Corticotropin-releasing Hormone Cells Research Articles

ACE2 overexpression in corticotropin-releasing-hormone cells offers protection against pulmonary hypertension.

Pulmonary hypertension (PH), characterized by elevated pulmonary pressure and right heart failure, is a systemic disease involving inappropriate sympathetic activation and an impaired gut-brain-lung axis. Global overexpression of angiotensin converting enzyme 2 (ACE2), a cardiopulmonary protective enzyme of the renin-angiotensin system, attenuates PH induced by chronic hypoxia. Neurons within the paraventricular nucleus of the hypothalamus (PVN) that synthesize corticotropin-releasing hormone (CRH) are activated by stressors, like hypoxia, and this activation augments sympathetic outflow to cardiovascular tissues. These data coupled with our observations that ACE2 overexpression in CRH cells (CRH-ACE2KI mice) decreases anxiety-like behavior via suppression of hypothalamic-pituitary-adrenal (HPA) axis activity by decreasing CRH synthesis, led us to hypothesize that selective ACE2 overexpression in CRH neurons would protect against hypoxia-induced PH. CRH-ACE2KI and WT male and female mice were exposed to chronic hypoxia (10%O2) or normoxia (21%O2) for 4 weeks in a ventilated chamber with continuous monitoring of oxygen and carbon dioxide concentrations (n = 7-10/group). Pulmonary hemodynamics were measured with Millar pressure catheters then tissues were collected for histological analyses. Chronic hypoxia induced a significant increase (36.4%) in right ventricular (RV) systolic pressure (RVSP) in WT mice, which was not observed in CRH-ACE2KI mice. No significant differences in RVSP were observed between male and female mice in any of the groups. Overexpression of ACE2 in CRH cells was protective against hypoxia-induced PH. Since the majority of expression of CRH is in brain nuclei such as paraventricular nucleus of the hypothalamus (PVN) and/or central nucleus of the amygdala (CeA) these data indicate that the protective effects of ACE2 are, at least in part, centrally mediated. This contributes to the systemic nature of PH disease and that CRH neurons may play an important role in PH.

Activated Wake Systems in Narcolepsy Type 1.

Narcolepsy type 1 (NT1) is assumed to be caused solely by a lack of hypocretin (orexin) neurotransmission. Recently, however, we found an 88% reduction in corticotropin-releasing hormone (CRH)-positive neurons in the paraventricular nucleus (PVN). We assessed the remaining CRH neurons in NT1 to determine whether they co-express vasopressin (AVP) to reflect upregulation. We also systematically assessed other wake-systems, since current NT1 treatments target histamine, dopamine, and norepinephrine pathways. In postmortem tissue of people with NT1 and matched controls, we immunohistochemically stained and quantified neuronal populations expressing: CRH and AVP in the PVN, and CRH in the Barrington nucleus; the key neuronal histamine-synthesizing enzyme, histidine decarboxylase (HDC) in the hypothalamic tuberomammillary nucleus (TMN); the rate-limited-synthesizing enzyme, tyrosine hydroxylase (TH), for dopamine in the mid-brain and for norepinephrine in the locus coeruleus (LC). In NT1, there was: a 234% increase in the percentage of CRH cells co-expressing AVP, while there was an unchanged integrated optical density of CRH staining in the Barrington nucleus; a 36% increased number of histamine neurons expressing HDC, while the number of typical human TMN neuronal profiles was unchanged; a tendency toward an increased density of TH-positive neurons in the substantia nigra compacta; while the density of TH-positive LC neurons was unchanged. Our findings suggest an upregulation of activity by histamine neurons and remaining CRH neurons in NT1. This may explain earlier reports of normal basal plasma cortisol levels but lower levels after dexamethasone suppression. Alternatively, CRH neurons co-expressing AVP neurons are less vulnerable. ANN NEUROL 2023;94:762-771.

Windows into stress: a glimpse at emerging roles for CRHPVN neurons.

