Corticotropin-releasing Factor Receptor Research Articles

Early-life social environment alters juvenile behavior and neuroendocrine function in a highly social cichlid fish

Early-life experiences can shape adult behavior, with consequences for fitness and health, yet fundamental questions remain unanswered about how early-life social experiences are translated into variation in brain and behavior. The African cichlid fish Astatotilapia burtoni, a model system in social neuroscience, is well known for its highly plastic social phenotypes in adulthood. Here, we rear juveniles in either social groups or pairs to investigate the effects of early-life social environments on behavior and neuroendocrine gene expression. We find that both juvenile behavior and neuroendocrine function are sensitive to early-life effects. Behavior robustly co-varies across multiple contexts (open field, social cue investigation, and dominance behavior assays) to form a behavioral syndrome, with pair-reared juveniles towards the end of syndrome that is less active and socially interactive. Pair-reared juveniles also submit more readily as subordinates. In a separate cohort, we measured whole brain expression of stress and sex hormone genes. Expression of glucocorticoid receptor 1a was elevated in group-reared juveniles, supporting a highly-conserved role for the stress axis mediating early-life effects. The effect of rearing environment on androgen receptor α and estrogen receptor α expression was mediated by treatment duration (1 vs. 5 weeks). Finally, expression of corticotropin-releasing factor and glucocorticoid receptor 2 decreased significantly over time. Rearing environment also caused striking differences in gene co-expression, such that expression was tightly integrated in pair-reared juveniles but not group-reared or isolates. Together, this research demonstrates the important developmental origins of behavioral phenotypes and identifies potential behavioral and neuroendocrine mechanisms.

Corticotropin Releasing Factor Type 1 and 2 Receptor Signaling in the Medial Prefrontal Cortex Modulates Binge-Like Ethanol Consumption in C57BL/6J Mice.

Corticotropin releasing factor (CRF) signaling via limbic CRF1 and 2 receptors (CRF1R and CRF2R, respectively) is known to modulate binge-like ethanol consumption in rodents. Though CRF signaling in the medial prefrontal cortex (mPFC) has been shown to modulate anxiety-like behavior and ethanol seeking, its role in binge ethanol intake is unknown. Here, we used “drinking-in-the-dark” (DID) procedures in male and female C57BL/6J mice to address this gap in the literature. First, the role of CRF1R and CRF2R signaling in the mPFC on ethanol consumption was evaluated through site-directed pharmacology. Next, we evaluated if CRF1R antagonist reduction of binge-intake was modulated in part through CRF2R activation by co-administration of a CRF1R and CRF2R antagonist. Intra-mPFC inhibition of CRF1R and activation of CRF2R resulted in decreased binge-like ethanol intake. Further, the inhibitory effect of the CRF1R antagonist was attenuated by co-administration of a CRF2R antagonist. We provide novel evidence that (1) inhibition of CRF1R or activation of CRF2R in the mPFC reduces binge-like ethanol intake; and (2) the effect of CRF1R antagonism may be mediated via enhanced CRF2R activation. These observations provide the first direct behavioral pharmacological evidence that CRF receptor activity in the mPFC modulates binge-like ethanol consumption.

Conditioned aversive memory associated with morphine withdrawal increases brain-derived neurotrophic factor in dentate gyrus and basolateral amygdala.

Morphine has been shown to increase the expression of brain-derived neurotrophic factor (BDNF) in the brain. However, little is known about the effect of conditioned naloxone-precipitated morphine withdrawal on BDNF and its precursor protein, proBDNF. We used the conditioned place aversion (CPA) paradigm to evaluate the role of corticotropin-releasing factor (CRF)/CRF1 receptor signaling on the BDNF expression and corticosterone plasma levels after CPA expression and extinction. Male mice were rendered dependent on morphine and injected acutely with naloxone before paired to confinement in a naloxone-associated compartment. The expression of BDNF and proBDNF in the dentate gyrus (DG) and basolateral amygdala (BLA) was measured in parallel with the corticosterone plasma levels with and without CRF1 receptor blockade. Mice subjected to conditioned naloxone-induced morphine withdrawal showed an increased expression of BDNF (in DG and BLA) in parallel with an enhancement of corticosterone plasma levels. These results demonstrated that BDNF expression together with the increased activity of hypothalamic-pituitary-adrenocortical (HPA) axis are critical to the acquisition of aversive memory. However, we have observed a decrease in corticosterone plasma levels and BDNF expression after CPA extinction reaffirming the importance of BDNF in the maintenance of aversive memory. In addition, the pre-treatment with the CRF1 receptor antagonist CP-154526 before naloxone conditioning session impaired morphine withdrawal-induced aversive memory acquisition, the increased corticosterone plasma levels, and the expression of BDNF observed after CPA expression in the DG and BLA. Altogether, present results are suggesting a clear connection between HPA axis and BDNF in the formation and extinction of aversive memory.

