CRF Expression Research Articles

Relaxin-3 regulates corticotropin-releasing factor gene expression in cultured rat hypothalamic 4B cells

The ancestral insulin/relaxin peptide superfamily member relaxin-3 is an important regulator of food intake and behaviors related to anxiety and motivation. Relaxin family peptide receptor 1 (RXFP1) and RXFP3 are expressed in the rat hypothalamic paraventricular nucleus (PVN). Corticotropin-releasing factor (CRF) is produced in the PVN in response to stressors and promotes adrenocorticotropic hormone secretion from the anterior pituitary. We hypothesized that relaxin-3 directly regulates Crf expression in the hypothalamus and investigated its effect on Crf expression in cultured hypothalamic 4B cells. Relaxin-3 increased Crf mRNA levels and stimulated Crf promoter activity. Both protein kinase A and C pathways contributed to relaxin-3-induced Crf promoter activity. Rxfp1 and Rxfp3 mRNA and their proteins were expressed in cultured hypothalamic 4B cells. Relaxin-3 decreased Rxfp1 mRNA and protein levels and increased Rxfp3 mRNA and protein levels. These results suggested that the action of relaxin-3 in cultured hypothalamic 4B cells may be regulated through both RXFP1 and RXFP3.

Involvement of the central hypothalamic-pituitary-adrenal axis in hair growth and melanogenesis among different mouse strains.

Stress has been demonstrated to play an important role in hair follicle function and the pathogenesis of some hair disorders. The central hypothalamic-pituitary-adrenal (HPA) axis is activated by stress stimuli, synthesizes and releases various components and eventually induces the pathogenesis and recurrence of peripheral diseases. Our aim is to compare the different responses under exposure of stress in hair follicle function among different mouse strains, and to detect the involvement of the central HPA axis after stress in hair follicle growth and melanogenesis. In this study, we exposed different mouse strains (C57BL/6, CBA/J, C3H/HeN, BALB/c and ICR) to a 21-day chronic restraint stress protocol and selected C57BL/6, CBA/J and BALB/c mice for further study because of their significant behavioral alterations. Then, we evaluated and compared the different responses and sensitivity to chronic restraint stress in hair follicle function and central HPA axis among the selected strains. The results showed that expression of POMC, CRF and GR mRNA and protein and serum levels of corticosterone were inhibited in response to stress. These findings suggested that chronic restraint stress may inhibit hair follicle growth and melanogenesis via regulating the key elements of the central HPA axis. In addition, the results revealed different mouse strains exhibit different responses in the central HPA axis and hair follicle after stress exposure. C57BL/6 might be the most sensitive strain among the three strains tested as well as an appropriate strain to study possible pathophysiological mechanisms by which the nervous system influences skin function and screen dermatological drugs suitable for psychotherapy. We believe the current study will provide some useful information for researchers who are interested in the bidirectional communication between the nervous and skin systems and the management of stress-induced cutaneous diseases.

Allostatic Load and Stress Physiology in European Seabass (Dicentrarchus labrax L.) and Gilthead Seabream (Sparus aurata L.).

The present study aimed to compare effects of increasing chronic stress load on the stress response of European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) to identify neuroendocrine functions that regulate this response. Fish were left undisturbed (controls) or exposed to three levels of chronic stress for 3 weeks and then subjected to an acute stress test (ACT). Chronic stress impeded growth and decreased feed consumption in seabass, not in seabream. In seabass basal cortisol levels are high and increase with stress load; the response to a subsequent ACT decreases with increasing (earlier) load. Basal cortisol levels in seabream increase with the stress load, whereas the ACT induced a similar response in all groups. In seabass and seabream plasma α-MSH levels and brain stem serotonergic activity and turnover were similar and not affected by chronic stress. Species-specific molecular neuro-regional differences were seen. In-situ hybridization analysis of the early immediate gene cfos in the preoptic area showed ACT-activation in seabream; in seabass the expression level was not affected by ACT and seems constitutively high. In seabream, expression levels of telencephalic crf, crfbp, gr1, and mr were downregulated; the seabass hypothalamic preoptic area showed increased expression of crf and gr1, and decreased expression of mr, and this increased the gr1/mr ratio considerably. We substantiate species-specific physiological differences to stress coping between seabream and seabass at an endocrine and neuroendocrine molecular level. Seabass appear less resilient to stress, which we conclude from high basal activities of stress-related parameters and poor, or absent, responses to ACT. This comparative study reveals important aquaculture, husbandry, and welfare implications for the rearing of these species.