The corticotropin-releasing hormone cells in the paraventricular nucleus of the hypothalamus (CRHPVN) control the slow endocrine response to stress. The synapses on these cells are exquisitely sensitive to acute stress, leveraging local signals to leave a lasting imprint on this system. Additionally, recent work indicates that these cells also play key roles in the control of distinct stress and survival behaviors. Here we review these observations and provide a perspective on the role of CRHPVN neurons as integrative and malleable hubs for behavioral, physiological, and endocrine responses to stress.

Effects of Growth Hormone Receptor Ablation in Corticotropin-Releasing Hormone Cells.

Corticotropin-releasing hormone (CRH) cells are the dominant neuronal population responsive to the growth hormone (GH) in the paraventricular nucleus of the hypothalamus (PVH). However, the physiological importance of GH receptor (GHR) signaling in CRH neurons is currently unknown. Thus, the main objective of the present study was to investigate the consequences of GHR ablation in CRH-expressing cells of male and female mice. GHR ablation in CRH cells did not cause significant changes in body weight, body composition, food intake, substrate oxidation, locomotor activity, glucose tolerance, insulin sensitivity, counterregulatory response to 2-deoxy-D-glucose and ghrelin-induced food intake. However, reduced energy expenditure was observed in female mice carrying GHR ablation in CRH cells. The absence of GHR in CRH cells did not affect anxiety, circadian glucocorticoid levels or restraint-stress-induced corticosterone secretion and activation of PVH neurons in both male and female mice. In summary, GHR ablation, specifically in CRH-expressing neurons, does not lead to major alterations in metabolism, hypothalamic–pituitary–adrenal axis, acute stress response or anxiety in mice. Considering the previous studies showing that central GHR signaling regulates homeostasis in situations of metabolic stress, future studies are still necessary to identify the potential physiological importance of GH action on CRH neurons.

Programming of Stress-Sensitive Neurons and Circuits by Early-Life Experiences.

Early-life experiences influence brain structure and function long-term, contributing to resilience or vulnerability to stress and stress-related disorders. Therefore, understanding the mechanisms by which early-life experiences program specific brain cells and circuits to shape life-long cognitive and emotional functions is crucial. We identify the population of corticotropin-releasing hormone (CRH)-expressing neurons in the hypothalamic paraventricular nucleus (PVN) as a key, early target of early-life experiences. Adverse experiences increase excitatory neurotransmission onto PVN CRH cells, whereas optimal experiences, such as augmented and predictable maternal care, reduce the number and function of glutamatergic inputs onto this cell population. Altered synaptic neurotransmission is sufficient to initiate large-scale, enduring epigenetic re-programming within CRH-expressing neurons, associated with stress resilience and additional cognitive and emotional outcomes. Thus, the mechanisms by which early-life experiences influence the brain provide tractable targets for intervention.

Carcinoid tumors of the thymus and Cushing's syndrome: Clinicopathologic features and current best evidence regarding the cell of origin of these unusual neoplasms

It is uncertain whether thymic neuroendocrine tumors (NET) associated with Cushing's syndrome (CS) produce corticotropin-releasing hormone (CRH) and adrenocorticotropin hormone (ACTH) and whether the thymus contains ACTH and/or CRH cells that could originate NET.The clinicopathologic features of 5 typical (TC) and 6 atypical carcinoids (ATC), 10 additional non-neoplastic thymi, 6 adrenal glands with bilateral nodular hyperplasia and 8 adrenal cortical adenomas were reviewed. Representative slides were immunostained for ACTH and CRH. Four (36.4%) of the 11 patients had CS. The incidence of Masaoka stage IV was higher (p < 0.0001) in patients with ATC than TC. Only 2 (18.1%) of the 11 patients were alive at follow-up. Ten NET were CRH immunoreactive and 6 were ACTH immunoreactive. Thymic NET with CS exhibited stronger immunoreactivity for ACTH and CRH than those without CS. Non-neoplastic thymi exhibited scattered ACTH and CRH immunoreactive cells. Normal adrenal cortex and glands with bilateral nodular hyperplasia showed diffuse CRH immunoreactivity while adrenal adenomas showed no or only focal CRH immunoreactivity. Literature review showed no association between thymic NET and adrenal adenomas.The thymus contains CRH and ACTH immunoreactive cells that are probably the origin of thymic NET. Neoplasms associated with CS exhibit strong immunoreactivity for both hormones, suggesting that CRH probably plays a role in the pathogenesis of CS. As adrenals with bilateral nodular hyperplasia exhibit diffuse CRH immunoreactivity and adrenal cortical adenomas either lack this finding or show few immunoreactive cells, this marker may be useful to distinguish these lesions.