Interactive effects of peripheral and central administration of LPS with inhibition of CRF receptors on food intake in neonatal chicks

Anorexia is a part of the acute phase response (APR). Lipopolysaccharide (LPS) is frequently used to mimic APR and induces anorexia. The mechanism underlying anorexia associated with APR in chicks is not well understood. In the present study, the possible involvement of corticotrophin-releasing factor (CRF) on anorexic effects of LPS in neonatal chicks was investigated. For this aim, different doses of LPS were administrated via both intracerebroventricular (ICV) and intraperitoneal (IP) routes in order to assess its effects on chick’s food intake. Subsequently, the effect of ICV injection of astressin, a CRF receptor antagonist, on anorexia induced by ICV and IP administration of LPS was investigated. Food intake was significantly decreased following either central or systemic administration of LPS. ICV co -injection of astressin and LPS significantly diminished anorexic effects of central LPS. However, anorexia induced by peripheral LPS was not attenuated by central injection of astressin. These data indicated that the brain CRF receptors are involved in central LPS-induced anorexia in chicks.

Localization of amyloid beta peptides to locus coeruleus and medial prefrontal cortex in corticotropin releasing factor overexpressing male and female mice.

A culmination of evidence from the literature points to the Locus Coeruleus (LC)-Norepinephrine system as an underappreciated and understudied area of research in the context of Alzheimer's Disease (AD). Stress is a risk factor for developing AD, and is supported by multiple clinical and preclinical studies demonstrating that amplification of the stress system disrupts cellular and molecular processes at the synapse, promoting the production and accumulation of the amyloid beta (Aβ42) peptide. Stress-induced activation of the LC is mediated by corticotropin releasing factor (CRF) and CRF receptors exhibit sex-biased stress signaling. Sex differences are evident in the neurochemical, morphological and molecular regulation of LC neurons by CRF, providing a compelling basis for the higher prevalence of stress-related disorders such as AD in females. In the present study, we examined the cellular substrates for interactions between Aβ and tyrosine hydroxylase a marker of noradrenergic somatodendritic processes in the LC, and Dopamine-β-Hydroxylase (DβH) in the infralimbic medial prefrontal cortex (ILmPFC) in mice conditionally overexpressing CRF in the forebrain (CRFOE) under a Doxycycline (DOX) regulated tetO promoter. CRFOE was sufficient to elicit a redistribution of Aβ peptides in the somatodendritic processes of the LC of male and female transgenic mice, without altering total Aβ42 protein expression levels. DOX treated groups exhibited lysosomal compartments with apparent lipofuscin and abnormal morphology, indicating potential dysfunction of these Aβ42-clearing compartments. In female DOX treated groups, swollen microvessels with lipid-laden vacuoles were also observed, a sign of blood-brain-barrier dysfunction. Finally, sex differences were observed in the prefrontal cortex, as females responded to DOX treatment with increased frequency of co-localization of Aβ42 and DβH in noradrenergic axon terminals compared to vehicle treated controls, while male groups showed no significant changes. We hypothesize that the observed sex differences in Aβ42 distribution in this model of CRF hypersignaling is based on increased responsivity of female rodent CRFR1 in the LC. Aβ42 production is enhanced during increased neuronal activation, therefore, the excitation of DOX treated female CRFOE LC neurons projecting to the mPFC may exhibit more frequent co-localization with Aβ due to increased neuronal activity of noradrenergic neurons.