Increased expression of CRF and CRF-receptors in dorsal striatum, hippocampus, and prefrontal cortex after the development of nicotine sensitization in rats

BackgroundNicotine addiction supports tobacco smoking, a main preventable cause of disease and death in Western countries. It develops through long-term neuroadaptations in the brain reward circuit by modulating intracellular pathways and regulating gene expression. This study assesses the regional expression of the transcripts of the CRF transmission in a nicotine sensitization model, since it is hypothesised that the molecular neuroadaptations that mediate the development of sensitization contribute to the development of addiction. MethodsRats received intraperitoneal nicotine administrations (0.4 mg/kg) once daily for either 1 day or over 5 days. Locomotor activity was assessed to evaluate the development of sensitization. The mRNA expression of CRF and CRF1 and CRF2 receptors was measured by qPCR in the ventral mesencephalon, ventral striatum, dorsal striatum (DS), prefrontal cortex (PFCx), and hippocampus (Hip). ResultsAcute nicotine administration increased locomotor activity in rats. In the sub-chronic group, locomotor activity progressively increased and reached a clear sensitization. Significant effects of sensitization on CRF mRNA levels were detected in the DS (increasing effect). Significantly higher CRF1 and CRF2 receptor levels after sensitization were detected in the Hip. Additionally, CRF2 receptor levels were augmented by sensitization in the PFCx, and treatment and time-induced increases were detected in the DS. Nicotine treatment effects were observed on CRF1R levels in the DS. ConclusionsThis study suggests that the CRF transmission, in addition to its role in increasing withdrawal-related anxiety, may be involved in the development of nicotine-habituated behaviours through reduced control of impulses and the aberrant memory plasticity characterising addiction.

Corticotropin-releasing factor regulates caspase-3 and may protect developing zebrafish from stress-induced apoptosis

The corticotropin-releasing factor (CRF) system is expressed in the earliest stages of zebrafish development, long before its canonical function in the endocrine stress response is realized, and yet its function during embryogenesis is unknown. We tested the hypothesis that CRF protects embryos from stress-induced apoptosis. Here we confirm that a 1 h heat shock applied at either 6 h post-fertilization (hpf) or 30 hpf elicits an increase in caspase-3 activity, a key effector of apoptosis. Temporal changes in the expression of crf and its binding protein (crf-bp) during recovery from heat shock indicate that the CRF system is responsive to stressors experienced as early as gastrulation. Next, we heat shocked embryos that were microinjected with crf mRNA, and showed that caspase-3 induction is significantly reduced in embryos that overexpress CRF relative to control embryos. In addition, incubating embryos in the presence of the CRF receptor type 1 (CRF-R1) antagonist, antalarmin, during recovery from heat shock significantly increased caspase-3 activity, suggesting that CRF regulates caspase-3 via a CRF-R1-dependent pathway. Finally, we show that most heat shock-induced mortality occurred during the first hour of recovery, long before a significant increase in caspase-3 activity was detected. Indeed, the delayed caspase-3 induction coincided with a mortality plateau, and neither CRF overexpression nor antalarmin treatment altered heat shock induced mortality, supporting previous in vitro evidence that CRF-mediated cytoprotection occurs through the slow and tightly controlled apoptotic pathway. This study provides novel in vivo evidence that CRF regulates stress-induced apoptosis in a vertebrate model species, and demonstrates for the first time a function for the CRF system in early development that precedes its role in the endocrine stress response.

Evaluation of Alcohol Preference and Drinking in msP Rats Bearing a Crhr1 Promoter Polymorphism.