2429 Optogenetic stimulation of corticotropin-releasing hormone expressing neurons in Barrington’s nucleus recapitulates the social stress voiding phenotype in mice

OBJECTIVES/SPECIFIC AIMS: Voiding postponement is common cause of LUT dysfunction in which children void infrequently with large volumes. This condition is modeled in mice that are subjected to social stress who show decreased voiding frequency and increased voided volumes along with increases in corticotropin-releasing hormone (CRH) expression in Barrington’s nucleus (BN) (i.e., the pontine micturition center). Optogenetics is a technique to selectively stimulate cells or neurons of interest via light activated channel receptors [i.e., channel-2 rhodopsin (ChR2)]. Here we examined the effects of optogenetic manipulation of CRH BN neurons on the in vivo voiding phenotype and urodynamics in awake mice. We hypothesized that stimulating these neurons at higher frequencies (10–50 Hz) would lead to CRH-dependent alterations in voiding phenotype (i.e., larger voided volumes and longer intermicturition intervals. METHODS/STUDY POPULATION: Double transgenic mice expressing ChR2 in CRH cells were generated using the Cre-lox recombinase system and had fiberoptic probes implanted into BN at 8 weeks of age. The mice also underwent simultaneous catheter placement into the bladder for in vivo cystometry. In vivo cystometry before and during optogenetic stimulation at various frequencies was performed 5–7 days postoperatively. Saline was perfused at 10 µL/minute and baseline stable voiding cycles were established. Bladder capacity, threshold pressure, voiding pressure, and voided volume were recorded at baseline and at each optogenetic setting. In some mice, the protocol was repeated in the presence of CRH antagonist (NBI 30775). RESULTS/ANTICIPATED RESULTS: Fiberoptic stimulation (470 nm at 25 and 50 Hz) produced a significant rise in the intermicturition interval, bladder capacities and increased void volumes. This effect was especially pronounced in females in whom bladder capacity and intermicturition interval more than doubled at 50 Hz stimulation. Fluoroscopic images confirmed complete bladder emptying with each void. The increased bladder capacity at higher frequencies (25 and 50 Hz) was CRH-dependent as injection of a CRH antagonist (NBI 30775) blocked the optogenetic effect. Control non-double mice showed no effects from optogenetic stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that optogenetic stimulation of CRH-expressing neurons in BN at high frequency (25 and 50 Hz) inhibits micturition and recapitulates the voiding phenotype seen in socially stressed mice (large, infrequent voids). Lower frequencies of optogenetic stimulation (2 and 10 Hz) had no effects on cystometry parameters or voiding phenotype. In addition, females had a greater response to optogenetic stimulation compared with males with larger bladder capacities and longer intermicturition intervals. The changes in voiding phenotype seen were CRH dependent as blockage of the CRH receptor prevented changes in cystometry parameters with optogenetic stimulation. Further elucidation of these and other neural subpopulations in BN are warranted to understand micturition and how it may be manipulated in disease states such as voiding postponement and acute urinary retention.

Convergence and Divergence of CRH Amacrine Cells in Mouse Retinal Circuitry.

