AMYGDALOID pERK1/2 IN CORTICOTROPIN-RELEASING HORMONE OVEREXPRESSING MICE UNDER BASAL AND ACUTE STRESS CONDITIONS S. SILBERSTEIN, a A. M. VOGL, b D. REFOJO, b S. A. SENIN, a W. WURST, b,c F. HOLSBOER, b J. M. DEUSSING b * AND E. ARZT a * a Laboratorio de Fisiología y Biología Molecular, Departamento de Fi- siología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and IFIBYNE-CONICET, Ciudad Universitaria, Pab. II, 1428 Buenos Aires, Argentina b Max Planck Institute of Psychiatry, Kraepelinstrae 2–10, 80804 Munich, Germany c Institute of Developmental Genetics, Helmholtz Zentrum München, Ger- man Research Center for Environmental Health, Munich, Germany Abstract—Corticotropin-releasing hormone (CRH) coordi- nates neuroendocrine and behavioral adaptations to stress. Acute CRH administration in vivo activates extracellular sig- nal-regulated kinase 1/2 (ERK1/2) in limbic brain areas, acting through the CRH receptor type 1 (CRH-R1). In the present study, we used CRH-COE-Cam mice that overexpress CRH in limbic-restricted areas, to analyze the effect of chronic CRH overexpression on ERK1/2 activation. By immunohistochem- istry and confocal microscopy analysis we found that pERK1/2 levels in the basolateral amygdala (BLA) were sim- ilar in control and CRH overexpressing mice under basal conditions. Acute stress caused comparably increased levels of corticosterone in both control (CRH-COE con -Cam) and CRH overexpressing (CRH-COE hom -Cam) animals. CRH-COE hom - Cam mice after stress showed reduced pERK1/2 immunore- activity in the BLA compared to CRH-COE hom -Cam animals under basal conditions. Radioligand binding and in situ hy- bridization revealed higher density of CRH-R1 in the amyg- dala of CRH-COE hom mice under basal conditions compared to control littermates. A significant reduction of the receptor levels was observed in this area after acute stress, suggest- ing that stress may trigger CRH-R1 internalization/downregu- lation in these CRH overexpressing mice. Chronic CRH over- expression leads to reduced ERK1/2 activation in response to acute stress in the BLA. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: HPA axis, CRH, CRF, CRH receptor, ERK. Corticotropin-releasing hormone (CRH) is a 41–amino acid peptide that exerts a key role in the adjustment of neu- roendocrine and behavioral adaptations to stress (Vale et al., 1981; de Kloet et al., 2005). Internal homeostasis or changes in external environmental stress are conveyed to the CNS by neurochemical pathways and are integrated at the hypothalamic level where they reach paraventricular CRH neurosecretory neurons controlling CRH secretion. CRH activates adrenocorticotropin (ACTH) secretion, which in turn stimulates corticosteroid release from the adrenal glands (Herman et al., 2003). Besides the hypo- thalamus, CRH is widely distributed in extrahypothalamic circuits of the CNS (Swanson et al., 1983) where it acts as a neuroregulator to develop and integrate a complex re- sponse to stress (Bale and Vale, 2004; de Kloet et al., 2005; Smith and Vale, 2006; Korosi and Baram, 2008). CRH exerts its actions by activating two distinct G pro- tein– coupled receptors, corticotropin-releasing hormone re- ceptor 1 and 2 (CRH-R1 and CRH-R2) that display different localization throughout the brain (Chalmers et al., 1995; Bit- tencourt and Sawchenko, 2000; Chen et al., 2000; Van Pett et al., 2000). CRH is a high affinity ligand for CRH-R1 and binds poorly to the CRH-R2 for which other ligands such as urocortin II and III have higher affinity (Grigoriadis, 2005; Hauger et al., 2006; Holsboer and Ising, 2008). Dysregulation of the CRH system and chronically ele- vated levels of CRH are implicated in human stress-related and affective disorders (Arborelius et al., 1999; Holsboer, 1999; de Kloet et al., 2005). In vivo studies in animal models show that the behavioral and hormonal effects of CRH can be ascribed to CRH-R1 (Smith et al., 1998; Timpl et al., 1998). Clinical studies in humans also support this view (Ising et al., 2007; Holsboer and Ising, 2008). A limbic-restricted CRH-R1 KO mouse line, in which the basal activity of the HPA axis remains intact, presents reduced anxiety (Muller et al., 2003) and diminished CRH signaling pathways (Refojo et al., 2005). Central administration of CRH in mice revealed phenotypic alterations reminiscent of symptoms of affective disorders (Dunn and Berridge, 1990). Accordingly, genetically modified mice that overexpress CRH exhibit altered responses in be- havioral tests (Stenzel-Poore et al., 1994; Groenink et al., 2002; Lu et al., 2008). CRH-Rs are linked to several intracellular signaling pathways beside the classical cAMP-PKA cascade (Bayatti et al., 2003; Papadopoulou et al., 2004; Teli et al., 2005). We first demonstrated that, upon CRH-R1 activa- tion, corticotrophs utilize extracellular signal-regulated ki- nase 1/2 (ERK1/2), which is downstream of PKA to mod- ulate POMC transcription (Kovalovsky et al., 2002). In previous in vivo studies we observed that the MAPKs ERK1/2 in specific limbic areas like hippocampus and basolateral amygdala (BLA), are activated by acute i.c.v. administration of CRH acting through CRH-R1. These brain structures are related to external environment *Corresponding author. Tel: +54-11-4576-3368-86; fax: +54-11- 4576-3321 (E. Arzt); Tel: +49-0-89-30622-639; fax: +49-0-89-30622- 639 (J. Deussing). Abbreviations: ACTH, adrenocorticotropin; BLA, basolateral amyg- dala; CRH, corticotropin-releasing hormone; CRH-R1, corticotropin- releasing hormone receptor 1; ERK1/2, extracellular signal-regulated kinase 1/2; PI, propidium iodide; T-PBS, 0.01% Tween 20 PBS. Neuroscience 159 (2009) 610 – 617 0306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.01.014 610