Amygdaloid pERK1/2 in corticotropin-releasing hormone overexpressing mice under basal and acute stress conditions

doi:10.1016/j.neuroscience.2009.01.014

Neuroscience

Volume 159, Issue 2, 17 March 2009, Pages 610-617

https://doi.org/10.1016/j.neuroscience.2009.01.014 Get rights and content

Abstract

Corticotropin-releasing hormone (CRH) coordinates neuroendocrine and behavioral adaptations to stress. Acute CRH administration in vivo activates extracellular signal-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 immunohistochemistry and confocal microscopy analysis we found that pERK1/2 levels in the basolateral amygdala (BLA) were similar 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 immunoreactivity in the BLA compared to CRH-COE^hom-Cam animals under basal conditions. Radioligand binding and in situ hybridization revealed higher density of CRH-R1 in the amygdala 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, suggesting that stress may trigger CRH-R1 internalization/downregulation in these CRH overexpressing mice. Chronic CRH overexpression leads to reduced ERK1/2 activation in response to acute stress in the BLA.

Section snippets

Animals and housing

The generation of conditional CRH overexpressing (CRH-COE-Cam) mice as well as their genetic features has been previously described (Lu et al., 2008). Briefly, CRH-COE mice were established by introducing a single copy of the murine CRH cDNA, which is preceded by a loxP-flanked transcriptional terminator, into the ubiquitously expressed ROSA26 (R26) locus (Zambrowicz et al., 1997). Forebrain-restricted overexpression was achieved by breeding CRH-COE mice to transgenic CamK2a-cre mice (

Acute stress in CRH overexpressing mice results in decreased pERK1/2 levels in the BLA

Since the acute administration of CRH induces a limbic-specific ERK activation pattern restricted to the hippocampus and amygdala (Refojo et al., 2005), we analyzed the effect of chronic CRH overexpression on pERK1/2 immunoreactivity in these limbic areas in both control (CRH-COE^con-Cam) and CRH overexpressing (CRH-COE^hom-Cam) mice on basal conditions and after 10 min of restraint stress. Brain slices of both genotypes in both experimental conditions were subjected to immunohistochemical

Discussion

In this work we evaluated for the first time pERK1/2 levels in a mouse model of limbic-restricted CRH overexpression. Previous work has shown that acute i.c.v. administration of CRH leads to a strong ERK1/2 activation in limbic areas (Refojo et al., 2005). Under basal conditions, CRH-COE^hom-Cam mice exhibit levels of ERK1/2 activation in the BLA similar to those observed in control littermates. Restraint stress did not change pERK1/2 levels in the BLA of control animals, although the stress

Acknowledgments

We would like to thank Ursula Habersetzer, Sabrina Meyr, Tanja Orschmann and Stefanie Unkmeir for excellent technical assistance. This work was supported by grants from the university of Buenos Aires (UBA), the CONICET and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina, as well as by the Bundesministerium für Bildung und Forschung within the framework of NGFN-Plus (01GS08151 and 01GS08155). Part of the study was supported by the Freedom to Discover Award given by the

References (57)

F. Ailing et al.
Role of extracellular signal-regulated kinase signal transduction pathway in anxiety
J Psychiatr Res
(2008)
E. Arzt et al.
CRF signaling: molecular specificity for drug targeting in the CNS
Trends Pharmacol Sci
(2006)
A.J. Dunn et al.
Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses?
Brain Res Brain Res Rev
(1990)
S.L. Gourley et al.
Acute hippocampal brain-derived neurotrophic factor restores motivational and forced swim performance after corticosterone
Biol Psychiatry
(2008)
L. Groenink et al.
HPA axis dysregulation in mice overexpressing corticotropin releasing hormone
Biol Psychiatry
(2002)
R. Hanstein et al.
Transgenic overexpression of corticotropin releasing hormone provides partial protection against neurodegeneration in an in vivo model of acute excitotoxic stress
Neuroscience
(2008)
J.P. Herman et al.
Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness
Front Neuroendocrinol
(2003)
F. Holsboer
The rationale for corticotropin-releasing hormone receptor (CRH-R) antagonists to treat depression and anxiety
J Psychiatr Res
(1999)
F. Holsboer et al.
Central CRH system in depression and anxiety—evidence from clinical studies with CRH1 receptor antagonists
Eur J Pharmacol
(2008)
A. Korosi et al.
The central corticotropin releasing factor system during development and adulthood
Eur J Pharmacol
(2008)

