Corticotropin releasing hormone (CRH) produces age-dependent limbic seizures in the infant rat. Both the phenotype and the neuroanatomic matrix of CRH-induced seizures resemble the seizures induced by the rigid glutamate analogue, kainic acid (KA), and by rapid amygdala kindling. The experiments described in this study tested the hypothesis that the in vivo proconvulsant effects of CRH require activation of ionotropic glutamate receptors. Non-competitive (+MK-801) or competitive (CGP-39551) antagonists of N-methyl-d-aspartate (NMDA) receptors decreased or eliminated the motor effects of CRH, but electrographic CRH-induced seizures were unaffected. Administration of CRH antagonists did not affect the acquisition or the maintenance of rapid kindling, which are mediated by NMDA and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor activation, respectively. CRH receptor blockers failed to alter the latency or duration of seizures induced by activation of KA receptors, and threshold doses of CRH and KA had additive effects. CRH given repeatedly decreased the convulsant threshold dose of KA, probably via injury to hippocampal neurons. These results suggest that CRH and glutamate increase neuronal excitability via independent mechanisms. Because the proconvulsant effects of CRH are highly specific to the developmental period, glutamate-receptor-independent, CRH-receptor mediated excitation may account for some of the enhanced susceptibility to seizures during this period.

Whereas genetic factors contribute crucially to brain function, early-life events, including stress, exert long-lasting influence on neuronal function. Here, we focus on the hippocampus as the target of these early-life events because of its crucial role in learning and memory. Using a novel immature-rodent model, we describe the deleterious consequences of chronic early-life 'psychological' stress on hippocampus-dependent cognitive tasks. We review the cellular mechanisms involved and discuss the roles of stress-mediating molecules, including corticotropin releasing hormone, in the process by which stress impacts the structure and function of hippocampal neurons.

Background and rationale

Infantile spasms (IS) are an age-specific seizure disorder occurring in 1:2,000 infants and associated with mental retardation in approximately 90% of affected individuals. The costs of IS in terms of loss of lifetime productivity and emotional and financial burdens on families are enormous. It is generally agreed that the seizures associated with IS respond poorly to most conventional anticonvulsants. In addition, in the majority of patients, a treatment course with high-dose corticotropin (ACTH) arrests the seizures completely within days, often without recurrence on discontinuation of the hormone. However, the severe side effects of ACTH require development of better treatments for IS. Based on the rapid, all-or-none and irreversible effects of ACTH and on the established physiological actions of this hormone, it was hypothesized that ACTH eliminated IS via an established neuroendocrine feedback mechanism involving suppression of the age-specific endogenous convulsant neuropeptide corticotropin-releasing hormone (CRH). Indeed, IS typically occur in the setting of injury or insult that activate the CNS stress system, of which CRH is a major component. CRH levels may be elevated in the IS brain, and the neuropeptide is known to cause seizures in infant rats, as well as neuronal death in brain regions involved in learning and memory. If 'excess' CRH is involved in the pathogenesis of IS, then blocking CRH receptors should eliminate both seizures and the excitotoxicity of CRH-receptor-rich neurons subserving learning and memory.

Patients and methods

With FDA approval, alpha-helical CRH, a competitive antagonist of the peptide, was given as a phase I trial to 6 infants with IS who have either failed conventional treatment or who have suffered a recurrence. The study was performed at the Clinical Research Center of the Childrens Hospital, Los Angeles. The effects of alpha-helical CRH on autonomic parameters (blood pressure, pulse, temperature, respiration) were determined. In addition, immediate and short-term effects on ACTH and cortisol and on electrolytes and glucose were examined. The potential efficacy of alpha-helical CRH for IS was studied, using clinical diaries and video EEG.

Results

alpha-Helical CRH, a peptide, did not alter autonomic or biochemical parameters. Blocking peripheral CRH receptors was evident from a transient reduction in plasma ACTH and cortisol. No evidence for the compound's penetration of the blood-brain barrier was found, since no central effects on arousal, activity or seizures and EEG patterns were observed. In addition, a striking resistance of the patients' plasma ACTH to the second infusion of alpha-helical CRH was noted.

Conclusions

Peptide analogs of CRH do not cross the blood-brain barrier, and their effects on peripheral stress hormones are transient and benign. Nonpeptide compouds that reach CNS receptors are required to test the hypothesis that blocking CRH receptors may ameliorate IS and its cognitive consequences.