Inhibitory interneurons sculpt the outputs of excitatory circuits to expand the dynamic range of information processing. In mammalian retina, >30 types of amacrine cells provide lateral inhibition to vertical, excitatory bipolar cell circuits, but functional roles for only a few amacrine cells are well established. Here, we elucidate the function of corticotropin-releasing hormone (CRH)-expressing amacrine cells labeled in Cre-transgenic mice of either sex. CRH cells costratify with the ON alpha ganglion cell, a neuron highly sensitive to positive contrast. Electrophysiological and optogenetic analyses demonstrate that two CRH types (CRH-1 and CRH-3) make GABAergic synapses with ON alpha cells. CRH-1 cells signal via graded membrane potential changes, whereas CRH-3 cells fire action potentials. Both types show sustained ON-type responses to positive contrast over a range of stimulus conditions. Optogenetic control of transmission at CRH-1 synapses demonstrates that these synapses are tuned to low temporal frequencies, maintaining GABA release during fast hyperpolarizations during brief periods of negative contrast. CRH amacrine cell output is suppressed by prolonged negative contrast, when ON alpha ganglion cells continue to receive inhibitory input from converging OFF-pathway amacrine cells; the converging ON- and OFF-pathway inhibition balances tonic excitatory drive to ON alpha cells. Previously, it was demonstrated that CRH-1 cells inhibit firing by suppressed-by-contrast (SbC) ganglion cells during positive contrast. Therefore, divergent outputs of CRH-1 cells inhibit two ganglion cell types with opposite responses to positive contrast. The opposing responses of ON alpha and SbC ganglion cells are explained by differing excitation/inhibition balance in the two circuits.SIGNIFICANCE STATEMENT A goal of neuroscience research is to explain the function of neural circuits at the level of specific cell types. Here, we studied the function of specific types of inhibitory interneurons, corticotropin-releasing hormone (CRH) amacrine cells, in the mouse retina. Genetic tools were used to identify and manipulate CRH cells, which make GABAergic synapses with a well studied ganglion cell type, the ON alpha cell. CRH cells converge with other types of amacrine cells to tonically inhibit ON alpha cells and balance their high level of excitation. CRH cells diverge to different types of ganglion cell, the unique properties of which depend on their balance of excitation and inhibition.

Coupling corticotropin-releasing-hormone and angiotensin converting enzyme 2 dampens stress responsiveness in male mice

This study used mice to evaluate whether coupling expression of corticotropin-releasing hormone (CRH) and angiotensin converting enzyme 2 (ACE2) creates central interactions that blunt endocrine and behavioral responses to psychogenic stress. Central administration of diminazene aceturate, an ACE2 activator, had no effect on restraint-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis; however, mice that ubiquitously overexpress ACE2 had reduced plasma corticosterone (CORT) and pituitary expression of POMC mRNA. The Cre-LoxP system was used to restrict ACE2 overexpression to CRH synthesizing cells and probe whether HPA axis suppression was the result of central ACE2 and CRH interactions. Within the paraventricular nucleus of the hypothalamus (PVN), mice with ACE2 overexpression directed to CRH had a ≈2.5 fold increase in ACE2 mRNA, which co-localized with CRH mRNA. Relative to controls, mice overexpressing ACE2 in CRH cells had a decreased CORT response to restraint as well as decreased CRH mRNA in the PVN and CEA and POMC mRNA in the pituitary. Administration of ACTH similarly increased plasma CORT, indicating that the blunted HPA axis activation that accompanies ACE2 overexpression in CRH cells is centrally mediated. Anxiety-like behavior was assessed to determine whether the decreased HPA axis activation was predictive of anxiolysis. Mice with ACE2 overexpression directed to CRH cells displayed decreased anxiety-like behavior in the elevated plus maze and open field when compared to that of controls. Collectively, these results suggest that exogenous ACE2 suppresses CRH synthesis, which alters the central processing of psychogenic stress, thereby blunting HPA axis activation and attenuating anxiety-like behavior.