A. Korosi et al.

Distribution and expression of CRF receptor 1 and 2 mRNAs in the CRF over-expressing mouse brain

Brain Res

(2006)

E. Meller et al.

Region-specific effects of acute and repeated restraint stress on the phosphorylation of mitogen-activated protein kinases

Brain Res

(2003)

Z. Merali et al.

Corticotropin-releasing hormone, arginine vasopressin, gastrin-releasing peptide, and neuromedin B alterations in stress-relevant brain regions of suicides and control subjects

Biol Psychiatry

(2006)

L. Minichiello et al.

Essential role for TrkB receptors in hippocampus-mediated learning

Neuron

(1999)

X. Qi et al.

The depressive-like behaviors are correlated with decreased phosphorylation of mitogen-activated protein kinases in rat brain following chronic forced swim stress

Behav Brain Res

(2006)

G.W. Smith et al.

Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development

Neuron

(1998)

L. Arborelius et al.

The role of corticotropin-releasing factor in depression and anxiety disorders

J Endocrinol

(1999)

T.L. Bale et al.

CRF and CRF receptors: role in stress responsivity and other behaviors

Annu Rev Pharmacol Toxicol

(2004)

N. Bayatti et al.

Brain region-specific neuroprotective action and signaling of corticotropin-releasing hormone in primary neurons

Endocrinology

(2003)

J.C. Bittencourt et al.

Do centrally administered neuropeptides access cognate receptors?An analysis in the central corticotropin-releasing factor system

J Neurosci

(2000)

D.T. Chalmers et al.

Localization of novel corticotropin-releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression

J Neurosci

(1995)

Y. Chen et al.

Immunocytochemical distribution of corticotropin-releasing hormone receptor type-1 (CRF(1))-like immunoreactivity in the mouse brain: light microscopy analysis using an antibody directed against the C-terminus

J Comp Neurol

(2000)

A. Dagerlind et al.

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Histochemistry

(1992)

E.R. de Kloet et al.

Stress and the brain: from adaptation to disease

Nat Rev Neurosci

(2005)

B. Di Benedetto et al.

Differential mRNA distribution of components of the ERK/MAPK signalling cascade in the adult mouse brain

J Comp Neurol

(2007)

Y. Dwivedi et al.

Reduced activation and expression of ERK1/2 MAP kinase in the post-mortem brain of depressed suicide subjects

J Neurochem

(2001)

H. Einat et al.

The role of the extracellular signal-regulated kinase signaling pathway in mood modulation

J Neurosci

(2003)

D.G. Flood et al.

Immunolocalization of the mitogen-activated protein kinases p42MAPK and JNK1, and their regulatory kinases MEK1 and MEK4, in adult rat central nervous system

J Comp Neurol

(1998)

Cited by (12)