Alcoholism is a pervasive societal problem, yet available pharmacotherapies fail to treat most sufferers. The type 1 corticotropin-releasing factor (CRF1) receptor has received much attention for its putative role in the progression to alcohol dependence, although at present its success in clinical trials has been limited. Two single-nucleotide polymorphisms in the rat Crhr1 promoter have been identified in the Marchigian substrain of Sardinian alcohol-preferring (msP) rats. Unlike other Wistar-derived alcohol-preferring lines, nondependent msP rats reduce their alcohol self-administration in response to CRF1 antagonists and show increased brain CRF1 expression. The current study tested the hypotheses that the A alleles in the Crhr1 promoter polymorphisms are: (1) unique to msP (vs. CRF1 antagonist-insensitive) alcohol-preferring lines and (2) associate with greater alcohol preference or intake. Two related polymorphisms were observed in which both loci on a given chromosome were either mutant variant (A) or wild-type (G) alleles within the distal Crhr1 promoter of 17/25 msP rats (68%), as compared to 0/23 Indiana P rats, 0/20 Sardinian alcohol-preferring rats bred at Scripps (Scr:sP) and 0/21 outbred Wistar rats. Alcohol consumption in msP rats did not differ according to the presence of Crhr1 A alleles, but greater alcohol preference (98%) was observed in A allele homozygous msP rats (AA) compared to msP rats with wild-type (GG, 91%) or heterozygous (GA, 91%) genotypes. The greater alcohol preference reflected decreased water intake, accompanied by reduced total calories consumed by AA rats. The data show that msP rats differentially possess mutant A variant alleles in the polymorphic promoter region of the Crhr1 gene that may differentially regulate consumption.

Distribution of corticotropin-releasing factor (CRF) receptor binding in the mouse brain using a new, high-affinity radioligand, [125 I]-PD-Sauvagine.

The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.

Bioactive forms of vitamin D selectively stimulate the skin analog of the hypothalamus-pituitary-adrenal axis in human epidermal keratinocytes

Ultraviolet radiation B stimulates both the production of vitamin D3 in the skin and the activation of the skin analog of the hypothalamic-pituitary-adrenal axis (HPA) as well as the central HPA. Since the role of vitamin D3 in the regulation of the HPA is largely unknown, we investigated the impact of 1,25(OH)2D3 and its noncalcemic analogs, 20(OH)D3 and 21(OH)pD, on the expression of the local HPA in human epidermal keratinocytes. The noncalcemic analogs showed similar efficacy to 1,25(OH)2D3 in stimulating the expression of neuropeptides, CRF, urocortins and POMC, and their receptors, CRFR1, CRFR2, MC1R, MC2R, MC3R and MC4R. Interestingly, unlike other secosteroids, the activity of 21(OH)pD did not correlate with induction of differentiation, suggesting a separate but overlapping mechanism of action. Thus, biologically active forms of vitamin D can regulate different elements of the local equivalent of the HPA with implications for the systemic HPA.

Differentiation of Keratinocytes Modulates Skin HPA Analog.

It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

Developmental Characteristics of the Hypothalamo-Hypophyseal-Adrenal System in Chronic Heterotypical Stress

Experimental studies of the developmental characteristics of the adaptation of the hypothalamo-hypophyseal-adrenal system (HHAS) to the actions of heterotypical stressors in the context of stress-associated behavioral reactions were studied. Sprague–Dawley rats aged three (young), six (adult), and 12 (aged) months (total number of animals = 36) were exposed to changing stressors for seven days, after which their behavioral reactions were assessed. Histological changes in the hypothalamus, hypophysis, and adrenals were studied and compared with age-matched controls, and immunochemical reactions to corticoliberin (CRF), adrenocorticotropic hormone (ACTH), ED1, PCNA, and caspase-3 were performed, with subsequent image analysis. In aged animals, as compared with young and adult animals, the levels of activation of the components of the HHAS provided evidence of dissociation in its central component and blockade of the adaptive desensitization typical of young and adult animals. In particular, aged animals showed a high level of CRF expression in the hypothalamus in stress, with a relatively low level of ACTH expression in the adenohypophysis and a high level of adrenal activity. Decreases in HHAS plasticity in aged rats as compared with other age groups corresponded to behavioral changes seen in these animals, demonstrating decreases in the ability of aged animals to adapt to chronic exposure to unpredictably changing stressors.

Changes in the CRF expression after chronic restraint stress in high (HR) and low (LR) anxiety rats

Changes in the CRF expression after chronic restraint stress in high (HR) and low (LR) anxiety rats

Activity of the hypothalamus-pituitary-interrenal axis (HPI axis) and immune response in carp lines with different susceptibility to disease.

