Research reportThe CRF1 receptor mediates the excitatory actions of corticotropin releasing factor (CRF) in the developing rat brain: in vivo evidence using a novel, selective, non-peptide CRF receptor antagonist
Introduction
Corticotropin releasing factor (CRF) is a 41 amino acid neuropeptide which activates target pituitary cells and neurons via specific membrane receptors 16, 38. CRF release during the hormonal response to stress induces the secretion of corticotropin (ACTH) from the pituitary, an action blocked by analogues which compete for the receptors 33, 39. In the central nervous system (CNS), CRF mediates `central' components of the stress response, activating neurons in the amygdala as well as locus ceruleus, hippocampus and cerebellum 1, 20, 21, 40. In the immature rat, synthetic CRF excites hippocampal neurons [35]and produces prolonged limbic seizures 3, 7. Electroencephalographic (EEG) mapping has been used to localize the origin of these seizures to the amygdala [4], and propagation of the seizures to the hippocampus has been demonstrated. The postsynaptic mechanisms of the limbic seizures induced by CRF, and the reason for the increased convulsant potency of the peptide during development have not been established.
CRF activates postsynaptic receptors on target neurons 16, 22. Two members of the CRF receptor family are currently known, and consist of membrane-spanning G-protein coupled molecules 14, 27, 30. The first, CRF1, is found in the CNS and the pituitary 32, 44, and is the candidate mediator for many of the endocrine effects of CRF. The second receptor, CRF2, is found in at least two splice forms in the rat (CRF2α and CRF2β; 27, 28). One of these subtypes, CRF2α, is found primarily in the CNS. Although the messenger ribonucleic acid (mRNA) distribution patterns for CRF1 and CRF2 in the adult rat brain are quite distinct, both genes are expressed in limbic neurons of the amygdala and the hippocampus [13]. Therefore, both types of CRF receptor are candidates for mediating the convulsant effects of CRF. In the developing rat brain, CRF receptors have been demonstrated in the amygdala and hippocampus via binding assays 24, 31. Recently, CRF1-mRNA has been localized to the amygdala and hippocampus of the developing rat [2].
The goal of this study was to determine the receptor type that is responsible for mediating the excitatory effects of CRF on limbic neurons. The study compared the effects of a novel, selective CRF1 blocker and of established non-selective CRF1/CRF2 receptor antagonists on the behavioral and electrographic seizures induced by CRF. In addition, the convulsive effects of a second endogenous ligand for both types of CRF receptor, urocortin, were eliminated by selective blocking of CRF1.
Section snippets
Animals
Infant rats (n=185) were offspring of time-pregnant, Sprague–Dawley rats. They were born in the University of California, Irvine (UCI) federally approved animal facility, kept on a 12 h light/dark cycle (lights on at 07.00 h) and given access to unlimited food and water. The time of birth of pups was determined every 12 h, and the day of birth was considered day 0. Litters were culled to 12 pups and mixed among experimental groups. Thus, for each experiment, controls were littermates of, or
Results
NBI 27914 given i.c.v. increased the latency to the onset of CRF-induced seizures and decreased their duration in a dose-dependent manner (Table 1, Fig. 1). As is evident from the table, increasing doses of NBI 27914 (0.33, 0.67 and 1.0 mg/kg) abolished the seizures entirely in 40, 62.5 and 67% of rats, respectively. In comparison, the i.c.v. pre-administration of the non-selective CRF receptor blocker α-helical CRF9–41 eliminated CRF-induced seizures in 33% of the animals. In rats implanted
Discussion
The current study demonstrates that a selective competitive antagonist of the CRF1 receptor type abolishes the neuronal excitation effect of CRF in developing rats. The behavioral and electroencephalographic seizures induced by CRF in neonatal (first postnatal week) and infant (second postnatal week) rats have been described and characterized previously 3, 4, 7, 8. CRF leads to seizures that originate in the amygdala and spread to other limbic structures [4]. The doses required for seizure
Acknowledgements
The technical assistance of Linda Schultz is appreciated. These studies were partially supported by NIH NS 28912 (T.Z.B.).
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