The neuropeptide corticotropin releasing hormone (CRH) is the central nervous system (CNS) transducer of stressful stimuli. Endogenous CRH is released from neuronal terminals in several central nervous system regions-for example, amygdala and hypothalamus-during stress, and exogenous CRH administration mimics stress-related behaviors and hormonal patterns. However, whereas the role of endogenous CRH as a stress neuromodulator has been established, recent findings suggest that the peptide also functions to influence cognitive, emotional, and neuroimmune functions by modulating neuronal communication in a number of circuits. Although anatomic and pharmacologic approaches have provided evidence for this wider spectrum of CRH actions, the discrete regions and specific circuits activated by CRH have not been fully elucidated. In this article, the authors report on the use of two complementary methods to discern specific regions and cell groups activated by the administration of CRH. Glucose metabolism analysis provided quantitative measures of CRH-induced activation, but at a regional resolution; expression of the immediate early gene c-fos permitted a single cell resolution, but underestimated the neuroanatomic extent of CRH-induced activation. Overlapping regions activated using both methods delineated discrete cortical, limbic. and motor pathways. Importantly, cell groups activated by CRH included those possessing either or both members of the CRH receptor family, suggesting that both receptors may mediate the effects of the endogenous ligand. In summary, CRH activates a broad but selective array of neuronal structures belonging to cortical, limbic, and motor circuits. These findings indicate that stress-related release of this peptide may contribute to a spectrum of important modulations of CNS function.

West syndrome (WS) is associated with diverse etiological factors. This fact has suggested that there must be a 'final common pathway' for these etiologies, which operates on the immature brain to result in WS only at the maturational state present during infancy. Any theory for the pathogenesis of WS has to account for the unique features of this disorder. For example, how can a single entity have so many etiologies? Why does WS arise only in infancy, even when a known insult had occurred prenatally, and why does it disappear? Why is WS associated with lasting cognitive dysfunction? And, importantly, why do these seizures--unlike most others--respond to treatment by a hormone, ACTH? The established hormonal role of ACTH in human physiology is to function in the neuroendocrine cascade of the responses to all stressful stimuli, including insults to the brain. As part of this function, ACTH is known to suppress the production of corticotropin releasing hormone (CRH), a peptide that is produced in response to diverse insults and stressors.The many etiologies of WS all lead to activation of the stress response, including increased production and secretion of the stress-neurohormone CRH. CRH has been shown, in infant animal models, to cause severe seizures and death of neurons in areas involved with learning and memory. These effects of CRH are restricted to the infancy period because the receptors for CRH, which mediate its action on neurons, are most abundant during this developmental period. ACTH administration is known to inhibit production and release of CRH via a negative feedback mechanism. Therefore, the efficacy of ACTH for WS may depend on its ability to decrease the levels of the seizure-promoting stress-neurohormone CRH.This CRH-excess theory for the pathophysiology of WS is consistent not only with the profile of ACTH effects, but also with the many different 'causes' of WS, with the abnormal ACTH levels in the cerebrospinal fluid of affected infants and with the spontaneous disappearance of the seizures. Furthermore, if CRH is responsible for the seizures, and CRH-mediated neuronal injury contributes to the worsened cognitive outcome of individuals with WS, then drugs which block the actions of CRH on its receptors may provide a better therapy for this disorder.

Neurogenesis of dentate gyrus granule cells is generally considered to be negatively regulated by glucocorticoids. We tested the hypothesis that exposure to low plasma corticosteroid levels starting in the early postnatal period enhances granule cell proliferation rate during adulthood. Rat pups were adrenalectomized (ADX) on postnatal day 10 and were then "clamped" throughout life at low corticosterone levels via oral supplementation. Neurogenesis was determined using BrdU immunochemistry at 3 and 12 months in clamped rats as compared with age-matched, sham-operated controls. Rate of neurogenesis did not differ between the groups at either 3 or 12 months. It was significantly lower in 12-month-old compared with 3-month-old rats, despite the presence of an age-dependent increase of plasma corticosterone only in the sham-ADX rats. Granule cell layer volume, granule cell density, and granule cell degeneration (determined using apoptotic markers) were indistinguishable in the two groups, further supporting the comparable rate of neurogenesis under differing chronic glucocorticoid levels. In addition, whereas acute deprivation of plasma glucocorticoids (adrenalectomy) in adult rats evoked a burst of granule cell neurogenesis, complete elimination of these hormones (by stopping hormone supplementation) in adult, early-life ADX/clamped rats did not. These data do not support a simple inverse relationship between chronic plasma glucocorticoid levels and granule cell neurogenesis. Specifically, chronic modulation of glucocorticoid levels commencing early in life evokes additional, adaptive, and compensatory mechanisms that contribute to the regulation of granule cell proliferation.

High affinity, gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) influence the availability of GABA, the main inhibitory neurotransmitter in the brain. Recent studies suggest a crucial role for GATs in maintaining levels of synaptic GABA in normal as well as abnormal (i.e., epileptic) adult brain. However, the role of GATs during development and specifically changes in their expression in response to developmental seizures are unknown. The present study examined GAT-1-immunolabeling in infant rats with two types of developmental seizures, one induced by corticotropin-releasing hormone (CRH) lasting about 2 h and the other by hyperthermia (a model of febrile seizures) lasting only 20 min. The number of GAT-1-immunoreactive (ir) neurons was increased in several forebrain regions 24 h after induction of seizures by CRH as compared to the control group. Increased numbers of detectable GAT-1-ir cell bodies were found in the hippocampal formation including the dentate gyrus and CA1, and in the neocortex, piriform cortex and amygdala. In contrast, hyperthermia-induced seizures did not cause significant changes in the number of detectable GAT-1-ir somata. The increase in GAT-1-ir somata in the CRH model and not in the hyperthermia model may reflect the difference in the duration of seizures. The brain regions where this increase occurs correlate with the occurrence of argyrophyllic neurons in the CRH model.