The stress response transmitted by the HPA axis is one of the best examples of neuroendocrine-immune interactions that are critical for survival. Analogous to the situation in mammals, the stress response in fish is characterized by the activation of the hypothalamo-pituitary-interrenal axis (HPI). Effects of cortisol on the fish immune system comply with findings in mammals and suggest that the differences in sensitivity to stress will influence the immune response and as a consequence of survival. Therefore, we studied the stress response and its immunity-related effects in four different carp lines (R3, R3xR8, K and R2) that display a differential pathogen susceptibility. Previous studies indicate that R3xR8 and R3 carp are susceptible to bacterial and parasite infection, while R2 and K are relatively resistant to infection. Interestingly, the most striking effect of stress on leukocyte composition and activity was observed in the pathogen-resistant K carp, even though no robust changes in gene expression of stress-involved factors were observed. In contrast, R3 carp showed no spectacular stress-induced changes in their immunological parameters with concurrent significant activation of the HPI axis. Upon stress, the R3 carp showed up-regulation of crf, pomc and gr2 gene expression in the hypothalamus. Furthermore in R3 carp, at all levels of the HPI axis, stress induced the highest up-regulation of il-1β gene expression. Although we are aware of the complexity of the interactions between stress and pathogen susceptibility and of the risk of interpretation based on correlations, it is noteworthy that the fish more susceptible to infection also exhibited the highest response to stress.

Scales tell a story on the stress history of fish.

Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol into the blood. Scientifically validated biomarkers to capture systemic cortisol exposure over longer periods of time are of utmost importance to assess chronic stress in governmental, wildlife, aquaculture and scientific settings. Here we demonstrate that cortisol in scales of common carp (Cyprinus carpio L.) is the long-sought biomarker for chronic stress. Undisturbed (CTR) and daily stressed (STRESS) carp were compared. Dexamethasone (DEX) or cortisol (CORT) fed fish served as negative and positive controls, respectively. Scale cortisol was quantified with a validated ultra-performance liquid chromatography tandem mass spectrometry method. An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish. Scale cortisol content reflects its accumulation in a stressor and time dependent manner and validates the scale cortisol content as biomarker for chronic stress. Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress. The expression of HPI key genes crf, pomc, and star were up-regulated in STRESS fish in the absence of a plasma cortisol response, as was the target gene of cortisol encoding subunit α1 of the Na+/K+-ATPase in gills. When lost, scales of fish regenerate fast. Regenerated scales corroborate our findings, offering (i) unsurpassed time resolution for cortisol incorporation and as such for stressful events, and (ii) the possibility to investigate stress in a well defined and controlled environment and time frame creating novel opportunities for bone physiological research. We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.

CRF2 Receptor Deficiency Eliminates the Long-Lasting Vulnerability of Motivational States Induced by Opiate Withdrawal.

Vulnerability to stressful life events is a hallmark of drug dependence that may persist long after cessation of drug intake and dramatically fuel key clinical features, such as deregulated up-shifted motivational states and craving. However, to date, no effective therapy is available for reducing vulnerability to stressful events in former drug users and drug-dependent patients, mostly because of poor knowledge of the mechanisms underlying it. In this study, we report that genetic inactivation of the stress-responsive corticotropin-releasing factor receptor-2 (CRF2-/-) completely eliminates the reemergence of increased nonrewarded nose-pokes, reflecting up-shifted motivational states, triggered by ethological environmental stressors long after cessation of morphine administration in mice. Accordingly, CRF2 receptor deficiency completely abolishes the increase in biomarkers of synthesis of major brain motivational substrates, such as ventral tegmental area (VTA) dopamine (DA) and amygdala γ-aminobutyric acid (GABA) systems, associated with the stress-induced reemergence of up-shifted motivational states long after opiate withdrawal. Nevertheless, neither CRF2 receptor deficiency nor long-term opiate withdrawal affects amygdala CRF or hypothalamus CRF expression, indicating preserved brain stress-coping systems. Moreover, CRF2 receptor deficiency does not influence the locomotor or the anxiety-like effect of long-term opiate withdrawal. Thus, the present results reveal an essential and specific role for the CRF2 receptor in the stress-induced reemergence of up-shifted motivational states and related alterations in brain motivational systems long after opiate withdrawal. These findings suggest new strategies for the treatment of the severe and long-lasting vulnerability that inexorably follows drug withdrawal and hinder drug abstinence.

