Neuronal activity and stress differentially regulate hippocampal and hypothalamic corticotropin-releasing hormone expression in the immature rat

Abstract

Corticotropin-releasing hormone, a major neuromodulator of the neuroendocrine stress response, is expressed in the immature hippocampus, where it enhances glutamate receptor-mediated excitation of principal cells. Since the peptide influences hippocampal synaptic efficacy, its secretion from peptidergic interneuronal terminals may augment hippocampal-mediated functions such as learning and memory. However, whereas information regarding the regulation of corticotropin-releasing hormone's abundance in CNS regions involved with the neuroendocrine responses to stress has been forthcoming, the mechanisms regulating the peptide's levels in the hippocampus have not yet been determined. Here we tested the hypothesis that, in the immature rat hippocampus, neuronal stimulation, rather than neuroendocrine challenge, influences the peptide's expression. Messenger RNA levels of corticotropin-releasing hormone in hippocampal CA1, CA3 and the dentate gyrus, as well as in the hypothalamic paraventricular nucleus, were determined after cold, a physiological challenge that activates the hypothalamic pituitary adrenal system in immature rats, and after activation of hippocampal neurons by hyperthermia. These studies demonstrated that, while cold challenge enhanced corticotropin-releasing hormone messenger RNA levels in the hypothalamus, hippocampal expression of this neuropeptide was unchanged. Secondly, hyperthermia stimulated expression of hippocampal immediate-early genes, as well as of corticotropin-releasing hormone. Finally, the mechanism of hippocampal corticotropin-releasing hormone induction required neuronal stimulation and was abolished by barbiturate administration. Taken together, these results indicate that neuronal stimulation may regulate hippocampal corticotropin-releasing hormone expression in the immature rat, whereas the peptide's expression in the hypothalamus is influenced by neuroendocrine challenges.

Fig. 1

Differential induction of hippocampal c-fos mRNA and Fos protein expression by two distinct physiological stimuli. Nine- or ten-day-old rats were killed 1 h following hyperthermia (C, D) or cold stress (E, F) and compared with laboratory controls (A, B). Film autoradiograms following ISH for detection of c-fos mRNA are shown in A, C and E. Little c-fos mRNA signal was observed in animals maintained in the laboratory for 2–4 h (A). Hyperthermia induced c-fos mRNA expression in the hippocampus (Hippo) and piriform cortex (PirCtx; C). Following cold exposure, c-fos mRNA was expressed in the PVN and piriform cortex, but not in the hippocampus (E). Fos protein immunoreactivity was evident in individual hippocampal neurons following hyperthermia (D), but not after cold (F) or in laboratory controls (B). Scale bar in F = 2 mm (A, C, E), 0.4 mm (B, D, F).

Fig. 2

Differential CRH mRNA expression in the hippocampus and PVN of immature rats. Top: CRH mRNA signal was detected using ISH in coronal sections through the hippocampus of 10-day-old rats. (A) Dark-field photomicrograph of an emulsion-dipped section, showing silver grains over a CRH mRNA-expressing neuron (arrow) located at the edge of CA3 stratum pyramidale (s.p.). (B–D) Representative film autoradiograms demonstrating CRH mRNA expression in the hippocampus of laboratory control rats (B), compared with those subjected to hyperthermia (C) or to cold exposure (D). Hyperthermia resulted in significant increases of CRH mRNA hybridization signal over the entire hippocampal formation (CA1 and CA3 subfields, and the DG). Magnifications: × 200 (A), × 15 (B–D). Bottom: representative photomicrographs of CRH mRNA expression in the PVN, as influenced by cold challenge (Cold), hyperthermia (HT) or pentobarbital (Pb) administration. Under both stress-free and laboratory control conditions (see Experimental Procedures), CRH mRNA signal was robust in the parvicellular division of the PVN. These levels were markedly enhanced 4 h after cold. In contrast, CRH mRNA levels were not appreciably altered 4, 8.5 or 24 h after hyperthermia. In addition, this transcript was not influenced by barbiturate administration. Magnification: × 9.

Fig. 3

Time-course and regional distribution of the effects of neuronal activation on CRH mRNA expression in the hippocampal formation and PVN. Semi-quantitative analysis of CRH mRNA hybridization signal over the DG and hippocampal CA3 or CA1, as well as the PVN, at specific time-points following hyperthermia. Sections were subjected to ISH and analysis as described in the Experimental Procedures. For hippocampal CRH expression (top three panels), ANOVA revealed significant effects of hyperthermia (CA1: P = 0.0004, F = 4.84, d.f. = 5; CA3: P = 0.0001, F = 5.95; DG: P = 0.0001, F = 5.47). Bonferroni’s post hoc test analysis revealed a significant increase (*P < 0.01; **P < 0.05) of CRH mRNA levels at all time-points (DG, CA3) or for 24 h (CA1) relative to laboratory control levels. Each bar represents the mean ± S.E.M. of six to 10 matched sections from each of at least three rat pups, providing a sample size of 18–30. Bottom panel: in contrast to the hippocampus, hypothalamic CRH mRNA expression was not significantly influenced by hyperthermia (ANOVA for effect of treatment revealed P > 0.05). Also, although a trend for reduced CRH mRNA levels may be apparent for the 1- and 48-h time-points, it did not reach statistical significance (Bonferroni’s multiple comparison tests).

Fig. 4

Hippocampal electrophysiological activation and up-regulation of CRH mRNA expression are abolished by barbiturate pre-administration. (A) Representative EEG recordings obtained via bipolar depth electrodes from the hippocampi of immature rats. Top left: the baseline theta-frequency, low-amplitude hippocampal EEG activity. The arrow shows a movement artifact. Top right: hyperthermia induced repetitive high-amplitude semi-rhythmic discharges (arrowheads), typical of electrographic seizures involving the hippocampus. The animal was motionless during these events. Pentobarbital (Pentobarb; 30 mg/kg, i.p.), given immediately prior to the onset of hyperthermia, altered the baseline activity by eliminating the theta rhythm and decreasing the EEG amplitude (left, bottom tracing). Importantly, when pentobarbital-treated animals were subjected to hyperthermia, electrographic seizures were not observed on the hippocampal EEG (right, bottom tracing), and no behavioral seizures occurred. Vertical scale: 50 μV; horizontal: 1 s. (B) Semi-quantitative analysis showing the effects of hyperthermia and barbiturates (pentobarbital, Pb) on CRH mRNA levels in the hippocampus. Bars represent CRH mRNA hybridization signal over the hippocampal formation (combined CA1, CA3 and DG) 8.5 h after hyperthermia alone or with pentobarbital pretreatment. Hyperthermia resulted in a significant increase relative to both the control and the pentobarbital-treated rats. *P < 0.05 (Student’s t-test), comparing hyperthermics to the laboratory controls and the hyperthermic (pentobarbital-treated) controls. Hippocampal CRH mRNA levels of animals injected with pentobarbital alone did not differ significantly from those who received an i.p. injection of vehicle (88 ± 5.5%; P = 0.52, Student’s t-test). Samples are the mean ± S.E.M. of a total of 30 matched sections from at least three animals per group.