N-3 Polyunsaturated Fatty Acids Attenuated Postpartum Depression Through MicroRNA-regulated Neuroendocrinergic and Serotonergic Mechanisms (P08-118-19)

ObjectivesThe purpose of the present study was to investigate the hypothesis that n-3 polyunsaturated fatty acids (PUFAs) had antidepressant effects through microRNA (miR)-regulated neurobiological systems in dams with postpartum depression (PPD) induced by pup separation (PS). MethodsAfter mating, the female rats were fed a modified AIN-93 G diet with 0% or 1% n-3 PUFAs relative to total energy intake during gestation and lactation periods. After birth, dams were separated from their pups for 15 min (control) or 180 min (PS) every day on postpartum day 2–14. There were four groups (n = 8/group): control, PS, n-3 PUFAs and PS + n-3 PUFAs. ResultsPS increased latencies to contact first pup and to retrieve all pup increased immobility but decreased climbing during forced swimming test, and decreased sucrose preference. PS increased activity of hypothalamic-pituitary-adrenal (HPA) axis including expression of corticotrophin releasing factor and glucocorticoid receptor, and levels of adrenocorticotropic hormone and corticosterone. PS also decreased serotonergic neurotransmission including expressions of cAMP response element binding protein (CREB), pCREB, brain-derived neurotrophic factor (BDNF) and serotonin 1A receptor, and level of serotonin. In addition, PS increased inflammation including expressions of TNF-α, IL-6, PGE2, and N-methyl-D-aspartate receptor (NMDAR) 2B. PS modified HPA-axis, serotonergic neurotransmission, inflammation and NMDAR 2B through miR-218, miR-132, miR-182 and miR-155. N-3 PUFAs modified depressive behaviors, HPA axis activity, serotonergic neurotransmission, inflammation through miR-218, miR-182 and miR-155. However, n-3 PUFAs had no effect on miR-17, miR-132, and NMDAR 2B. ConclusionsThe present study suggested that the antidepressant-like effects of n-3 PUFAs could be due to regulation of HPA axis activity, serotonergic neurotransmission and inflammation via miR-218, miR-182 and miR-155, rather than NMDAR. Funding SourcesThis work was supported by the National Research Foundation of Korea grant funded by the Korea government (NRF-2018R1A2B6002486).

Conditioned Aversion and Neuroplasticity Induced by a Superagonist of Extrasynaptic GABAA Receptors: Correlation With Activation of the Oval BNST Neurons and CRF Mechanisms.

THIP (gaboxadol), a superagonist of the δ subunit-containing extrasynaptic GABAA receptors, produces persistent neuroplasticity in dopamine (DA) neurons of the ventral tegmental area (VTA), similarly to rewarding drugs of abuse. However, unlike them THIP lacks abuse potential and induces conditioned place aversion in mice. The mechanism underlying the aversive effects of THIP remains elusive. Here, we show that mild aversive effects of THIP were detected 2 h after administration likely reflecting an anxiety-like state with increased corticosterone release and with central recruitment of corticotropin-releasing factor corticotropin-releasing factor receptor 1 (CRF1) receptors. A detailed immunohistochemical c-Fos expression mapping for THIP-activated brain areas revealed a correlation between the activation of CRF-expressing neurons in the oval nucleus of the bed nuclei of stria terminalis and THIP-induced aversive effects. In addition, the neuroplasticity of mesolimbic DA system (24 h after administration) and conditioned place aversion by THIP after four daily acute sessions were dependent on extrasynaptic GABAA receptors (abolished in δ-GABAA receptor knockout mice) and activation of the CRF1 receptors (abolished in wildtype mice by a CRF1 receptor antagonist). A selective THIP-induced activation of CRF-expressing neurons in the oval part of the bed nucleus of stria terminalis may constitute a novel mechanism for inducing plasticity in a population of VTA DA neurons and aversive behavioral states.