Preimplantation maternal stress impairs embryo development by inducing oviductal apoptosis with activation of the Fas system.

What are the mechanisms by which the preimplantation restraint stress (PIRS) impairs embryo development and pregnancy outcome? PIRS impairs embryo development by triggering apoptosis in mouse oviducts and embryos,and this involves activation of the Fas system. Although it is known that the early stages of pregnancy are more vulnerable than later stages to prenatalstress, studies on the effect of preimplantation stress on embryo developmentare limited. Furthermore, the mechanisms by which psychological stress impairs embryo development are largely unknown. These issues are worth exploring using the mouse PIRS models because restraint of mice is an efficient experimental procedure developed for studies of psychogenic stress. Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in FasL in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female and male mice were used 8-10 weeks and 10-12 weeks after birth, respectively. Female mice showing vaginal plugs were paired by weight and randomly assigned to restraint treatments or as controls. For restraint treatment, an individual mouse was put in a micro-cage with food and water available. Control mice remained in their cages with food and water during the time treated females were stressed. Female mice were exposed to PIRS for 48 h starting from 16:00 on the day of vaginal plug detection. At the end of PIRS, levels of glucorticoids (GC), corticotropin-releasing hormone (CRH)and redox potential were measured in serum, while levels of GC, GC receptor (GR), CRH, CRH receptor (CRHR), Fas and Fas ligand (FasL) protein, mRNAs for brain derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), oxidative stress (OS) and apoptosis were examined in oviducts. Preimplantation development and levels of GR, Fas, redox potential and apoptosis were observed in embryos recovered at different times after the initiation of PIRS. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts. Compared to those in control mice, while the number of blastocysts/mouse (5.0±0.7 versus 11.1±0.5), cell number/blastocyst (49.1±1.3 versus 61.5±0.9), percentages of term pregnancy (37.5% versus 90.9%) and litter size (3.7±0.1versus 9.6±0.6) decreased, blood CRH (560±23 versus 455±37 pg/ml), cortisol (27.3±3.4 versus 5±0.5 ng/ml) and OS index (OSI: 2.8 versus 1.7) increased significantly (all P<0.05) following PIRS. In the oviduct, while levels of CRH (1175±85 versus 881±33 pg/100 mg), cortisol (28.9±1.7 versus14±4 ng/g), CRHR (2.3±0.3 versus 1.0±0.0), FasL (1.31±0.06 versus 1.08±0.05 ng/g), Fas (1.42±0.13 versus 1.0±0.0) and apoptotic cells (19.1±0.5% versus 8.4±0.4%) increased, levels of GR proteins (0.67±0.14 versus 1.0±0.0) and Igf-1 (0.6±0.09 versus 1.0±0.0) and Bdnf (0.73±0.03 versus 1.0±0.0) mRNAs decreased significantly (all P<0.05 versus control) after PIRS. Mouse embryos expressed GR and Fas at all stages of preimplantation development and embryo OS (GSH/GSSG ratio: 0.88±0.03 versus 1.19±0.13) and annexin-positive cells (blastocysts: 31.4±3.8% versus 10.96±3.4%) increased significantly (P<0.05) following PIRS. Furthermore, the detrimental effects of PIRS on embryo development and oviductal apoptosis were much reduced in gld mice. Thus, PIRS triggered apoptosis in oviductal cells with activation of the Fas/FasL system. The apoptotic oviductal cells promoted embryo apoptosis with reduced production of IGF-1 and BDNF and increased production of FasL. Although important, the conclusions were drawn from limited results obtained using a single model in one species and thus they need further verification using other models and/or in other species. Furthermore, as differences in stressed samples were modest and sometimes not significant between gld and wild-type mice whereas differences between control and stressed samples were always present within gld mice, it is deduced that signaling pathways other than the Fas/FasL system might be involved as well in the PIRS-triggered apoptosis of oviducts and embryos. The data are important for studies on the mechanisms by which psychological stress affects female reproduction, as FasL expression has been observed in human oviduct epithelium. Not applicable. This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

The Severity of Acute Stress Is Represented by Increased Synchronous Activity and Recruitment of Hypothalamic CRH Neurons.

