Chronic salt-loading reduces basal excitatory input to CRH neurons in the paraventricular nucleus and accelerates recovery from restraint stress in male mice

Abstract

Neurons synthesizing corticotrophin-releasing hormone (CRH) in the paraventricular nucleus of the hypothalamus (PVN) are activated during acute stress and act via the hypothalamic-pituitary-adrenal (HPA) axis to increase systemic levels of corticosterone (CORT). Recent data indicates that CRH neurons in the PVN are inhibited by acute salt-loading, and that this inhibition blunts the response to restraint stress as measured by increases in plasma CORT. The current study evaluates the effects of chronic rather than acute salt-loading on stress-induced activation of the HPA axis. Relative to euhydrated controls, chronic salt-loading over a 5-day period elevated plasma sodium and fluid intake without eliciting hypovolemia or substantial alterations in food intake or body weight. Chronic salt-loading also decreased expression of CRH mRNA in the anterior but not posterior portion of the PVN. Similarly, whole cell patch clamp recordings revealed that salt-loading effectively decreases spontaneous excitatory input to CRH neurons in the PVN without altering spontaneous inhibitory input. Generally consistent with these observations, chronic salt attenuated HPA axis activation as indicated by a significant reduction of plasma CORT during recovery from restraint stress.

Figure 1

Fluid consumption and the effects on indices of hydration. Mice with access to 2% NaCl (shaded circles) instead of water (open squares) drank significantly more (time x treatment interaction; [F(_{4, 115}) = 3.14, (P<0.05)]) each day after the first 48 h (A). Mice maintained on 2% NaCl had higher pNa⁺ on Day 5 compared with Day 1 (B; P<0.05). Hematocrit (C) and plasma proteins (D) were similar between groups. Water n=12, 2% NaCl n=13.

Fig. 2

Daily food intake and body mass measurements. The consumption of chow was similar between salt-loaded mice and water-drinking controls as there were no significant differences in daily intakes (A), however, there was a main effect of treatment [F(1, 115) = 9.53, (P<0.05)]. Body weights did not differ significantly (B). Water n=12, 2% NaCl n=13.

Fig. 3

Representative images of PVN sections processed for in situ hybridization of digoxigenin-conjugated probe and CRH mRNA. Comparable sections from mice in the Water (A, C) and 2% NaCl (B, D) groups. Salt-loading was associated with a decrease in density at Bregma −0.70 mm, but did not differ at Bregma (E, P<0.05). Water n=4–6, 2% NaCl n=4. Scale bars = 100 μm. 3v, 3rd ventricle.

Fig. 4

Chronic salt-loading reduces spontaneous excitatory input to CRH positive neurons in PVN without altering spontaneous inhibitory input. A) Representative raw data traces illustrating isolated sEPSCs in CRH positive neurons voltage clamped at −70 mV (See Methods). B) Summary data for sEPSC experiments. Mean sEPSC frequency was 5.2 ± 1.5 Hz in neurons from control animals vs. 0.9 ± 0.2 Hz in neurons from salt loaded animals (asterisk indicates p=0.03, n=6, 7 respectively). Mean sEPSC amplitude was 9.1 ± 1.1 pA in neurons from control animals vs. 10.3 ± 0.6 pA in neurons from salt loaded animals (p=0.3, n=6, 7 respectively). C) Representative raw data traces illustrating isolated sIPSCs in CRH positive neurons voltage clamped at −70 mV (See Methods). D) Summary data for sIPSC experiments. Mean sIPSC frequency was 4.6 ± 0.8 Hz in neurons from control animals vs. 3.4 ± 1.1 Hz in neurons from salt loaded animals (p=0.4, n=6 in both groups). Mean sEPSC amplitude was 27.0 ± 5.74 pA in neurons from control animals vs. 30.0 ± 4.30 pA in neurons from salt loaded animals (p=0.7, n=6 in both groups).

Fig. 5

Chronic salt-loading reduces the CORT response to restraint during the recovery period. The integrated CORT response was not significantly reduced. *p<0.05. AUC=Area Under the Curve. Error bars indicate SEM.