Corticotropin-releasing factor depolarizes rat lateral vestibular nuclear neurons through activation of CRF receptors 1 and 2

Corticotropin-releasing factor (CRF) is a neuropeptide mainly synthesized in the hypothalamic paraventricular nucleus and has been traditionally implicated in stress and anxiety. Intriguingly, genetic or pharmacological manipulation of CRF receptors affects locomotor activity as well as motor coordination and balance in rodents, suggesting an active involvement of the central CRFergic system in motor control. Yet little is known about the exact role of CRF in central motor structures and the underlying mechanisms. Therefore, in the present study, we focused on the effect of CRF on the lateral vestibular nucleus (LVN) in the brainstem vestibular nuclear complex, an important center directly contributing to adjustment of muscle tone for both postural maintenance and the alternative change from the extensor to the flexor phase during locomotion. The results show that CRF depolarizes and increases the firing rate of neurons in the LVN. Tetrodotoxin does not block the CRF-induced depolarization and inward current on LVN neurons, suggesting a direct postsynaptic action of the neuropeptide. The CRF-induced depolarization on LVN neurons was partly blocked by antalarmin or antisauvagine-30, selective antagonists for CRF receptors 1 (CRFR1) and 2 (CRFR2), respectively. Furthermore, combined application of antalarmin and antisauvagine-30 totally abolished the CRF-induced depolarization. Immunofluorescence results show that CRFR1 and CRFR2 are co-localized in the rat LVN. These results demonstrate that CRF excites the LVN neurons by co-activation of both CRFR1 and CRFR2, suggesting that via the direct modulation on the LVN, the central CRFergic system may actively participate in the central vestibular-mediated postural and motor control.

Striatal Cholinergic Interneurons Are a Novel Target of Corticotropin Releasing Factor.

Cholinergic interneurons (CINs) are critical regulators of striatal network activity and output. Changes in CIN activity are thought to encode salient changes in the environment and stimulus-response-outcome associations. Here we report that the stress-associated neuropeptide corticotropin releasing factor (CRF) produces a profound and reliable increase in the spontaneous firing of CINs in both dorsal striatum and nucleus accumbens (NAc) through activation of CRF type 1 receptors, production of cAMP and reduction in spike accommodation in male mice. The increase of CIN firing by CRF results in the activation muscarinic acetylcholine receptors type 5, which mediate potentiation of dopamine transmission in the striatum. This study provides critical mechanistic insight into how CRF modulates striatal activity and dopamine transmission in the NAc to likely account for CRF facilitation of appetitive behaviors.SIGNIFICANCE STATEMENT Although the presence of CRF receptors in the dorsal and ventral striatum has been acknowledged, the cellular identity and the functional consequences of receptor activation is unknown. Here we report that striatal cholinergic interneurons express CRF-R1 receptors and are acutely activated by the neuropeptide CRF that is released in response to salient environmental stimuli. Cholinergic interneurons make <1% of the cells in the striatum but are critical regulators of the striatal circuitry and its output. CRF's fast and potent activation of cholinergic interneurons could have far reaching behavioral implications across motivated behaviors controlled by the striatum.

Protective effects of urocortin 2 against caerulein-induced acute pancreatitis.

Because little is known about the role of corticotropin-releasing factor (CRF) agonists in regulating responses in pancreatitis, we evaluated the effects of urocortin 2 (UCN2) and stressin1 in caerulein-induced acute pancreatitis (AP) model in rats. Male rats were pretreated with UCN2 or stressin1 for 30 min followed by induction of AP with supraphysiologic doses of caerulein. Serum amylase and lipase activity, pancreatic tissue necrosis, immune cell infiltrate, nuclear factor (NF)-κB activity, trypsin levels, and intracellular Ca2+ ([Ca2+]i) were ascertained. UCN2, but not stressin1 attenuated the severity of AP in rats. UCN2, but not stressin1, reduced serum amylase and lipase activity, cell necrosis and inflammatory cell infiltration in AP. NF-κB activity in pancreatic nuclear extracts increased in AP and UCN2 treatment reduced caerulein-induced increases in NF-κB activity by 42%. UCN2 treatment prevented caerulein-induced degradation of IκB-α in the cytosolic fraction as well as increased levels of p65 subunit of NF-κB in the cytosolic fraction. Pancreatic UCN2 levels decreased in AP compared with saline. UCN2 evoked [Ca2+]i responses in primary acinar cells and abolished caerulein-evoked [Ca2+]i responses at 0.1nM, and decreased by ~50% at 1.0nM caerulein. UCN2 stimulation resulted in redistribution of a portion of F-actin from the apical to the basolateral pole. UCN2 prevented the massive redistribution of F-actin observed with supraphysiologic doses of caerulein. UCN2, but not stressin1 attenuated severity of an experimental pancreatitis model. The protective effects of UCN2, including anti-inflammatory and anti-necrotic effects involve activation of the CRF2 receptor, [Ca2+]i signaling, and inhibition of NF-κB activity.