Stressors trigger adaptive responses in the body that are essential for survival. How the brain responds to acute stressors of varying intensity in an intact animal, however, is not well understood. We address this question using two-photon Ca(2+) imaging in larval zebrafish with transgenically labeled corticotropin-releasing hormone (CRH) cells, which represent a major regulator of the stress axis. We show that stressor strength-dependent responses of CRH neurons emerge via an intensity-dependent increase in the activity of individual CRH cells, and by an increase in the pool of responsive CRH cells at the population level. Furthermore, we report striking synchronicity among CRH neurons even across hemispheres, which suggests tight intrahypothalamic and interhypothalamic coordination. Thus, our work reveals how CRH neurons respond to different levels of acute stress in vivo.

Activation of the mouse odorant receptor 37 subsystem coincides with a reduction of novel environment-induced activity within the paraventricular nucleus of the hypothalamus.

Within the main olfactory system of mammals, a unique subsystem exists that is comprised of sensory neurons expressing odorant receptors (ORs) of the OR37 subfamily. These receptors are exclusive for mammals and are highly conserved across species. The mouse OR37 receptor subtypes A, B and C were shown to be activated by the long-chain aliphatic aldehydes pentadecanal, hexadecanal and heptadecanal, respectively. The search for biological sources of these compounds showed that bodily secretions from conspecifics activated the OR37A, B and C glomerulus. At the same time, the activity of cells in a target region of projection neurons from OR37 glomeruli, the paraventricular nucleus of the hypothalamus (PVN), was reduced compared with controls (clean test box). A large number of the activated cells in the PVN of mice that were placed into a clean test box were corticotropin-releasing hormone cells, indicating an induction of the stress axis due to the novel environment. The much lower number of activated cells of mice in a box enriched with bodily secretions from conspecifics indicated a reduced stress response. As bodily secretions from conspecifics activated the OR37 system and simultaneously reduced stress-induced activation of the PVN, it was tested whether the ligands for OR37 receptors could induce this effect. Indeed, a similarly reduced activity in the PVN was found in mice kept in a clean test box and exposed to a mixture of the OR37 ligands delivered via an air stream. These data indicate that the OR37 system may play a role in mediating a phenomenon called social buffering.

Neurotensin and CRH Interactions Augment Human Mast Cell Activation

Stress affects immunity, but the mechanism is not known. Neurotensin (NT) and corticotropin-releasing hormone (CRH) are secreted under stress in various tissues, and have immunomodulatory actions. We had previously shown that NT augments the ability of CRH to increase mast cell-dependent skin vascular permeability in rodents. Here we show that NT triggered human mast cell degranulation and significantly augmented CRH-induced vascular endothelial growth factor (VEGF) release. Investigation of various signaling molecules indicated that only NF-κB activation was involved. These effects were blocked by pretreatment with the NTR antagonist SR48692. NT induced expression of CRH receptor-1 (CRHR-1), as shown by Western blot and FACS analysis. Interestingly, CRH also induced NTR gene and protein expression. These results indicate unique interactions among NT, CRH, and mast cells that may contribute to auto-immune and inflammatory diseases that worsen with stress.

Synaptic regulation of the hypothalamic-pituitary-adrenal axis and its modulation by glucocorticoids and stress.

Dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis has been implicated in a range of affective and stress-related disorders. The regulatory systems that control HPA activity are subject to modulation by environmental influences, and stressful life events or circumstances can promote subsequent HPA dysregulation. The brain is a major regulator of the HPA axis, and stress-induced plasticity of the neural circuitry involved in HPA regulation might constitute an etiological link between stress and the development of HPA dysregulation. This review focuses on the synaptic regulation of neuroendocrine corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus, which are the cells through which the brain predominantly exerts its influence on the HPA axis. CRH neuronal activity is largely orchestrated by three neurotransmitters: GABA, glutamate, and norepinephrine. We discuss our current understanding of the neural circuitry through which these neurotransmitters regulate CRH cell activity, as well as the plastic changes in this circuitry induced by acute and chronic stress and the resultant changes in HPA function.

