Aversive and appetitive events evoke the release of corticotropin-releasing hormone and bombesin-like peptides at the central nucleus of the amygdala

Abstract

There is wide agreement that corticotropin-releasing hormone (CRH) systems within the brain are activated by stressful stimuli. There is also mounting evidence for the role of bombesin (BN)-like peptides in the mediation of the stress response. To date, however, the extent to which other stimuli increase the activity of these peptidergic systems has received little attention. In the present investigation we validated and used in vivo microdialysis sampling followed by ex vivo radioimmunoassays to monitor the release of CRH and BN-like peptides during appetitive (food intake) and stressful (restraint) events. It is demonstrated for the first time that the in vivo release of CRH and BN-like peptides at the central nucleus of the amygdala was markedly increased by both stressor exposure and food ingestion. In fact, the meal-elicited rise of CRH release was as great as that associated with 20 min of restraint stress. Paralleling these findings, circulating ACTH and corticosterone levels were also increased in response to both food intake and restraint. Contrary to the current views, these results indicate that either food ingestion is interpreted as a "stressful" event by certain neural circuits involving the central amygdala or that the CRH- and BN-related peptidergic systems may serve a much broader role than previously envisioned. Rather than evoking feelings of fear and anxiety, these systems may serve to draw attention to events or cues of biological significance, such as those associated with food availability as well as those posing a threat to survival.

Fig. 1.

HPLC separation and identification of endogenous CRH and BN-like immunoreactivity in microdialysis samples.A, HPLC profile ir-CRH material from dialysates collected from two separate animals (open andfilled circles, respectively). The boxed arrow indicates the elution time for synthetic CRH using UV detection. B, HPLC profile of endogenous ir-BN-like material eluting from the pooled dialysate compared with the elution time for synthetic GRP_1–27 and GRP_18–27(boxed arrows).

Fig. 2.

Restraint stress-induced release of CRH at the central nucleus of the amygdala as measured by in vivomicrodialysis. Dialysate samples were collected uninterrupted throughout the experiment, and samples were pooled every 30 min. After collection of five baseline samples, rats in the stress group (n = 8–10) were hand-restrained (filled circles) for 30 min episodes on two separate occasions (shaded vertical bars, stress 1 and stress 2). The rats in the no-stress (control) group (n = 5–7) were left undisturbed throughout (open circles). The five baseline values from each of the subjects were averaged and defined as 100%. All values were then expressed as a percent of that baseline. Basal CRH values for the no stress and stress groups were 2.77 ± 0.11 and 2.31 ± 0.39 fmol/sample, respectively.

Fig. 3.

Anatomical localization of the microdialysis membranes aimed at the central nucleus of the amygdala in the stress study. A series of consecutive brain sections bearing the trace of microdialysis probes were stained, the most ventral location of the probe tip was determined, and a 2.5 mm line was drawn vertically, tracing the estimated location of the active region of the probe.Solid vertical lines with pinheadsrepresent probes of animals included in the no-stress control group. The off-site probes (broken vertical line withpinhead) were excluded from analysis. The probe placements of animals included in the stress group are identified withsolid vertical lines, whereas those considered off-site and excluded from analysis are depicted by broken vertical lines.

Fig. 4.

Restraint stress-induced release of BN-like peptides from the central nucleus of the amygdala as measured byin vivo microdialysis. Dialysate samples were collected uninterrupted throughout the experiment, and samples were pooled every 30 min. After collection of five baseline samples, rats in the stress group (n = 10) were hand-restrained (filled circles) for 20 min episodes on two separate occasions (shaded bars, stress 1and stress 2). The rats in the no stress control group (n = 7) were left undisturbed (open circles). The five baseline values from each of the subjects were averaged and defined as 100%. All values were then expressed as a percent of that baseline. Basal values for BN immunoreactive peptides were 3.01 ± 0.42 and 3.28 ± 0.33 fmol/sample for the stress and no-stress groups, respectively.

Fig. 5.

Meal-elicited release of CRH and BN-like peptides at the central nucleus of the amygdala. Microdialysate samples were collected continually and pooled every 30 min for 5 hr. The quantity of food ingested during each 30 min bin was noted. The 30 min period before meal initiation was considered the preprandial period, and the 30 min sample preceding this was considered the baseline. The postprandial period was the 30 min period after meal termination. The baseline value was defined as 100%, and all other values were expressed as a percentage of this value. The meal-related CRH changes are presented at the top, whereas the changes in BN-like peptides are shown at the bottom. The basal values for CRH and BN-like peptides were 0.95 ± 0.12 and 0.77 ± 0.07 fmol/sample, respectively.

Fig. 6.

Anatomical localization of the microdialysis membranes aimed at the central nucleus of the amygdala in animals involved in the feeding study. Probe placements were identified and represented as described in Figure 3. Probe placements of animals included in the analysis (n = 7) are identified with solid vertical lines, whereas those of animals excluded from analysis attributable to misalignment (off-site,n = 3) are depicted by broken vertical lines.

Fig. 7.

Effects of food ingestion on circulating ACTH and corticosterone levels. Rats equipped with carotid cannula were used in this study. Blood samples (400 μl) were drawn remotely at 20, 10, and 0 min (just before food presentation). Rats were then offered graham wafers for 10 min (shaded bar). Blood samples were collected at 5, 15, 30, and 60 min after food presentation. Plasma ACTH levels are expressed as picograms per milliliter and presented as mean ± SEM (top). Plasma corticosterone levels are expressed as micrograms per 100 ml and presented as mean ± SEM (bottom).