Most human seizures occur early in life,consistent with established excitability-promoting features of the developing brain. Surprisingly, the majority of developmental seizures are not spontaneous but are provoked by injurious or stressful stimuli. What mechanisms mediate'triggering' of seizures and limit such reactive seizures to early postnatal life? Recent evidence implicates the excitatory neuropeptide, corticotropin-releasing hormone (CRH). Stress activates expression of the CRH gene in several limbic regions, and CRH-expressing neurons are strategically localized in the immature rat hippocampus, in which this neuropeptide increases the excitability of pyramidal cells in vitro. Indeed, in vivo, activation of CRH receptors--maximally expressed in hippocampus and amygdala during the developmental period which is characterized by peak susceptibility to 'provoked' convulsions--induces severe, age-dependent seizures. Thus, converging data indicate that activation of expression of CRH constitutes an important mechanism for generating developmentally regulated, triggered seizures, with considerable clinical relevance.

Age-appropriate acute stress, such as cold exposure, provokes the secretion of corticotropin releasing factor (CRF) from the hypothalamus, leading to a robust increase of plasma corticosterone in the immature rat. This activation of the hypothalamic-pituitary-adrenal system is accompanied by a stress-induced increase of steady-state CRF-mRNA expression in the hypothalamic paraventricular nucleus (PVN). In the current study, we analysed changes in CRF-mRNA expression in the PVN and the central nucleus of the amygdala (ACe) in the immature rat in response to a single episode of cold stress and three repeated exposures to this same stressor. CRF-mRNA expression in the PVN increased after a single, but not repeated exposures to cold stress, while repeated acute stress increased the content of the CRF peptide in the anterior hypothalamus. In the ACe, repeated episodes of cold stress resulted in increased expression of CRF-mRNA. These findings indicate a differential regulation of CRF gene expression in the PVN and ACe of the immature rat by single and repeated acute stress.

Purpose

The gamma-aminobutyric acid (GABA) degradation blocker gamma-vinyl-GABA (VGB) is used clinically to treat seizures in both adult and immature individuals. The mechanism by which VGB controls developmental seizures is not fully understood. Specifically, whether the anticonvulsant properties of VGB arise only from its elevation of brain GABA levels and the resulting activation of GABA receptors, or also from associated mechanisms, remains unresolved. Corticotropin-releasing hormone (CRH), a neuropeptide present in many brain regions involved in developmental seizures, is a known convulsant in the immature brain and has been implicated in some developmental seizures. In certain brain regions, it has been suggested that CRH synthesis and release may be regulated by GABA. Therefore we tested the hypothesis that VGB decreases CRH gene expression in the immature rat brain, consistent with the notion that VGB may decrease seizures also by reducing the levels of the convulsant molecule, CRH.

Methods

VGB was administered to immature, 9-day-old rats in clinically relevant doses, whereas littermate controls received vehicle.

Results

In situ hybridization histochemistry demonstrated a downregulation of CRH mRNA levels in the hypothalamic paraventricular nucleus but not in other limbic regions of VGB-treated pups compared with controls. In addition, VGB-treated pups had increased CRH peptide levels in the anterior hypothalamus, as shown by radioimmunoassay.

Conclusions

These findings are consistent with a reduction of both CRH gene expression and secretion in the hypothalamus, but do not support an indirect anticonvulsant mechanism of VGB via downregulation of CRH levels in limbic structures. However, the data support a region-specific regulation of CRH gene expression by GABA.

Adrenalectomy in the mature rat leads to death of granule cells in the dentate gyrus of the hippocampal formation. The mechanisms underlying this cell death have not been fully clarified: It has been considered that the granule cells require adrenal steroids for their survival, since corticosterone replacement prevents their death. However, adrenalectomy-induced loss of negative feedback also increases levels of corticotropin releasing hormone (CRH) in several limbic brain regions. CRH is known to induce neuronal death in hippocampal regions rich in CRH receptors. This study tested the hypothesis that adrenalectomy-induced granule cell death is mediated via the enhanced activation of CRH receptors. The extent of granule cell degeneration was compared among 4 groups of young adult male rats: Sham-adrenalectomy controls, adrenalectomized rats, adrenalectomized rats infused with a CRH antagonist from the onset of steroid deprivation to the time of sacrifice, and adrenalectomized rats infused with vehicle only. (9-41)-alpha-helical CRH was administered using an osmotic pump into the cerebral ventricles. Adrenalectomy led to robust granule cell degeneration, which was maximal in the suprapyramidal blade of the dentate gyrus. Infusion of the CRH antagonist in doses shown to block CRH actions on limbic neurons did not decrease the number of degenerating granule cells compared with the untreated or vehicle-infused adrenalectomized groups. Therefore, blocking the actions of CRH does not prevent adrenalectomy-induced granule cell death, consistent with a direct effect of corticoids on the survival of these neurons.