β‐Endorphin Neuronal Cell Transplant Reduces Corticotropin Releasing Hormone Hyperresponse to Lipopolysaccharide and Eliminates Natural Killer Cell Functional Deficiencies in Fetal Alcohol Exposed Rats

Natural killer (NK) cell dysfunction is associated with hyperresponse of corticotropin releasing hormone (CRH) to immune challenge and with a loss of beta-endorphin (BEP) neurons in fetal alcohol exposed animals. Recently, we established a method to differentiate neural stem cells into BEP neurons using cyclic adenosine monophosphate (cAMP)-elevating agents in cultures. Hence, we determined whether in vitro differentiated BEP neurons could be used for reversing the compromised stress response and immune function in fetal alcohol exposed rats. To determine the effect of BEP neuron transplants on NK cell function, we implanted in vitro differentiated BEP neurons into the paraventricular nucleus of pubertal and adult male rats exposed to ethanol or control in utero. The functionality of transplanted BEP neurons was determined by measuring proopiomelanocortin (POMC) gene expression in these cells and their effects on CRH gene expression under basal and after lipopolysaccaride (LPS) challenge. In addition, the effectiveness of BEP neurons in activating NK cell functions is determined by measuring NK cell cytolytic activity and interferon-gamma (IFN-gamma) production in the spleen and in the peripheral blood mononuclear cell (PBMC) following cell transplantation. We showed here that when these in vitro differentiated BEP neurons were transplanted into the hypothalamus, they maintain biological functions by producing POMC and reducing the CRH neuronal response to the LPS challenge. BEP neuronal transplants significantly increased NK cell cytolytic activity in the spleen and in the PBMC and increased plasma levels of IFN-gamma in control and fetal alcohol exposed rats. These data further establish the BEP neuronal regulatory role in the control of CRH and NK cell cytolytic function and identify a possible novel therapy to treat stress hyperresponse and immune deficiency in fetal alcohol exposed subjects.

Corticotropin-releasing hormone and the blood-brain-barrier

Increased blood-brain-barrier (BBB) permeability precedes any clinical or pathologic signs and is critical in the pathogenesis of multiple sclerosis (MS) and brain metastases. CD4+ TH1 cells mediate demyelination in MS, but how they get sensitized and enter the brain to induce brain inflammation remains obscure. TH2 cytokines associated with allergic disorders have recently been implicated in MS, while genes upregulated in MS plaques include the mast cell-specific tryptase, the IgE receptor (Fc-epsilon-RI) and the histamine-1 receptor. Mast cell specific tryptase is elevated in the CSF of MS patients, induces microvascular leakage and stimulates protease-activated receptors (PAR), leading to widespread inflammation. BBB permeability, MS and brain metastases appear to worsen in response to acute stress that leads to the local release of corticotropin-releasing hormone (CRH), which activates brain mast cells to selectively release IL-6, IL-8 and vascular endothelial growth factor (VEGF). Acute stress increases BBB permeability that is dependent on CRH and mast cells. Acute stress shortens the time of onset of experimental alleric encephalomyelitis (EAE) that does not develop in W/W mast cell deficient or CRH -/- mice. Brain mast cell inhibition and CRHR antagonists offer novel therapeutic possibilities.