Stress and nicotine during adolescence disrupts adult hippocampal‐dependent learning and alters stress reactivity

Adolescence represents increased susceptibility to stress that increases risk for nicotine dependence. The present study examined the interactive effects of brief exposure to stress (shipping/transportation or experimentally induced) and chronic nicotine during adolescence on cognitive function and stress reactivity in adulthood. Adolescent (P31), but not young adult (P47), C57BL/6J mice had higher levels of corticosterone after shipping vs mice bred onsite. Shipped preadolescent (P23) and adolescent (P38) mice, but not those bred onsite, exposed to nicotine showed deficits in contextual fear learning when tested in adulthood. Adult learning deficits were replicated in adolescent mice bred onsite, exposed to experimentally induced stress, and administered chronic nicotine. Stress and nicotine during adolescence resulted in higher expression of hippocampal glucocorticoid receptors and corticotropin-releasing factor receptors and blunted restraint induced CORT release in adulthood. Importantly, studies examining adolescent behavior in mice should consider stress influences outcomes.

The transcripts of CRF and CRF receptors under fasting stress in Dabry’s sturgeon (Acipenser dabryanus Dumeril)

Dabry’s sturgeon (Acipenser dabryanus Dumeril, 1868) belongs to Sturgeon and is distributed throughout the mainstream of the upper Yangtze River. While there is little research onphysiological mechanism of Dabry’s sturgeon, such as feeding regulation by the CRF system. At present, CRF is thought to regulate feeding via CRF receptors (CRF-Rs) in several mammals, but relatively few studies of CRF and feeding exist in teleosts. Herein, the transcripts of CRF and CRF-Rs under fasting stress in Dabry’s sturgeon (Acipenser dabryanus Dumeril) have been explored. A full length Dabry’s sturgeon CRF cDNA of 953 bp was identified, which contained a 447 bp open reading frame (ORF). A partial CRF-R1 cDNA of 1053 bp and CRF-R2 cDNA of 906 bp corresponding to the coding sequences (CDS) was obtained. In addition, analysis of the tissue distribution of CRF and CRF-Rs mRNAs revealed they were widely distributed in the central and peripheral nervous systems. Furthermore, periprandial (preprandial and postprandial), fasting, and re-feeding experiments revealed CRF mRNA was significantly increased 1 h and 3 h after feeding and CRF and CRF-Rs transcripts were significantly decreased after 10 days fasting, and significantly increased on re-feeding on day 10. These results suggest that CRF and CRF-Rs might regulate feeding by acting as satiety factors.

Sex Differences in Neuroplasticity- and Stress-Related Gene Expression and Protein Levels in the Rat Hippocampus Following Oxycodone Conditioned Place Preference

Prescription opioid abuse is a serious public health issue. Recently, we showed that female and male Sprague–Dawley rats acquire conditioned place preference (CPP) to the mu opioid receptor agonist oxycodone. Anatomical analysis of the hippocampus from these rats unveiled sex differences in the opioid system in a way that would support excitation and opiate associative learning processes especially in females. In this study, we examined the expression and protein densities of opioid, plasticity, stress and related kinase and signaling molecules in the hippocampus of female and male rats following oxycodone CPP. Oxycodone CPP females have: a) increases in ARC (activity regulated cytoskeletal-associated protein)-immunoreactivity (ir) in CA3 pyramidal cells; b) decreases in Npy (neuropeptide Y) gene expression in the medial hippocampus but higher numbers of NPY-containing hilar interneurons compared to males; c) increases in Crhr2 (corticotropin releasing factor receptor 2) expression in CA2/3; d) increases in Akt1 (AKT serine/threonine kinase 1) expression in medial hippocampus; and e) decreases in phosphorylated MAPK (mitogen activated protein kinase)-ir in CA1 and dentate gyrus. Oxycodone CPP males have: a) increases in Bdnf (brain derived-neurotrophic factor) expression, which is known to be produced in granule cells, relative to females; b) elevated Mapk1 expression and pMAPK-ir in the dentate hilus which harbors newly generated granule cells; and c) increases in CRHR1-ir in CA3 pyramidal cell soma. These sex-specific changes in plasticity, stress and kinase markers in hippocampal circuitry parallel previously observed sex differences in the opioid system after oxycodone CPP.