Influence of Jianpi Huashi granule on CRF and CRFR1 expression in the brain-gut axis of rats with diarrhea-predominant irritable bowel syndrome

目的：从结肠、海马及下丘脑中促肾上腺皮质激素释放因子（corticotropin releasing factor,CRF）和CRF受体1（CRF receptor 1,CRFR1）角度探讨健脾化湿颗粒改善腹泻型肠易激综合征（diarrhea-predominant irritable bowel syndrome,D-IBS）模型大鼠结肠运动和内脏敏感性的作用机制.方法：采用番泻叶灌胃结合束缚应激法建立D-I B S大鼠模型,应用健脾化湿颗粒进行干预,采用酶联免疫法（ELISA）检测大鼠结肠中CRF含量,采用免疫组织化学法检测结肠中CRFR1及海马、下丘脑中CRF,CRFR1阳性表达,采用RT-PCR法检测结肠、海马中CRF m RNA和CRFR1 m RNA的表达水平.结果：与正常组相比,模型组结肠中CRF含量（67.1±3.8 vs 36.0±3.0）,海马、下丘脑中CRF阳性表达（0.23±0.02 vs 0.09±0.01,0.17±0.02 v s 0.09±0.01）明显升高（P〈0.01）;结肠、海马、下丘脑中C R F R1阳性表达（0.17±0.01 vs 0.03±0.01,0.20±0.02 vs 0.09±0.01,0.19±0.02 vs 0.07±0.01）明显升高（P〈0.01）;结肠、海马中C R F m RNA和CRFR1 m RNA的表达（结肠：0.89±0.04 vs 0.09±0.01,1.09±0.09 vs 0.21±0.04;海马：0.56±0.01 vs 0.15±0.05,1.26±0.14 vs 0.23±0.06）显著升高（P〈0.01）.与模型组相比,各治疗组结肠、海马中C R F（51.0±3.4,54.6±4.1,45.1±4.7,43.3±3.9 vs 67.1±3.8;0.18±0.02,0.19±0.02,0.15±0.02,0.11±0.01 vs 0.23±0.02）显著下降（P〈0.01）,阳性对照组、中、高剂量组下丘脑中CRF（0.15±0.02,0.13±0.01,0.12±0.01 vs 0.17±0.02）下降显著（P〈0.05,P〈0.0 1）;阳性对照组、中、高剂量组结肠、海马、下丘脑中C R F R1表达（结肠：0.10±0.01,0.08±0.01,0.05±0.01 vs 0.17±0.01;海马：0.16±0.01,0.14±0.02,0.13±0.01 vs 0.20±0.02;下丘脑：0.15±0.02,0.13±0.01,0.11±0.01 vs 0.19±0.02）下降显著（P〈0.05,P〈0.01）;结肠中CRF m RNA表达（0.63±0.04,0.76±0.06,0.32±0.06,0.13±0.03 v s 0.89±0.04）及中、高剂量组海马中CRF m RNA表达（0.76±0.11,0.67±0.10 v s 1.09±0.09�

Stress in Atlantic salmon: response to unpredictable chronic stress.

Combinations of stressors occur regularly throughout an animal's life, especially in agriculture and aquaculture settings. If an animal fails to acclimate to these stressors, stress becomes chronic, and a condition of allostatic overload arises with negative results for animal welfare. In the current study, we describe effects of exposing Atlantic salmon parr to an unpredictable chronic stressor (UCS) paradigm for 3 weeks. The paradigm involves exposure of fish to seven unpredictable stressors three times a day. At the end of the trial, experimental and control fish were challenged with yet another novel stressor and sampled before and 1 h after that challenge. Plasma cortisol decreased steadily over time in stressed fish, indicative of exhaustion of the endocrine stress axis. This was confirmed by a lower cortisol response to the novel stressor at the end of the stress period in chronically stressed fish compared with the control group. In the preoptic area (POA) and pituitary gland, chronic stress resulted in decreased gene expression of 11βhsd2, gr1 and gr2 in the POA and increased expression of those genes in the pituitary gland. POA crf expression and pituitary expression of pomcs and mr increased, whereas interrenal gene expression was unaffected. Exposure to the novel stressor had no effect on POA and interrenal gene expression. In the pituitary, crfr1, pomcs, 11βhsd2, grs and mr were down-regulated. In summary, our results provide a novel overview of the dynamic changes that occur at every level of the hypothalamic-pituitary gland-interrenal gland (HPI) axis as a result of chronic stress in Atlantic salmon.