Sustained glucocorticoid exposure recruits cortico-limbic CRH signaling to modulate endocannabinoid function
2016, Psychoneuroendocrinology
Citation Excerpt :
In the current study, however, given that sustained CORT exposure had no effect on amygdala 2-AG levels, but CRH overexpression did significantly increase amygdala 2-AG concentrations, it appears that certain biological changes associated with the CRH overexpressing mice may have contributed to these effects. The line of mutant mice we employed have higher levels of CRHR1 mRNA and protein expression in the basolateral amygdala (Lu et al., 2008; Silberstein et al., 2009), therefore a heightened CRHR1 signaling capacity may have contributed to the 2-AG differences observed in these mice. We also previously reported that intracerebroventricular administration of CRH was capable of increasing 2-AG content within the amygdala, although the temporal nature of this effect (2 h from onset) was much more delayed than the CRH-induced effects on FAAH/AEA dynamics.
Sustained exposure to stress or corticosteroids is known to cause changes in brain endocannabinoid (eCB) signaling, such that tissue contents of the eCBs N-arachidonylethanolamine (AEA) are generally reduced while 2-arachidonoylglycerol (2-AG) levels increase. These changes in eCB signaling are important for many of the aspects of chronic stress, such as anxiety, reward sensitivity and stress adaptation, yet the mechanisms mediating these changes are not fully understood. We have recently found that the stress-related neuropeptide corticotropin-releasing hormone (CRH), acting through the CRH type 1 receptor (CRHR1), can reduce AEA content by increasing its hydrolysis by the enzyme fatty acid amide hydrolase (FAAH) as well as increase 2-AG contents. As extra-hypothalamic CRH is upregulated by chronic corticosteroid or stress exposure, we hypothesized that increased CRH signaling through CRHR1 contributes to the effects of chronic corticosteroid exposure on the eCB system within the amygdala and prefrontal cortex. Male rats were exposed to 7 days of systemic corticosterone capsules, with or without concurrent exposure to a CRHR1 antagonist, after which we examined eCB content. Consistent with previous studies in the amygdala, sustained corticosterone exposure increases CRH mRNA in the prefrontal cortex. As was shown previously, FAAH activity was increased and AEA contents were reduced within the amygdala and prefrontal cortex following chronic corticosterone exposure. Chronic corticosterone exposure also elevated 2-AG content in the prefrontal cortex but not the amygdala. These corticosteroid-driven changes were all blocked by systemic CRHR1 antagonism. Consistent with these data indicating sustained increases in CRH signaling can mediate the effects of chronic elevations in corticosteroids, CRH overexpressing mice also exhibited increased FAAH-mediated AEA hydrolysis in the amygdala and prefrontal cortex compared to wild type. CRH overexpression increased 2-AG content in the amygdala, but not the prefrontal cortex. These data indicate that chronic elevations in CRH signaling, as is seen following exposure to chronic elevations in corticosterone or stress, drive persistent changes in eCB function. As reductions in AEA signaling mediate the effects of CRH and chronic stress on anxiety, these data provide a mechanism linking these processes.
CRHR1 mediates p53 transcription induced by high altitude hypoxia through ERK 1/2 signaling in rat hepatic cells
2013, Peptides
Citation Excerpt :
The outcome of the GPCR pathway is the phosphorylation of a series of transcription factors and then transcriptional regulation of a series of genes. Although the CRHR1 pathway in rat liver has not been fully elucidated so far, CRH has been demonstrated to activate p-ERK 1/2 through CRHR1 in the central nervous system [34,38,46]. Our study also showed that CRHR1 on liver cells activated p-ERK 1/2 (Fig. 5B).
We have previously reported that hypoxia activates corticotrophin-releasing hormone (CRH) and the expression of its type-1 receptor (CRHR1) and induces disorders of the brain–endocrine–immune network. p53 is activated by hypoxia and involved in tumorigenesis and apoptosis. Whether CRHR1 regulates p53 transactivation to further influence apoptotic genes remains unclear. Here, we showed that hypoxia at a simulated altitude of 5 km or 7 km for 8 and 24 h increased p53 protein and mRNA, and reduced apoptotic bax and IGFBP3 gene expression while upregulating the cell-arrest gene p21 for 8 h in rat liver cells. The upregulation of p53 mRNA and downregulation of bax mRNA induced by hypoxia were blocked by pretreatment with the specific CRHR1 antagonist CP-154,526, but the downregulation of IGFBP3 and upregulation of p21 mRNA were not. Furthermore, CRH stimulated p53 mRNA via the ERK 1/2 pathway in the BRL-3A cell line and this was blocked by the ERK 1/2 antagonist U0126. These data provide novel evidence that the CRHR1-triggered ERK 1/2 pathway is involved in the activation of p53 and suppression of the apoptotic bax gene by hypoxia in rat liver.
Personality traits in rats predict vulnerability and resilience to developing stress-induced depression-like behaviors, HPA axis hyper-reactivity and brain changes in pERK1/2 activity
2012, Psychoneuroendocrinology
Citation Excerpt :
In addition, we evaluated the expression levels of the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in different brain regions. We focused on ERK1/2 because it plays an important role as an effector molecule in the actions of CRF in different brain regions, including the hippocampus and the amygdala (Refojo et al., 2005; Silberstein et al., 2009) and, in addition, because it has been suggested to play a role in stress, memory, plasticity and depression (Mazzucchelli and Brambilla, 2000; Shen et al., 2004; Qi et al., 2006; Reul and Chandramohan, 2007; Silberstein et al., 2009). A total of 156 adult male Sprague-Dawley rats (Charles River Laboratories, France) were used (approximately three months old and body weight of 250–275 g at the beginning of the experiment).