The stress neurohormone corticotropin releasing factor (CRF) activates at least two receptor types. Expression of corticotropin releasing factor receptor type II (CRF2) has been demonstrated in the hypothalamic ventromedial nucleus (VMH) of the adult and developing rat, but the physiological functions of VMH-CRF2 have not been elucidated. The VMH has been documented as an important participant in the regulation of food intake and its interactions with the hypothalamic-pituitary-adrenal axis and circadian rhythms. Regulation of VMH-CRF2 may thus play a role in the interplay of physiological alterations in metabolic state with the neuroendocrine and anorexic effects of CRF. This study determined the regulation of CRF2-mRNA expression in infant rats by the physiological consequences of maternal deprivation, i.e., fasting and stress. Using in situ hybridization, maternally deprived pups had an average 62% reduction of VMH-CRF2-mRNA levels compared with stress-free controls. Maternal deprivation also resulted in elevated plasma corticosterone levels (3.8 +/- 0.3 vs. 1.3 +/- 0.1 microg/dl) and an average 5.7% body weight loss. This study demonstrates that maternal deprivation, via fasting and HPA activation, leads to a dramatic decrease of CRF2-mRNA levels in the VMH. These results are consistent with a role for CRF2 activation in mediating some of the complex interactions of CRF (or urocortin) with regulation of food intake in the developing rat.

Maternal deprivation (MDep) of neonatal rats significantly influences the hypothalamic-pituitary-adrenal (HPA) axis. This study hypothesized that GR-mRNA modulation constituted an early, critical mechanism for the acute effects of MDep on neuroendocrine stress-responses. GR-mRNA hybridization signal in hippocampal CA1, hypothalamic paraventricular nucleus (PVN) and frontal cortex was significantly reduced immediately following 24 h MDep. In amygdala, cingulate cortex, PVN and CA1, apparent gender-dependent MDep effects on GR-mRNA expression were observed, without significant differences in absolute levels. Thus, rapid, region-specific MDep effects on GR-mRNA expression in HPA-regulating areas are shown, consistent with involvement of GR-expression in mechanisms of MDep influence on HPA tone.

The physiological consequences of activating corticotropin-releasing factor receptor type 2 (CRF2) are not fully understood. The neuroanatomic distribution of this CRF receptor family member is consistent with roles in mediating the actions of CRF and similar ligands on food intake control and integrative aspects of stress-related behaviors. However, CRF2 expression in the adult rat is not influenced by stress, corticosterone (CORT), or food intake. In immature rat we have demonstrated striking downregulation of CRF2mRNA in hypothalamic ventromedial nucleus (VMH) after 24 hr of maternal deprivation, a paradigm consisting of both physiological/psychological stress and food deprivation. The current study aimed to distinguish which element or elements of maternal deprivation govern CRF2mRNA expression by isolating the effects of food intake and discrete maternal sensory cues on CRF2mRNA levels in VMH and in reciprocally communicating amygdala nuclei. In maternally deprived pups, CRF2mRNA levels in VMH and basomedial (BMA) and medial (MEA) amygdala nuclei were 62, 72, and 102% of control levels, respectively. Sensory inputs of grooming and handling as well as of the pups' own suckling activity-but not food intake-fully restored CRF2mRNA expression in VMH. In contrast, all manipulations tended to increase CRF2mRNA levels in BMA of maternally deprived rats, and surrogate grooming increased CRF2mRNA expression significantly above that of nondeprived controls. CRF2mRNA expression was not influenced significantly by plasma adrenocorticotropic hormone (ACTH) and CORT levels. Thus, in the immature rat, (1) CRF2 expression is regulated differentially in hypothalamic and amygdala regions, and (2) CRF2mRNA levels in VMH are governed primarily by maternal or suckling-derived sensory input rather than food intake or peripheral stress hormones. These findings indicate a region-specific regulation of CRF2mRNA, supporting the participation of the receptor in neurochemically defined circuits integrating sensory cues to influence specific behavioral and visceral functions.

The mechanisms of the molecular and neuroendocrine responses to stress in the immature rat have been a focus of intense investigation. A principal regulator of the these responses in both mature and developing rat is the neuropeptide corticotropin releasing hormone (CRH), and levels of hypothalamic CRH mRNA are enhanced by stress. In vitro, transcription of the CRH gene is governed by binding of the phosphorylated form of cAMP responsive element binding protein (pCREB) to the promoter. Here we tested the hypothesis that rapid, stress-induced CRH transcription occurred during the first two postnatal weeks, and is associated with pCREB expression. The time-course of induction of unedited, heteronuclear CRH RNA (CRH hnRNA) was examined in hypothalamic paraventricular nucleus (PVN) of immature rats subjected to both modest and strong acute stressors using in situ hybridization; pCREB abundance was determined in individual neurons in specific PVN sub-nuclei using immunocytochemistry and unbiased quantitative analysis. CRH hnRNA signal was negligible in PVN of immature rats sacrificed under stress-free conditions, but was readily detectable within 2 min, and peaked at 15 min, in PVN of stressed animals. Enhanced pCREB immunoreactivity was evident within 2 min of stress onset, and was enhanced specifically in stress-responsive, CRH-expressing medial parvocellular neurons. These data support the notion that, already during early postnatal life, stress induces rapid CREB phosphorylation, interaction of pCREB-containing transcription complexes with the CRE element of the CRH gene promoter, and initiation of CRH hnRNA production in stress-responsive neurons of rat PVN.