A sexually dimorphic distribution of corticotropin-releasing factor receptor 1 in the paraventricular hypothalamus

Sex differences in neural structures are generally believed to underlie sex differences reported in anxiety, depression, and the hypothalamic–pituitary–adrenal axis, although the specific circuitry involved is largely unclear. Using a corticotropin-releasing factor receptor 1 (CRFR1) reporter mouse line, we report a sexually dimorphic distribution of CRFR1 expressing cells within the paraventricular hypothalamus (PVN; males > females). Relative to adult levels, PVN CRFR1-expressing cells are sparse and not sexually dimorphic at postnatal days 0, 4, or 21. This suggests that PVN cells might recruit CRFR1 during puberty or early adulthood in a sex-specific manner. The adult sex difference in PVN CRFR1 persists in old mice (20–24 months). Adult gonadectomy (6 weeks) resulted in a significant decrease in CRFR1-immunoreactive cells in the male but not female PVN. CRFR1 cells show moderate co-expression with estrogen receptor alpha (ERα) and high co-expression with androgen receptor, indicating potential mechanisms through which circulating gonadal hormones might regulate CRFR1 expression and function. Finally, we demonstrate that a psychological stressor, restraint stress, induces a sexually dimorphic pattern of neural activation in PVN CRFR1 cells (males >females) as assessed by co-localization with the transcription/neural activation marker phosphorylated CREB. Given the known role of CRFR1 in regulating stress-associated behaviors and hormonal responses, this CRFR1 PVN sex difference might contribute to sex differences in these functions.

Chronic stress increases susceptibility to food addiction by increasing the levels of DR2 and MOR in the nucleus accumbens.

Background: Stress-related obesity might be related to the suppression of the hypothalamic-pituitary- adrenocortical axis and dysregulation of the metabolic system. Chronic stress also induces the dysregulation of the reward system and increases the risk of food addiction, according to recent clinical findings. However, few studies have tested the effect of chronic stress on food addiction in animal models.Purpose: The objective of this study was to identify whether chronic stress promotes food addiction or not and explore the possible mechanisms.Method: We applied adaily 2 hrsflashing LED irradiation stress to mice fed chow or palatable food to mimic the effect of chronic stress on feeding. After 1 month of chronic stress exposure, we tested their binge eating behaviors, cravings for palatable food, responses for palatable food, and compulsive eating behaviors to evaluate the effect of chronic stress on food addiction-like behaviors. We detected changes in the levels of various genes and proteins in the nucleus accumbens (NAc), ventral tegmental area (VTA) and lateral hypothalamus using qPCR and immunofluorescence staining, respectively.Results: Behaviors results indicated chronic stress obviously increased food addiction score (FAS) in the palatable food feeding mice. Moreover, the FAS had astrong relationship with the extent of the increase in body weight. Chronic stress increased the expression of corticotropin-releasing factor receptor 1(CRFR1) was increased in the NAc shell and core but decreased in the VTA of the mice fed with palatable food. Chronic stress also increased expression of both dopamine receptor 2 (DR2) and mu-opioid receptor (MOR) in the NAc.Conclusion: Chronic stress aggravates the FAS and contributed to the development of stress-related obesity. Chronic stress drives the dysregulation of the CRF signaling pathway in the reward system and increases the expression of DR2 and MOR in the nucleus accumbens.

Molecular, Morphological, and Functional Characterization of Corticotropin-Releasing Factor Receptor 1-Expressing Neurons in the Central Nucleus of the Amygdala.

