High salt intake enhances swim stress-induced PVN vasopressin cell activation and active stress coping

Abstract

Purpose: Stress contributes to many psychiatric disorders; however, responsivity to stressors can vary depending on previous or current stress exposure. Relatively innocuous heterotypic (differing in type) stressors can summate to result in exaggerated neuronal and behavioral responses. Here we investigated the ability of prior high dietary sodium chloride (salt) intake, a dehydrating osmotic stressor, to enhance neuronal and behavioral responses of mice to an acute psychogenic swim stress (SS). Further, we evaluated the contribution of the osmo-regulatory stress-related neuropeptide arginine vasopressin (VP) in the hypothalamic paraventricular nucleus (PVN), one of only a few brain regions that synthesize VP. The purpose of this study was to determine the impact of high dietary salt intake on responsivity to heterotypic stress and the potential contribution of VPergic-mediated neuronal activity on high salt-induced stress modulation, thereby providing insight into how dietary (homeostatic) and environmental (psychogenic) stressors might interact to facilitate psychiatric disorder vulnerability.

Approach: Salt loading (SL) with 4% saline for 7 days was used to dehydrate and osmotically stress mice prior to exposure to an acute SS. Fluid intake and hematological measurements were taken to quantify osmotic dehydration, and serum corticosterone levels were measured to index stress axis activation. Immunohistochemistry (IHC) was used to stain for the immediate early gene product c-Fos to quantify effects of SL on SS-induced activation of neurons in the PVN and extended amygdala - brain regions that are synaptically connected and implicated in responding to osmotic stress and in modulation of SS behavior, respectively. Lastly, the role of VPergic PVN neurons and VP type 1 receptor (V1R) activity in the amygdala in mediating effects of SL on SS behavior was evaluated by quantifying c-Fos activation of VPergic PVN neurons and, in functional experiments, by nano-injecting the V1R selective antagonist dGly[Phaa1,d-tyr(et), Lys, Arg]-VP bilaterally into the amygdala prior to the SS.

Findings: SL increased serum osmolality (P < 0.01), which positively correlated with time spent mobile during, and time spent grooming after a SS (P < 0.01, P < 0.01), and SL increased serum corticosterone levels (P < 0.01). SL alone increased c-Fos immunoreactivity among PVN neurons (P = .02), including VP positive neurons (P < 0.01). SL increased SS-induced c-Fos activation of PVN neurons as well (P < 0.01). In addition, SL and SS each increased the total number of PVN neurons that were immunoreactive for VP (P < 0.01). An enhancing effect of SL and SS was observed on c-Fos positive cell counts in the central (P = .02) and basolateral (P < 0.01) nuclei of the amygdala and bilateral nano-injections of V1R antagonist into the amygdala reduced time spent mobile both in salt loaded and control mice during SS (P < 0.05, P < 0.05).

Summary: Taken together, these data indicate that neuronal and behavioral responsivity to an acute psychogenic stressor is potentiated by prior exposure to high salt intake. This synergistic effect was associated with activation of PVN VP neurons and depended, in part, on activity of V1 receptors in the amygdala. Findings provide novel insight into neural mechanisms whereby prior exposure to a homeostatic stressor such as osmotic dehydration by excessive salt intake increases responsivity to a perceived stress. These experiments show that high dietary salt can influence stress responsivity and raise the possibility that excessive salt intake could be a contributing factor in the development of stress-related psychiatric disorders.

Keywords: Arginine vasopressin; Dehydration; Hyperosmolality; Paraventricular nucleus; Psychiatric; Salt diet; Stress coping.

Figure 1

Chronic SL increases active coping in response to a acute swim stress. Summary data of time spent mobile during (A) and time spent grooming after (B) an acute SS. Data are mean ± SEM. ^**P < 0.01 significant difference from tap water controls with Dunnett’s post hoc multiple comparisons test after a one-factor ANOVA. Sample size per data point are as follows: (A) n = 9 – 11; (B) n = 8 – 11.

Figure 2

Salt loading increases PVN VP neuron recruitment in response to acute swim stress. Representative images of c-Fos (red), VP (green), or c-Fos/VP co-immunoreactivity (double labelled-yellow) in the PVN of mice that underwent SL without (A) or with (B) subsequent exposure to acute SS. (C) Summary data of c-Fos positive cell counts. (D) Summary data of VP positive cell counts. (E) Percent of activated (c-Fos positive) VP cells (double labelled- yellow). White arrows indicate cells co-stained for c-Fos and VP. Data are mean ± SEM. ^*P < 0.05 significant difference from within swim treatment tap water controls; ^**P < 0.01 significant difference from within swim treatment tap water controls using Bonferroni’s post hoc multiple comparisons test after a two-factor (SL, swim stress) ANOVA. Sample size per data point are n = 4 – 5.

Figure 3

SL increases recruitment of neurons in the basolateral and central nucleus of the amygdala in response to acute swim stress. Representative images of c-Fos positive cells in the basolateral (BLA) (A) and central (CeA) (C) nucleus of the amygdala in mice that either underwent SL alone, acute SS alone, or SL with acute SS. Dotted line represents outline of BLA and CeA. (B, D) Summary data of c-Fos positive cell counts from the BLA (B) and CeA (D). Data are mean ± SEM. ^*P < 0.05 significant difference from within swim treatment tap water controls determined using Bonferroni’s post hoc multiple comparisons test after a two-factor (SL, swim stress) ANOVA. Sample size per data point are n = 3 – 4.

Figure 4

Effect of intra-amygdala nano-injections of V1R antagonist on SL-induced active coping behavior. (A) Time (s) spent mobile during SS after bilateral nano-injections of a VP V1R antagonist into the amygdala. (B) Cage crossings in an observation chamber over 15 min as a measure of generalized locomotor activity. (C) Histological verification of nano-injection sites in the CeA and BLA. Image shows cannula track (track damage), optic nerve (opt) injection site (inj. site), and amygdala sub regions; Data are mean ± SEM. **P < 0.01 significant difference from within drug treatment group compared to tap water controls with Bonferroni’s post hoc multiple comparisons test. ^{# #} P < 0.01 indicated significant main effect of V1R antagonist with a two-factor ANOVA. Sample size per data point are n = 5 – 8.