Emerging evidence indicates that certain behavioral traits, such as anxiety, are associated with the development of depression-like behaviors after exposure to chronic stress. However, single traits do not explain the wide variability in vulnerability to stress observed in outbred populations. We hypothesized that a combination of behavioral traits might provide a better characterization of an individual's vulnerability to prolonged stress. Here, we sought to determine whether the characterization of relevant behavioral traits in rats could aid in identifying individuals with different vulnerabilities to developing stress-induced depression-like behavioral alterations. We also investigated whether behavioral traits would be related to the development of alterations in the hypothalamic-pituitary-adrenal axis and in brain activity – as measured through phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) – in response to an acute stressor following either sub-chronic (2 weeks) or chronic (4 weeks) unpredictable stress (CUS). Sprague-Dawley rats were characterized using a battery of behavioral tasks, and three principal traits were identified: anxiety, exploration and activity. When combined, the first two traits were found to explain the variability in the stress responses. Our findings confirm the increased risk of animals with high anxiety developing certain depression-like behaviors (e.g., increased floating time in the forced swim test) when progressively exposed to stress. In contrast, the behavioral profile based on combined low anxiety and low exploration was resistant to alterations related to social behaviors, while the high anxiety and low exploration profile displayed a particularly vulnerable pattern of physiological and neurobiological responses after sub-chronic stress exposure. Our findings indicate important differences in animals’ vulnerability and/or resilience to the effects of repeated stress, particularly during initial or intermediate levels of stress exposure, and they highlight that the behavioral inhibition profile of an animal provides a particular susceptibility to responding in a deleterious manner to stress.
Facilitating influence of stress on the consolidation of fear memory induced by a weak training: Reversal by midazolam pretreatment
2011, Behavioural Brain Research
Citation Excerpt :
The present results also show a slight enhancement of p-ERK2 expression in the BLA of naive stressed animals, indicating that prior exposure to restraint still activates the ERK signaling pathway, at least one day later. This activation is in agreement with a growing number of evidence showing that diverse types of acute stressors result in ERK activation in various brain structures, including the basolateral amygdala [46,53,58,60,61]. In most of these cases, the ERK1/2 levels were determined temporally close to the stressful experience.
It is well known that an emotionally arousing experience usually results in a robust and persistent memory trace. The present study explored the potential mechanisms involved in the influence of stress on the consolidation of a contextual fear memory in animals subjected to a weak fear training protocol, and whether pretreatment with intra-basolateral amygdala or systemic administration of midazolam (MDZ) prevents the potential stress-induced influence on fear memory formation. A previous restraint session facilitated fear retention, this effect was not due to a sensitized effect of restraint on the footshock experience. MDZ, both systemically or intra-basolateral amygdala infusion prior to the restraint, attenuated the stress-induced promoting influence on fear memory formation. In addition, stress exposure activated the ERK1/2 pathway in basolateral amygdala (BLA) after the weak training procedure but not after the immediate footshock protocol. Similar to our behavioral findings, MDZ attenuated stress-induced elevation of phospho-ERK2 (p-ERK2) in BLA following the acquisition session. Given that the activation of ERK1/2 pathway is essential for associative learning, we propose that stress-induced facilitation of p-ERK2 in BLA is an important mechanism for the promoting influence of stress on the consolidation of contextual fear memory.
Characterization of the B-Raf interactome in mouse hippocampal neuronal cells
2011, Journal of Proteomics
Citation Excerpt :
Furthermore, since – unlike the ubiquitous Raf-1 – B-Raf is predominantly expressed in the nervous system [15], it constitutes a determining factor in the ability of neurons to activate ERK [16]. ERK activity in the central nervous system is linked to a variety of crucial molecular processes [17] and, in limbic areas, it was found to be modulated under different stress-related conditions in vivo [18,19]. More than 30 mutations in B-Raf have been associated with human cancers [20,21].
B-Raf links a variety of extracellular stimuli downstream of cell surface receptors, constituting a determining factor in the ability of neurons to activate ERK. A detailed study of the B-Raf interactome is necessary to clarify the intricacy of B-Raf-dependent signal transduction. We used a mouse hippocampal cell line (HT22) that expresses B-Raf at high levels, to identify B-Raf associated proteins under endogenous expression conditions, avoiding artificial interactions from overexpression studies. We used stringent procedures to co-immunoprecipitate proteins that specifically associate with endogenous B-Raf with the help of gel electrophoresis separation and off-line LC-MALDI-MS/MS proteomic analysis. Our stringent protein identification criteria allowed confident identification of B-Raf interacting proteins under non-stimulating conditions. The presence of previously reported B-Raf interactors among the list of proteins identified confirms the quality of proteomic data. We identified tubulin and actin as B-Raf interactors for the first time, among structural and accessory proteins of cell cytoskeleton, molecular chaperones (Hsc70, GRP78), and cellular components involved in aspects of mRNA metabolism and translation. Interactions were validated in HT22 cells and in the neuronal cell line Neuro-2a providing further evidence that the identified proteins are B-Raf interactors, which constitute a basis for understanding MAPK pathway regulation in neurons.
Characterization of a pachymedusa dacnicolor-sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies
2015, Journal of Pharmacology and Experimental Therapeutics