The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF, Crh) acting at cognate type 1 receptors (CRF1, Crhr1) modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1–) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1– neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1– neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1– neurons, which may have important implications for the transduction of CRF signaling in the CeA.

Role of the posterodorsal medial amygdala in predator odour stress-induced puberty delay in female rats.

Puberty onset is influenced by various factors, including psychosocial stress. The present study investigated cat‐odour stress on puberty onset and oestrous cyclicity in rats. Female weanling rats were exposed to either soiled cat litter or fresh unused litter for 10 consecutive days. Following vaginal opening (VO), rats were smeared for 14 days to determine oestrous cyclicity. Anxiety‐like behaviour was assessed using standard anxiety tests. Brains were collected to determine corticotrophin‐releasing factor (CRF), CRF receptor 1 (CRF‐R1) and CRF receptor 2 (CRF‐R2) mRNA in the paraventricular nucleus (PVN), as well as the central nucleus of the amygdala (CEA) and the medial nucleus of the amygdala (MEA). Cat odour delayed VO and first oestrus, disrupted oestrous cycles and caused anxiogenic responses. Cat odour elicited increased CRF mRNA expression in the PVN but not in the CeA. CRF‐R1 and CRF‐R2 mRNA levels in the PVN and CeA were unaffected by cat odour; however, CRF‐R1 mRNA levels were decreased in the MeA. The role of CRF signalling in the MeA, particularly its posterodorsal subnucleus (MePD), with respect to pubertal timing was directly examined by unilateral intra‐MePD administration of CRF (0.2 nmol day‐1 for 14 days) via an osmotic mini‐pump from postnatal day 24 and was shown to delay VO and first oestrus. These data suggest that CRF signalling in the MePD may be associated with predator odour‐induced puberty delay.

SUN-479 Role of β-arrestin2 in ERK 1/2 Activation Mediated by Corticotropin-Releasing Factor Receptor Type 1

The Corticotropin-Releasing Factor (CRF) is a neuropeptide released by the hypothalamus in response to stress. CRF activates the CRF1 and CRF2 receptors, which are members of the G protein-coupled receptor (GPCR) superfamily. Although it has been shown that the CRF1 receptor activates ERK 1/2 cascade in different cell models [1] however, to date is not well understood the roles of upstream pathways associated with this effects. β-arrestin2 is an adapter protein that participates in the regulation of GPCRs and has important functions in cell signaling by mediating activation of multiple transductional pathways, including Src and MAPK [2]. In the present work, we investigated the role of β-arrestin2 in the activation of MAPK by induction of 100 nM CRF. We used mouse embryonic fibroblast cells that lack β-arrestin2 (MEF KO-β-arrestin2), transfected with the CRF1 receptor that lack β-arrestin2 (MEF KO-β-arrestin2) as model. Our data revealed that β-arrestin2 is involved in the activation of ERK 1/2 since we detected lower (P<0.05) phosphorylation of ERK 1/2 in the KO-β-arrestin2 cells after 5 min CRF of stimulationCRF. Using MEF cells from double-knockout mice, lacking β-arrestin1 and β-arrestin2 (MEF 2KO-β-arrestin1/2), we found that β-arrestin1 is not involved in ERK 1/2 activation induced by CRF because there was no change on phosphorylation of ERK 1/2 between MEF KO-β-arrestin2 and 2KO-β-arrestin1/2 cells. The pretreatment with the EGFR inhibitor AG1478 promoted a decreased (P<0.05) ERK 1/2 phosphorylation in control cells. Interestingly, no EGFR activation was detected in the KO-β-arrestin2 cells. These results indicate that β-arrestin2 is necessary for the CRF1 receptor-mediated EGFR transactivation mechanism that induces ERK 1/2 phosphorylation. We also found that the transactivation of EGFR by CRF stimulation is independent of metalloproteinase activity through the pretreatment with the metalloproteinase inhibitor GM6001. To determine the role of Src in the CRF-mediated ERK activation, we pretreated the cells with the Src-specific inhibitor PP2. We observed that PP2 promoted a significant decrease (P<0.05) in CRF-mediated ERK activation. We also found a reduction (P<0.05) in Src phosphorylation at Y416 in MEF KO-β-arrestin2 cells stimulated with 5 min of CRF, suggesting that β-arrestin2 is involved in Src activation. Together, our data demonstrate that β-arrestin2 is an important player during the CRF1-dependent activation of the ERK 1/2 pathway. Reference: [1]Hauger et al., Ann. N. Y. Acad. Sci. (2009) 1179: 120-143.[2]Luttrell et al., Proc. Natl. Acad. Sci. (2001) 98: 2449-2454Sources of Research Support: UC MEXUS-CONACYT grant for collaborative projects to JAO-R and RLH; CONACYT grant to JAO-R; Merit Review grant; MIRECC of VISN22, and CESMH from the Department of VA; NIH grant to RLH. GKP-M was supported by CONACYT fellowship grant.