It is well established that adrenal stress hormone-induced activation of the basolateral complex of the amygdala (BLA) influences memory consolidation. The present experiments investigated the involvement of corticotropin-releasing hormone (CRH) in the BLA in modulating memory consolidation. Bilateral infusions of the CRH receptor antagonist [9-41]-alpha-helical CRH (0.3, 1.0, or 3.0 microg in 0.2 microl) administered into the BLA of male Sprague-Dawley rats immediately after aversively motivated inhibitory avoidance training produced dose-dependent impairment of 48-h retention performance. Because the CRH receptor antagonist infusions did not impair retention when administered into the BLA 3 h after training, the retention impairment selectively was due to time-dependent influences on memory consolidation. Furthermore, because immediate posttraining infusions of [9-41]-alpha-helical CRH into the adjacent central nucleus of the amygdala (CEA) were ineffective, the effect selectively involved the BLA. Immunocytochemistry showed that the aversive training stimulus of a single, brief footshock increased CRH levels in the CEA. These findings indicate that activation of CRH receptors in the BLA, likely by training-induced release of endogenous peptide originating from the CEA, participates in mediating stress effects on memory consolidation.

In addition to regulating the neuroendocrine stress response, corticotropin-releasing hormone (CRH) has been implicated in both normal and pathological behavioral and cognitive responses to stress. CRH-expressing cells and their target neurons possessing CRH receptors (CRF1 and CRF2) are distributed throughout the limbic system, but little is known about the regulation of limbic CRH receptor function and expression, including regulation by the peptide itself. Because CRH is released from limbic neuronal terminals during stress, this regulation might play a crucial role in the mechanisms by which stress contributes to human neuropsychiatric conditions such as depression or posttraumatic stress disorder. Therefore, these studies tested the hypothesis that CRH binding to CRF1 influenced the levels and mRNA expression of this receptor in stress-associated limbic regions of immature rat. Binding capacities and mRNA levels of both CRF1 and CRF2 were determined at several time points after central CRH administration. CRH downregulated CRF1 binding in frontal cortex significantly by 4 h. This transient reduction (no longer evident at 8 h) was associated with rapid increase of CRF1 mRNA expression, persisting for >8 h. Enhanced CRF1 expression-with a different time course-occurred also in hippocampal CA3, but not in CA1 or amygdala, CRF2 binding and mRNA levels were not altered by CRH administration. To address the mechanisms by which CRH regulated CRF1, the specific contributions of ligand-receptor interactions and of the CRH-induced neuronal stimulation were examined. Neuronal excitation without occupation of CRF1 induced by kainic acid, resulted in no change of CRF1 binding capacity, and in modest induction of CRF1 mRNA expression. Furthermore, blocking the neuroexcitant effects of CRH (using pentobarbital) abolished the alterations in CRF1 binding and expression. These results indicate that CRF1 regulation involves both occupancy of this receptor by its ligand, as well as "downstream" cellular activation and suggest that stress-induced perturbation of CRH-CRF1 signaling may contribute to abnormal neuronal communication after some stressful situations.

Febrile seizures (FSs) constitute the most prevalent seizure type during childhood. Whether prolonged FSs alter limbic excitability, leading to spontaneous seizures (temporal lobe epilepsy) during adulthood, has been controversial. Recent data indicate that, in the immature rat model, prolonged FSs induce transient structural changes of some hippocampal pyramidal neurons and long-term functional changes of hippocampal circuitry. However, whether these neuroanatomical and electrophysiological changes promote hippocampal excitability and lead to epilepsy has remained unknown. By using in vivo and in vitro approaches, we determined that prolonged hyperthermia-induced seizures in immature rats caused long-term enhanced susceptibility to limbic convulsants that lasted to adulthood. Thus, extensive hippocampal electroencephalographic and behavioral monitoring failed to demonstrate spontaneous seizures in adult rats that had experienced hyperthermic seizures during infancy. However, 100% of animals developed hippocampal seizures after systemic administration of a low dose of kainate, and most progressed to status epilepticus. Conversely, a minority of normothermic and hyperthermic controls had (brief) seizures, none developing status epilepticus. In vitro, spontaneous epileptiform discharges were not observed in hippocampal-entorhinal cortex slices derived from either control or experimental groups. However, Schaeffer collateral stimulation induced prolonged, self-sustaining, status epilepticus-like discharges exclusively in slices from experimental rats. These data indicate that hyperthermic seizures in the immature rat model of FSs do not cause spontaneous limbic seizures during adulthood. However, they reduce thresholds to chemical convulsants in vivo and electrical stimulation in vitro, indicating persistent enhancement of limbic excitability that may facilitate the development of epilepsy.