Study of Molecular Changes Induced by Stress
Molecular Changes in Response to Stress Induced by Corticotropin Releasing Hormone Administration and Role of Catecholamines in these Processes
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
3 Approved without Funding
Ministry of Education and Science of Georgia / Beritashvili Institute of Physiology, Georgia, Tbilisi
- Finch University of Health Sciences, USA, IL, North Chicago\nLouisiana State University / School of Medicine in Shreveport, USA, CA, Shreveport\nIowa State University, USA, IA, Ames\nGeorge Mason University, USA, VA, Fairfax
Project summaryThe focus of this proposal is to use intracerebroventricular administration of corticotropin-releasing hormone (CRH) as a model of controlled stress to clarify the role of central stressor (CRH – imposed) on gene expression, and neuroendocrine and immune function and to determine whether the catecholamine response, associated with stress, plays a key role in changes of these parameters. The working hypothesis is that catecholamines as end-products of the activation of the stress system, are among the important mediators of the stress response in rats, causing adverse affects on endocrine, and immune responses, and, that administration of the beta-adrenergic receptor antagonist, nadolol, or a CRH antagonist, such as alpha-helical CRH9-41, prior to stress will block the stress-induced changes in gene expression, and endocrine, and immune functions. To obtain comparative pattern of stress response, the expression of the same set of candidate genes and some biochemical changes will be also investigated after footshock stress.
Objectives of this grant application focus on elucidating:
Objective 1. To determine the specific effect of CRH on the expression of various immediate early gens (Fos, cyclic AMP response element binding protein CREB; Jun and egr-1) in brain structures involved in stress (hypothalamus, limbic areas, neocortex, and pituitary) and identify brain region(s) most actively responding to stress. In control experiments CRH will be administered alone and in combination with the antagonist, alpha-helical CRH9-41 or the beta-adrenergic receptor antagonist, nadolol.
Objective 2. On the basis of results of Objective 1, we will study the specific changes in gene expression in certain brain structures (maximum 3-4) by polymerase chain reaction (PCR)-Select DNA subtraction after CRH administration, both alone and in combination with antagonists. Identified candidate genes will be investigated further by involvement of their protein products in the neurochemical processes in the stress response. To obtain comparative pattern in gene expression after stress, the expression of the same candidate genes will be investigated after footshock stress in the same brain structures.
Objective 3. In vivo determination of the effects of CRH, administered alone or in combination with its antagonist, alpha-helical CRH9-41 or the beta-adrenergic receptor antagonist, nadolol, on adrenocorticotropin (ACTH) and corticosterone release in rats, as well as immune suppression, and CRH-containing neurones in brain regions, involved in the central regulation of stress responses (hypothalamus, limbic areas, neocortex, and pituitary).
Objective 4. In vivo determination of the effects of CRH antagonist, alpha-helical CRH9-41 or beta-adrenergic receptor antagonist, nadolol, on adrenocorticotropin (ACTH) and corticosterone release in rats, and immune suppression during footshock.
Objective 5. In vitro experiments to determine the molecular mechanism of CRH, CRH-antagonist, and catecholamines action on hypothalamus/limbic system and pituitary cells in culture using a calcium imaging technique (Fura-2). Primary cell cultures from neonatal rat anterior pituitary and hypothalamus/limbic system will be established and the rapid changes in pituitary and hypothalamus/limbic system neurones (Ca2+) will be monitored
The members of the core team have a high level of knowledge in methods of immunocytochemistry, electron microscopy, molecular biology, biochemistry. Some of their studies concern to the problem of influence of different forms of stress on Central Nervous System (list of selected publications are enclosed).
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