Role of the dorsomedial hypothalamus in glucocorticoid-mediated feedback inhibition of the hypothalamic-pituitary-adrenal axis

Abstract

Previous studies suggest that multiple corticolimbic and hypothalamic structures are involved in glucocorticoid-mediated feedback inhibition of the hypothalamic-pituitary-adrenal (HPA) axis, including the dorsomedial hypothalamus (DMH), but a potential role of the DMH has not been directly tested. To investigate the role of the DMH in glucocorticoid-mediated negative feedback, adult male Sprague Dawley rats were implanted with jugular cannulae and bilateral guide cannulae directed at the DMH, and finally were either adrenalectomized (ADX) or were subjected to sham-ADX. ADX rats received corticosterone (CORT) replacement in the drinking water (25 μg/mL), which, based on initial studies, restored a rhythm of plasma CORT concentrations in ADX rats that was similar in period and amplitude to the diurnal rhythm of plasma CORT concentrations in sham-ADX rats, but with a significant phase delay. Following recovery from surgery, rats received microinjections of either CORT (10 ng, 0.5 μL, 0.25 μL/min, per side) or vehicle (aCSF containing 0.2% EtOH), bilaterally, directly into the DMH, prior to a 40-min period of restraint stress. In sham-ADX rats, bilateral intra-DMH microinjections of CORT, relative to bilateral intra-DMH microinjections of vehicle, decreased restraint stress-induced elevation of endogenous plasma CORT concentrations 60 min after the onset of intra-DMH injections. Intra-DMH CORT decreased the overall area under the curve for plasma CORT concentrations during the intermediate time frame of glucocorticoid negative feedback, from 0.5 to 2 h following injection. These data are consistent with the hypothesis that the DMH is involved in feedback inhibition of HPA axis activity at the intermediate time frame.

Keywords: 5-HT1A; HPA axis; automated blood sampling; corticosterone; dorsomedial hypothalamus; negative feedback; serotonin.

Figure 1

Timelines for Experiments 1, 2, and 3. (A) Experiment 1. Experiment 1 was designed to determine the amplitude and time course of restraint-induced activation of the hypothalamic-pituitary-adrenal axis using the automated blood sampling system. Automated blood sampling was started at 0830 h on Day 10 and the shaded bars represent the 3 baseline (light gray), 4 restraint (gray), and 5 post-restraint (dark gray) samples. The gray arrow indicates that restraint started at 0900 h. (B) Experiment 2. Experiment 2 was designed to determine the pattern of plasma corticosterone (CORT) concentrations in adrenalectomized (ADX) rats drinking CORT-replacement solution (25 µg/ml CORT and 0.45% HBC in 0.9% saline), relative to diurnal rhythm of plasma CORT concentrations in adrenal-intact rats (sham-ADX), drinking tap water containing 0.45% HBC, as well as the rate of clearance of exogenous CORT in ADX rats once CORT-replacement solution was removed. Automated blood sampling was initiated at 0900 h on Day 10. Corticosterone-replacement solution was removed at 0900 h on Day 11, 24 hours before the onset of restraint. The shaded bars on Day 12 represent the 3 baseline (light gray), 4 restraint (gray), and 8 post-restraint (dark gray) samples, respectively. The gray arrow indicates that restraint started at 0900 h. (C) Experiment 3. Experiment 3 was designed to determine the effects of intra-dorsomedial hypothalamus (DMH) CORT infusion on restraint stress-induced HPA axis activity. Rats were habituated to insertion of injection cannulae and jugular cannulae were flushed with heparinized saline daily at 0900 h starting on Day 5 and ending on Day 10 (represented by black arrows). Corticosterone-replacement solution was removed from ADX rats 24 hours prior to the beginning of sampling and replaced with 0.9% saline containing 0.45% HBC. The shaded bars on Day 11 represent the 3 baseline samples (light gray), 2 samples during intra-DMH injection and placement of rats into restraint and 4 samples during (gray), and 7 post-restraint samples (dark gray), respectively. The gray arrow indicates that restraint started at 0900 h. Abbreviations: ADX, adrenalectomized; CORT, corticosterone; DMH, dorsomedial hypothalamus; HBC, hydroxypropyl-beta-cyclodextrin.

