Heat-Stress Preconditioning Attenuates Behavioral Responses to Psychological Stress: The Role of HSP-70 in Modulating Stress Responses

Abstract

Exposure to high ambient temperature is a stressor that influences both biological and behavioral functions and has been previously shown to have an extensive impact on brain structure and function. Physiological, cellular and behavioral responses to heat-stress (HS) (40-41 °C, 2 h) were evaluated in adult male Sprague-Dawley rats. The effect of HS exposure before predator-scent stress (PSS) exposure (i.e., HS preconditioning) was examined. Finally, a possible mechanism of HS-preconditioning to PSS was investigated. Immunohistochemical analyses of chosen cellular markers were performed in the hippocampus and in the hypothalamic paraventricular nucleus (PVN). Plasma corticosterone levels were evaluated, and the behavioral assessment included the elevated plus-maze (EPM) and the acoustic startle response (ASR) paradigms. Endogenous levels of heat shock protein (HSP)-70 were manipulated using an amino acid (L-glutamine) and a pharmacological agent (Doxazosin). A single exposure to an acute HS resulted in decreased body mass (BM), increased body temperature and increased corticosterone levels. Additionally, extensive cellular, but not behavioral changes were noted. HS-preconditioning provided behavioral resiliency to anxiety-like behavior associated with PSS, possibly through the induction of HSP-70. Targeting of HSP-70 is an attractive strategy for stress-related psychopathology treatment.

Keywords: anxiety; heat shock protein-70; heat-stress; hyperthermia; hypothalamus-pituitary-adrenal axis; preconditioning.

Figure 1

Study design–Experiment 1: The effect of heat-stress on physiological, cellular and behavioral responses. After a habituation period of seven days, animals were subjected to heat stress (HS) (40 ± 1 °C, 2 h). Control (thermoneutral, TN) animals were kept at room temperature (22 ± 1 °C) for the same period. Body mass and rectal temperature (Tre) were measured at baseline and every hour of the protocol. Core temperature was measured using telemetry throughout the entire protocol. After completion of the protocol, animals were sacrificed and their brains were collected for immunohistochemical analysis of chosen cellular markers. Trunk blood was used for circulating corticosterone measurement (corticosterone measurement). In a cohort of the animals, behavioral assessments were performed either immediately (acute response) or seven days after the HS exposure (delayed response) (Behavioral tests).

Figure 2

Physiological response to heat stress. (a) The dynamic change of core body temperature (Tb) over time during HS. (b) The change in body mass (BM) as percent change from baseline over time during HS. The red line represents the temperature inside the chamber along the experiment. Results displayed as mean ± S.E.M. * < 0.001.

Figure 3

Cellular markers under basal thermoneutral naive and after heat stress. Immunoreactive-cell numbers and representative immunohistochemical images of HSP-70, GR, AVP, BDNF, NPY, COX-2 and Iba-1 positive cells within the CA1, CA3 and the DG of the hippocampus and the hypothalamic PVN (a–e). Results displayed as mean ± S.E.M.

Figure 3

Cellular markers under basal thermoneutral naive and after heat stress. Immunoreactive-cell numbers and representative immunohistochemical images of HSP-70, GR, AVP, BDNF, NPY, COX-2 and Iba-1 positive cells within the CA1, CA3 and the DG of the hippocampus and the hypothalamic PVN (a–e). Results displayed as mean ± S.E.M.

Figure 3

Cellular markers under basal thermoneutral naive and after heat stress. Immunoreactive-cell numbers and representative immunohistochemical images of HSP-70, GR, AVP, BDNF, NPY, COX-2 and Iba-1 positive cells within the CA1, CA3 and the DG of the hippocampus and the hypothalamic PVN (a–e). Results displayed as mean ± S.E.M.

Figure 4

Acute and delayed behavioral response to heat stress. Acute and delayed behavioral response to HS. A.U = arbitrary units. Results displayed as mean ± S.E.M. No significant differences were noted in any of the elevated plus maze (EPM) (a–g) and the acoustic startle response (ASR) (h,i) parameters between the groups.

Figure 5

Study design–Experiment 2: The effect of HS-preconditioning on resiliency to psychological stress. After a habituation period of seven days, animals were subjected to HS and immediately were exposed to psychological stress, the predator-scent stress (PSS) (HS + PSS). Control animals were exposed to PSS only. Body mass and body temperature were measured at baseline, at every hour of the protocol and after 2 h at a room temperature (22 ± 1 °C). Behavior was assessed on day seven to examine the delayed behavioral response of HS-preconditioning to PSS.

Figure 6

Behavioral response of HS-preconditioning to PSS (a–i). Behavioral response to HS-preconditioning to PSS. A.U = arbitrary units. Results displayed as mean ± S.E.M.

Figure 6

Behavioral response of HS-preconditioning to PSS (a–i). Behavioral response to HS-preconditioning to PSS. A.U = arbitrary units. Results displayed as mean ± S.E.M.

Figure 7

Study design–Experiment 3: The role of heat-shock protein (HSP)-70 in modulating the protective behavioral response to predator-scent stress (PSS). (a) L-glutamine: Animals were fed with the amino acid L-glutamine (0.375 g/kg) or vehicle via gavage for three days. On day three, animals were exposed to PSS and behavior was assessed on day seven. (b) Doxazosin: animals were injected with the α1-adrenoceptor antagonist Doxazosin (1.5 mg/kg i.p) or saline. Thirty minutes after the injection, animals were exposed to the HS and immediately after to the PSS. Body temperature and body mass were measured at baseline and every hour of the protocol. Behavior was assessed on day seven.

Figure 8

Behavioral response to manipulations of HSP-70 levels (a–i). Behavioral response to manipulations of HSP-70 levels. A.U = arbitrary units. Results displayed as mean ± S.E.M.