View all citing articles on Scopus

View full text

Molecular NeuroscienceAmygdaloid pERK1/2 in corticotropin-releasing hormone overexpressing mice under basal and acute stress conditions

Abstract

Section snippets

Animals and housing

Acute stress in CRH overexpressing mice results in decreased pERK1/2 levels in the BLA

Discussion

Acknowledgments

J Psychiatr Res

Trends Pharmacol Sci

Brain Res Brain Res Rev

Biol Psychiatry

Biol Psychiatry

Neuroscience

Front Neuroendocrinol

J Psychiatr Res

Eur J Pharmacol

Eur J Pharmacol

Brain Res

Brain Res

Biol Psychiatry

Neuron

Behav Brain Res

Neuron

The role of corticotropin-releasing factor in depression and anxiety disorders

J Endocrinol

CRF and CRF receptors: role in stress responsivity and other behaviors

Annu Rev Pharmacol Toxicol

Brain region-specific neuroprotective action and signaling of corticotropin-releasing hormone in primary neurons

Endocrinology

Do centrally administered neuropeptides access cognate receptors?An analysis in the central corticotropin-releasing factor system

J Neurosci

Localization of novel corticotropin-releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression

J Neurosci

Immunocytochemical distribution of corticotropin-releasing hormone receptor type-1 (CRF(1))-like immunoreactivity in the mouse brain: light microscopy analysis using an antibody directed against the C-terminus

J Comp Neurol

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Histochemistry

Stress and the brain: from adaptation to disease

Nat Rev Neurosci

Differential mRNA distribution of components of the ERK/MAPK signalling cascade in the adult mouse brain

J Comp Neurol

Reduced activation and expression of ERK1/2 MAP kinase in the post-mortem brain of depressed suicide subjects

J Neurochem

The role of the extracellular signal-regulated kinase signaling pathway in mood modulation

J Neurosci

Immunolocalization of the mitogen-activated protein kinases p42MAPK and JNK1, and their regulatory kinases MEK1 and MEK4, in adult rat central nervous system

J Comp Neurol

Molecular Neuroscience
Amygdaloid pERK1/2 in corticotropin-releasing hormone overexpressing mice under basal and acute stress conditions