Figure 2

Bilateral injection cannulae placements for Experiment 3. The four panels were adapted from a stereotaxic atlas of the rat brain (Paxinos and Watson, 1998). The dorsomedial hypothalamus (DMH) was defined based on Fontes et al. (2011). Open and closed circles represent cannulae placements considered inside the DMH for sham-adrenalectomized (ADX)/vehicle-treated rats and sham-ADX/corticosterone (CORT)-treated rats, respectively. Abbreviations: 3V, 3^rd ventricle; ADX, adrenalectomized; CORT, corticosterone; DA, dorsal hypothalamic area; DMC, dorsomedial hypothalamic nucleus, compact part; DMD, dorsomedial hypothalamic nucleus, dorsal part; DMV, dorsomedial hypothalamic nucleus, ventral part; f, fornix; mt, mammillothalamic tract; PH, posterior hypothalamic area; VMH, ventromedial hypothalamic nucleus. Scale bar, 1 mm.

Figure 3

Baseline and restraint-induced increases in plasma corticosterone (CORT) concentrations (ng/ml) in rats in Experiment 1 (n = 7). The break between the −10 and 10 min time points is due to intentional omission of automated sampling at that time point. The gray bar between time points 0 and 40 represents the time period during which rats were restrained. Data are presented as means ± SEM (n = 7). Within group comparisons: b, p < 0.01; c, p < 0.001, generated by a linear mixed model statistical analysis.

Figure 4

Plasma corticosterone (CORT) concentrations (ng/ml) in adrenalectomized (ADX) (n = 8) and sham-ADX (n = 8) rats over the course of Experiment 2. (A) The data illustrated before the break in the x-axis illustrate the diurnal rhythm of plasma CORT concentrations, measured at 4 h intervals, during a 48 h period. The white and black horizontal bars along the x-axis represent the light cycle phase (white indicates the light phase, black indicates the dark phase). The vertical gray bar represents the time at which CORT-replacement solution (25 µg/ml CORT in 0.9% saline containing 0.45% hydroxypropyl-beta-cyclodextrin (HBC)) was replaced with 0.9% saline containing 0.45% HBC for ADX rats 24 h prior to the onset of restraint. The data after the break in the x-axis illustrate baseline, restraint, and post-restraint samples taken at 10 min intervals. (B) Graph illustrating plasma CORT concentrations in a representative sham-ADX rat. (C) Graph illustrating plasma CORT concentrations in a representative ADX rat. Data in A represent means ± SEM of plasma CORT concentrations for each treatment group. Between-group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001. Within-group comparisons: a = p < 0.05, c = p < 0.001. Abbreviations: ADX, adrenalectomy; CORT, corticosterone; HBC, hydroxypropyl-beta-cyclodextrin.

Figure 5

Effects of bilateral intra-dorsomedial hypothalamus (DMH) microinjections of corticosterone (CORT) or vehicle in sham-adrenalectomized (sham-ADX) rats exposed to a 40 min period of restraint stress on plasma CORT concentrations (ng/ml). All rats included in the data analysis had bilateral cannulae placements within the DMH. A) Open and closed circles represent sham-ADX/vehicle-treated (n = 10) and sham-ADX/CORT-treated (n = 8) rats, respectively. Three baseline samples were taken at −10, −20, and −30 min, immediately prior to intra-DMH CORT microinjection and restraint. The vertical light gray bar indicates the window of time from −10 to 0 min during which rats were handled for insertion of injection cannulae and initiation of the intra-DMH microinjections. The vertical dark gray bar represents the time from 0 to 10 min taken to complete the intra-DMH microinjections, remove the injection cannulae, and put the rats into the restraint tube. Data were normalized so that the first sample with elevated plasma CORT concentration (>50 ng/ml) was at the 10 min time point. The horizontal black bar indicates the 40-min restraint period, from 10 min to 50 min. B) Graph illustrating the area under the curve for the intermediate (30–120 min) feedback timeframe. CORT-treated rats showed a significantly attenuated HPA axis response during the intermediate feedback timeframe (p < 0.01). Abbreviations: ADX, adrenalectomy; AUC, area under the curve; CORT, corticosterone. Between-group comparisons: *p < 0.05, **p < 0.01, Fisher’s protected least significant difference (LSD) test.