Midazolam treatment before re-exposure to contextual fear reduces freezing behavior and amygdala activity differentially in high- and low-anxiety rats

The aim of this study was to examine the effects of benzodiazepine (midazolam) administration on rat conditioned fear responses and on local brain activity (c-Fos and CRF expressions) of low- (LR) and high- (HR)anxiety rats after the first and second contextual fear test sessions. The animals were divided into LR and HR groups based on the duration of their conditioned freezing response in the first contextual fear test. The fear-re-conditioned LR and HR animals (28days later) had increased freezing durations compared with those durations during the first conditioned fear test. These behavioral effects were accompanied by increased c-Fos expression in the medial amygdala (MeA), the basolateral amygdala (BLA), and the paraventricular hypothalamic nuclei and elevated CRF expression in the MeA. All these behavioral and immunochemical effects of fear re-conditioning were stronger in the LR group compared with the effects in the HR group. Moreover, in the LR rats, the re-conditioning led to decreased CRF expression in the primary motor cortex (M1) and to increased CRF expression in the BLA. The pretreatment of rats with midazolam before the second exposure to the aversive context significantly attenuated the conditioned fear response, lowered the serum corticosterone concentration, decreased c-Fos and CRF expressions in the MeA and in the BLA, and increased CRF complex density in M1 area only in the LR group. These studies have demonstrated that LR rats are more sensitive to re-exposure to fear stimuli and that midazolam pretreatment was associated with modified brain activity in the amygdala and in the prefrontal cortex in this group of animals. The current data may facilitate a better understanding of the neurobiological mechanisms responsible for individual differences in the psychopathological processes accompanying some anxiety disorders characterized by stronger reactivity to re-exposure to stressful challenges, e.g., posttraumatic stress disorder.

Overexpression of corticotropin releasing factor in the central nucleus of the amygdala advances puberty and disrupts reproductive cycles in female rats.

Prolonged exposure to environmental stress activates the hypothalamic-pituitary-adrenal (HPA) axis and generally disrupts the hypothalamic-pituitary-gonadal axis. Because CRF expression in the central nucleus of the amygdala (CeA) is a key modulator in adaptation to chronic stress, and central administration of CRF inhibits the hypothalamic GnRH pulse generator, we tested the hypothesis that overexpression of CRF in the CeA of female rats alters anxiety behavior, dysregulates the HPA axis response to stress, changes pubertal timing, and disrupts reproduction. We used a lentiviral vector to increase CRF expression site specifically in the CeA of preweaning (postnatal day 12) female rats. Overexpression of CRF in the CeA increased anxiety-like behavior in peripubertal rats shown by a reduction in time spent in the open arms of the elevated plus maze and a decrease in social interaction. Paradoxically, puberty onset was advanced but followed by irregular estrous cyclicity and an absence of spontaneous preovulatory LH surges associated with proestrous vaginal cytology in rats overexpressing CRF. Despite the absence of change in basal corticosterone secretion or induced by stress (lipopolysaccharide or restraint), overexpression of CRF in the CeA significantly decreased lipopolysaccharide, but not restraint, stress-induced suppression of pulsatile LH secretion in postpubertal ovariectomized rats, indicating a differential stress responsivity of the GnRH pulse generator to immunological stress and a potential adaptation of the HPA axis to chronic activation of amygdaloid CRF. These data suggest that the expression profile of this key limbic brain CRF system might contribute to the complex neural mechanisms underlying the increasing incidence of early onset of puberty on the one hand and infertility on the other attributed to chronic stress in modern human society.

Effect of DOCA/salt hypertension on CRF expression in the amygdala and the autonomic stress response in conscious rats.