Increased protein and mRNA expression of corticotropin-releasing factor (CRF), decreased CRF receptors and CRF binding protein in specific postmortem brain areas of teenage suicide subjects

Overactivity of hypothalamic-pituitary-adrenal (HPA) axis function has been implicated in depression and suicidal behavior. This is based on the observation of an abnormal dexamethasone (DEX) and DEX-adrenocorticotropic hormone (ACTH) test in patients with depression and suicidal behavior. Recently, some studies have also found abnormalities of glucocorticoid receptors (GR), mineralocorticoid receptors (MR), corticotropin releasing factor (CRF), CRF receptors (CRF-R) and CRF binding protein (CRF-BP) in depressed and suicidal patients. Some investigators have also observed increased levels of CRF in the cerebrospinal fluid (CSF) and altered levels of MR, GR and CRF in the postmortem brain of depressed and suicidal subjects. We have earlier reported decreased protein and mRNA expression of GR and GILZ, a chaperone protein, in the postmortem brain of teenage suicide subjects. We have further studied CRF and its receptors in different areas of the postmortem brain of suicide subjects, i.e., the prefrontal cortex (PFC), hippocampus (HIPPO), subiculum and amygdala (AMY) from teenage suicide subjects. The CRF and its receptors were determined in the PFC (Brodmann area 9), HIPPO, subiculum and different amygdaloid nuclei from 24 normal control subjects and 24 teenage suicide subjects. Protein expression of CRF, its receptors and CRF-BP was determined by immunolabeling using the Western blot technique and mRNA expression was determined by real-time PCR (qPCR) technique. We found that the mRNA levels of CRF were significantly increased in the PFC, in the central amygdaloid nucleus (CeAMY) and in the subiculum. mRNA levels of CRF-R1 and CRF-BP were significantly decreased in the PFC. We did not find any changes in the HIPPO of any of the CRF components we studied. When we compared the protein expression of CRF components we found that CRF was significantly increased and CRF-R1, CRF-R2 and CRF-BP significantly decreased in the PFC. On the other hand, there were no changes in the protein expression of CRF components in the HIPPO. Our results in the postmortem brain suggest that, as found by clinical studies in the CSF, there are significant alterations of CRF and its receptors in the postmortem brain of teenage suicide subjects. These alterations of CRF and its components were region-specific, as changes were not generally observed in the HIPPO.

Helicobacter pylori Vacuolating Cytotoxin A Causes Anorexia and Anxiety via Hypothalamic Urocortin 1 in Mice

Helicobacter pylori (Hp) infection is related to the pathogenesis of chronic gastric disorders and extragastric diseases. Here, we examined the anorexigenic and anxiogenic effects of Hp vacuolating cytotoxin A (VacA) through activation of hypothalamic urocortin1 (Ucn1). VacA was detected in the hypothalamus after peripheral administration and increased Ucn1 mRNA expression and c-Fos-positive cells in the hypothalamus but not in the nucleus tractus solitarius. c-Fos and Ucn1-double positive cells were detected. CRF1 and CRF2 receptor antagonists suppressed VacA-induced anxiety and anorexia, respectively. VacA activated single paraventricular nucleus neurons and A7r5 cells; this activation was inhibited by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. VacA causes anorexia and anxiety through the intracellular PLC-PKC pathway, migrates across the blood-brain barrier, and activates the Ucn1-CRF receptor axis.