Stress induces CRF release in the paraventricular nucleus, and both CRF and GABA release in the amygdala
Introduction
Corticotropin-releasing factor (CRF), a 41 amino acid peptide, has a long-established functional role in stress reactivity, primarily at the hypothalamic level, controlling pituitary release of adrenocorticotropin hormone (ACTH), which in turn results in adrenal release of glucocorticoids, including cortisol [20], [42], [43], [44], [45]. This hypothalamic–pituitary–adrenal (HPA) axis is regarded as the fundamental and traditionally documented system of stress response [3]. CRF in the HPA arises from neurones with cell bodies in the paraventricular nucleus of the hypothalamus (PVN) and axon terminals in the median eminence [13], [14], [17]. Within the PVN itself, there are also short loop axons releasing CRF.
Beyond HPA regulation, CRF also appears important in other brain areas with widespread distribution of CRF immunoreactive terminals and binding sites through the brain. This includes areas such as the amygdala [25], [26], [27], hippocampus and the locus ceruleus [12]. These areas appear to contribute to various behavioural aspects of the stress response and may also be involved in feedback to the HPA. The amygdala has extensive connections with areas of the hypothalamus, including the PVN, and interactions seem highly likely in coordinating stress responses [28]. Similarly, there is a high extent of feedback from the hippocampus to the PVN via the medial hippocampal tract and lateral septum [18]. Work utilising a mouse gene knockout model [28] suggests that CRF receptors in the limbic system drive aspects of the stress response independently of the HPA axis and also strongly modulate HPA responsiveness.
New probe techniques have allowed rapid and direct measurements of both cortisol and CRF in different brain areas of animals behaving in both stressful and nonstressful environments [8], [9]. Similar measurement techniques for systemic hormones have also been used with some success [5], [6]. These probes are solid-state immunosensors in which current produced by the activation of a bound ligand conjugated to a redox species is measured at a set voltage. The bound ligand competes with the analyte of interest for binding sites, resulting in a current change inversely proportional to the presence of the analyte of interest. Rapid and frequent measurements offer the opportunity to longitudinally track CRF changes in the brain and their relationship to hormonal variation. This same method of approach has been applied successfully to monitoring amino acid neurotransmitters, including gamma amino butyric acid (GABA) [7].
Using these methods, amygdala CRF was demonstrated, in sheep, to increase with stress. It was also shown that circulating cortisol contributed feedback to the responsiveness of the amygdala to further stressors tested some time later [9]. The neurohormone actions of CRF at the level of the brain appear regulated by glucocorticoids in a complex and often indirect fashion [14], [37], [38]. Fast-rate sensitive feedback and delayed concentration level feedback have both been repeatedly demonstrated [4], [15], [39]. Stress exposure appears able to result in either a feedback inhibition or a feed-forward sensitisation towards a further stress [4], [15], [22], [38]. Corticosteroid feedback has been demonstrated at both the PVN and amygdala level and other brain areas also show CRF regulation by glucocorticoids [31], [34]. Adrenalectomy produces tonic hypersecretion of CRF in the locus cereleus, presumably due to a loss of inhibitory glucocorticoid tone [12] while prior stress exposure can sensitise locus cereleus neurones to CRF. Glucocorticoid feedback thus appears a critical variable in predicting subsequent stress responsiveness and may be involved in dysfunctional stress response in conditions, such as depression and anxiety [29], [30], [40]. Regulation is extremely complex and is still relatively poorly understood. As well as a circadian rhythm that can interact with stress responsiveness, cortisol secretion also shows pulsatile ultradian characteristics across time in both rodents [46], [47] and in sheep [16]. In rodents, these ultradian rhythms have a considerable influence on the magnitude of stress response [46], [47]. Simultaneous monitoring of CRF in a number of brain areas would assist in developing an overall model of stress control and feedback.
Numerous other transmitter systems also contribute to stress, including serotonin and catecholamines. The GABA system has been recognised as important for some time and traditionally has provided a model for many anxiolytic pharmacological developments [39]. Corticosterones have been demonstrated to regulate characteristics of GABA receptors in the hippocampus [31] and pharmacological antagonism of GABA in the PVN increases CRF secretion [1], suggesting GABA is involved in CRF control. GABA may also be important in such control at the amygdala level as changes in extracellular GABA in this brain area have been noted with conditioned fear stressors [42] and in cardiovascular responses to stress [41]. Understanding this overlay with CRF and glucocorticoids into a system of control would also be useful in constructing a model of brain–stress response.
The aims of this study were to measure extracellular CRF changes in both the PVN and amygdala simultaneously during exposure to a stress, and to also measure GABA activity in the amygdala. The purpose was to establish temporal relationships between these systems and to a systemic change in cortisol. A second purpose was to explore changes in these systems relative to repeat stress situations. To evaluate the potential for causal involvement, both GABA and CRF in the amygdala were manipulated pharmacologically immediately prior to stress impositions and any effect on both the immediate following stressor and a stressor 2 days later was examined.
Section snippets
Animals and surgery
Thirty-six female sheep (ewes) were used in the study. At the time of study, they were 18- to 20-month-old Romney crossbreds, with live weights between 37 and 42 kg. They were separated from a main flock 3 weeks prior to experimentation and run together until experimental completion, with ad libitum access to pasture and water.
One week prior to commencing experimentation, animals were anaesthetised with a combination of 2% (W/V) xylazine hydrochloride (Rompun, Bayer, Germany) and zolazepam
Results
Data are presented as mean±S.E. unless otherwise stated.
Discussion
This study demonstrates for the first time that, in sheep, PVN CRF levels change with a stressor and relate in time to other aspects of the stress response, including venous cortisol changes, and amygdala CRF responses. With imposition of a stressor, clear changes were seen in both amygdala and PVN CRF prior to changes in venous cortisol. Two discernible peaks were seen in the amygdala, one rapid and of short duration followed by a second peak that mirrored in time the rise in venous cortisol.
Acknowledgements
I acknowledge support from the Foundation for Research, Science and Technology, New Zealand and the New Zealand Royal Society Marsden Fund. This work was performed with animal ethics approval.
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