The effects of DOCA/salt treatment on amygdala-area CRF gene expression and the autonomic response to air jet stress (AJS) were evaluated in conscious male Sprague Dawley (SD) rats. Fifteen days of DOCA/salt treatment significantly increased resting arterial pressure (AP), decreased resting heart rate (HR) and significantly reduced regional CRF mRNA compared to controls (23±7% vs. 100±26%) independent of changes in regional CRF receptor expression. Twenty min of AJS elicited a rise in AP (~15mmHg) that was similar in both DOCA/salt animals (n=11) and controls (n=6). Alternatively, increases in HR were significantly different in the DOCA/salt animals compared to controls; including one group of DOCA/salt animals (n=5) which responded with an attenuated HR response at the onset of AJS (low-responders) and a second group (n=6) which demonstrated an elevated HR response to AJS (high-responders), specifically during the last 10min of AJS. The divergent HR responses to AJS in the DOCA/salt animals were linked to differences in resting heart rate variability. During recovery HR returned to baseline within 10min in both control and the low responder DOCA group but indicators of spontaneous baroreflex gain only increased significantly in controls. HR in the high-responder DOCA animals did not return to baseline during the same period. These results show that DOCA/salt treatment triggers downregulation of CRF gene expression in the region of the amygdala and significantly alters the HR response to acute stress but does not alter the pressor response to stress compared to normotensive controls.

Early life stress: nature and nurture.

Recent and still nascent understanding of epigenetic marks, how they occur and are modified, has been an enormous boon to studies in the fields of endocrinology and neuroscience. Because the genetic DNA sequence does not usually change during the lifetime of the individual, it had been difficult to understand how marked and persistent changes in gene transcription rates occur in response to changes in the environment and experiences of the individual. Although not the only mechanism, hiding and exposing regulatory transcription sites on DNA through epigenetic changes in chromatin folding and unfolding allow much of the great flexibility that we observe occurring in biological processes. Methylation of cytosine residues in cytosine-phosphoguanine dinucleotides is known to result in either silencing or augmenting gene activity (see Figure 1) (1, 2). Methylated DNA can be bound by methyl-CpG-binding domain proteins. Methyl-CpG-binding domain protein then recruits additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins that modify histones, thereby forming compact, inactive chromatin, making genes inaccessible for transcription. The link between DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpGbinding protein 2 (MeCP2) has been implicated in Rett syndrome (3). In an article published in this issue of Endocrinology, Wu et al (4) show that the long-lasting effects of early life stress (ELS) inmousepups that result in increasedpituitary proopiomelanocortin mRNA, ACTH and corticosterone secretion are mediated by the reduction of methylation in a specific CpG site on the distal POMC promoter (4). Methylation of the CpG 6–8 site was shown to reduce POMC activity in vitro and in vivo and was accompanied by high levels of MeCP2, which selectively bound the CpG 6–8 methylation site. MeCP2 recruits corepressors to decrease POMC gene activity. The effect of methylation in CpG sites on transcription, in this case, is inhibitory. The authors did a very thorough job in determining the effects of ELS on pituitary POMC synthesis, although they did not determine what cellular event initiated the hypomethylation and low MeCP2 at the CpG6–8 site. However, hypomethylation of the distal POMC promoter is just one effect of ELS on gene methylation and on regulation of the hypothalamo-pituitary-adrenal axis, as the authors showed in an earlier study on the brains of the same mice that had been subjected to neonatal stress (5). In that study, they found that, through acting at a specific CpG methylation site, MeCP2 persistently down-regulated Avp expression in parvocellular cells in the paraventricular nuclei (PVN). ELS decreased MeCP2 binding to methylcytosines in the promoter region but did not alter DNA methylation. In this case, the authors show that it was likely that neuronal activity resulted in Ca -dependent phosphorylation of MeCP2 which reduced its binding to specific methylated cytosines. There was no effect on corticotropin-releasing factor mRNA (5). That is both interesting and somewhat surprising, because there are high levels of MeCP2 in parvocellular neurons in the PVN, and mice bearing a truncated MeCP2 gene (Mecp2) have elevated CRF mRNA in the PVN, amygdala, and bed nuclei of the stria terminalis (6). Earlier studies have shown that the effect of ELS on CRF expression depends on the control group that is used to compare with the group exposed to 3 hours per day of maternal separation (7–9). In untouched, unhandled litters, which was the control group in Refs. 4, 5, adults have similar CRF expression levels compared with rats exposed to ELS of 3-hour separation. However, if the reference control group is either animals experiencing normal vi-