent freezing during the 20 minute observation period. (<b>D</b>) Shuttle box escape latencies for one block of 2 FR-1 trials (FR-1) and five blocks of 5 FR-2 trials (FR-2). (<b>E</b>) The mean escape latency for all 25 FR-2 escape trials. Data represent group means ± SEM. * p<0.05 relative to 0.3 mM groups and off-site control group; Φ p<0.05 relative to 2.0 mM sedentary group.</p

Adrenergic regulation of exosomal Hsp72.

<p>Adult male Fisher 344 rats were either injected intraperitoneally with the α₁-ADR antagonist prazosin (2.0 mg/kg) 30 min prior to exposure to tail shock stress (Stress) or left undisturbed. Plasma analysis reveals that prazosin administration A. significantly attenuates stress-induced elevations of Hsp72 as well as B. lactate dehydrogenase activity, a marker of cell death. Exosomes were fractionated with ExoQuick in the stressed rats, revealing that prazosin administration C. had no effect on CD63 particle concentrations, but D. significantly attenuates Hsp72 concentration in both the exosome depleted and exosome enriched fractions. Results are expressed in means ± SE; 6–8 rats/condition. *indicates significant difference when compared to control rats (p<0.05). #indicates significant difference when compared to no treatment group or exosome depleted fraction (p<0.05). Two-way ANOVA with Fisher PLSD post hoc test was used.</p

Six weeks of voluntary wheel running reduces the increase in fear behavior produced by 5-HT_2C receptor activation in the region of the lateral/basolateral amygdala (BLA).

<p>Following 6 weeks of voluntary wheel running (Run) or no running (Sedentary), rats received intra-BLA microinjections of either saline or the selective 5-HT_2C receptor agonist CP-809101 (0.3 mM, 2.0 mM, or 6.0 mM). Shockelicited freezing and shuttle box escape latency were measured sequentially in shuttle boxes 15 minutes later. (<b>A</b>) Cannula placement within the amygdala. Sedenatry rats are denoted with black triangles, physically active rats are denoted with gray triangles, and off-site placements are denoted with an <b>X</b>. Brain illustrations adapted from Paxinos and Watson (published in the Rat Brain in Stereotaxic Coordinates, 4^th ed., Copyright Elsevier (1998)). Numbers left of illustrations refer to distance from Bregma (mm). (<b>B</b>) Mean freezing behavior presented in 2 minute blocks (pre-FR-1 scores are not different and therefore overlap). Error bars are ommited for clarity. (<b>C</b>) The mean percent shock-elicited freezing for the entire 20 minute observation period. (<b>D</b>) Shuttle box escape latencies for one block of 2 FR-1 trials (FR-1) and five blocks of 5 FR-2 trials (FR-2). Error bars are omitted for clarity. (<b>E</b>) The mean escape latency for all 25 FR-2 escape trials. Data represent group means ± SEM. * p<0.05 relative to Offsite Control, Sedentary/Saline, Sedentary/0.3 mM, Run/2.0 mM, and Run/6.0 mM groups. Φ p<0.05 relative to the Sedentary/2.0 mM group. θ p<0.05 relative to the Run/2.0 mM group.</p

Confirmation of successful plasma exosome enrichment from male Fisher 344 rats.

<p>A. TEM demonstrates successful isolation of exosome-sized particles (∼80 nm) from the plasma with ExoQuick precipitation. B. TEM shows how pre-treating plasma with Thromboplastin-D prior to exosome isolation with ExoQuick removes clouding factors and prevents vesicle aggregation. C. Exosome enriched and exosome depleted plasma fractions from stressed rats pre-treated with Thromboplastin-D were characterized for size distribution and quantitated by NanoSight LM10, using light scatter from a 532 nm green laser. ELISA analyses reveal that ExoQuick exosome isolation successfully enriches exosomes as marked by known exosome markers. Exposing rats to tail shock stress (Stress) had no impact on the rate of plasma exosome release compared to non-stressed rats (Control) as measured by: D. the tetraspanin CD63, E. the adhesion molecule Rab5b, F. and the intestinal exosome marker A33. G. Analysis of IL-6 confirms activation of the stress response and specificity of ExoQuick for exosome associated proteins. These data suggest that stress does not impact the rate of exosome release into the plasma. Proteomic results are expressed in means ± SE; 6–8 rats/condition. *indicates significant difference when compared to control rats (p<0.05). #indicates significant difference when compared to exosome depleted fraction (p<0.05). Two-way ANOVA with Fisher PLSD post hoc test was used.</p

Six weeks of voluntary wheel running decreases 5-HT_2C mRNA expression in the amygdala.

<p>(<b>A</b>) The region of the amygdala as shown by Paxinos and Watson (published in the Rat Brain in Stereotaxic Coordinates, 4^th ed., Copyright Elsevier (1998)). (<b>B</b>) Representative autoradiographic coronal section through the region of the striatum (Bregma – 3.14 mm) in a sedentary rat processed with <i>in situ</i> hybridization for 5-HT_2CR messenger ribonucleic acid (mRNA). (<b>C</b>) Representative autoradiographic coronal section through the region of the amygdala (Bregma – 3.14 mm) in a physically active rat (Run) processed with <i>in situ</i> hybridization for 5-HT_2CR mRNA. (<b>D</b>) Relative levels of 5-HT_2C receptor (mRNA) in the lateral amygdala (LA), basolateral amygdala (BLA), central amygdala (CeA), and lateral ventricle (LV) of sedentary rats or rats allowed voluntary access to running wheels for 6 weeks (Run). Values represent mean integrated density ± SEM. * p≤0.05 relative to respective sedentary groups.</p

Impact of acute stressor and adrenergic regulation of plasma exosomes miRNA.

<p>Exposure to tail shock stress (Stress) significantly reduced plasma exosome miR-142-52 and -203. Intraperitoneal administration of the α₁-ADR antagonist prazosin (2.0 mg/kg) 30 min prior to Stress attenuated the stress-induced down-regulation of miR-142-5p, but not miR-203. *indicates significant fold change when compared to plasma exosomes from control rats in the absence of prazosin (p<0.05). #indicates significant effect of prazosin (p<0.05). Two-way ANOVA with Fisher PLSD post hoc test was used.</p