Corticotropin-releasing hormone neurons in the paraventricular nucleus of the human hypothalamus in subjects with normal and abnormal sex hormone status

In order to determine the role of peripheral sex hormone levels in the expression of corticotropin-releasing hormone (CRH) neurons, we investigated by means of immunocytochemistry the number of CRH neurons in the hypothalamic paraventricular nucleus (PVN) in postmortem material of young and old subjects with normal and abnormal sex hormone conditions. Clinical data and nuclear androgen receptor (AR) immunoreactivity (ir) in the hypothalamic medial mamillary nucleus (MMN) were used as an estimate for changes in circulating sex hormone levels. It was confirmed that nuclear AR-ir staining in the MMN is a good estimate for abnormal peripheral androgen levels and that the CRH neurons in the hypothalamic PVN are activated with age in male subjects. Male subjects with lower testosterone levels due to castration showed significantly fewer CRH neurons than age-matched male controls. Castrated male-to-female (MF) transsexuals with estrogen replacement showed normal numbers of CRH neurons, while one male case, which had high estrogen levels due to an estrogen-producing tumor, even showed a high number of CRH neurons in the PVN. Results of the present study suggested that the activation of CRH neurons during aging in male subjects results from factors other than changes in endogenous peripheral sex hormone levels. Since estrogens can prevent the drop of CRH cell number following castration in MF transsexuals, and more CRH neurons were found in the case of an estrogen-producing tumor, estrogen seems to play a stimulating role in the activity of CRH neurons in the PVN. The stimulatory effect of androgens on CRH neurons seems to occur via an indirect effect following aromatization into estrogens.

Systemic Interleukin-1β stimulates the simultaneous release of norepinephrine in the paraventricular nucleus and the median eminence

Interleukin-1β (IL-1β), a cytokine with pronounced central effects such as fever, anorexia, analgesia, etc., is also known to activate the hypothalamo–pituitary–adrenal (HPA) axis. Corticotropin releasing hormone (CRH) neurons located in the hypothalamus are important for HPA activation. The cell bodies of CRH neurons are located in the paraventricular nucleus (PVN) and their terminals are present in the median eminence (ME). Although the catecholamines, norepinephrine (NE) and dopamine (DA) are believed to be crucial factors in the stimulation of CRH neurons, it is not clear if they affect the cell bodies or terminals of these neurons to cause HPA activation. This study was done to determine if IL-1β affects NE and DA release at the level of CRH cell bodies or their terminals. Adult male Sprague–Dawley rats were implanted with two push–pull cannulae, one in the PVN and another in the ME, and were subjected to push–pull perfusion. They were treated either with 0, 1 or 5 μg of IL-1β. Perfusates were collected for 2 h after treatment and analyzed for NE concentrations using HPLC-EC. NE levels in the control and low dose groups did not change significantly during the entire period of observation both in the PVN and ME. In contrast, treatment with 5 μg of IL-1β produced a marked increase in NE release in the PVN at 20 and 40 min post-treatment. NE release in the ME increased from 10 to 140 min post-treatment. There were no significant changes in the release of DA from both these areas. These results indicate that IL-1β increases NE levels both in the PVN and in the ME and this could be a possible mechanism by which it stimulates the HPA axis.

Inhibition of Cell Migration by Corticotropin-Releasing Hormone (CRH) in Human Natural Killer Cell Line, NK-92MI

Background: Natural killer (NK) cells are CD3 (-) CD14 (-) CD56 (+) lymphocytes. They play an important role in the body's innate immune response. They can induce spontaneous killing of cancer cells or virus-infected cells via the Fas/Fas ligand or the granzyme/perforin systems. The corticotropin-releasing hormone (CRH) is an important regulator for the body's stress response. It promotes proliferation and migration of various cancer cells through the CRH type 1 receptor under stress, and also inhibits NK or T cell activity. However, the relationship of CRH and NK cell migration to the target has not been confirmed. Herein, we study the effect of CRH on NK cell migration. Methods: We used the human NK cell line, NK-92MI, and tested the expression of CRH receptor type 1 on NK-92MI by RT-PCR. This was to examine the effect of CRH on tumor and NK cell migration, thus NK cells (NK-92MI) were incubated with or without CRH and then each CRH treated cell's migration ability compared to that of the CRH untreated group. Results: We confirmed that CRH receptor type 1 is expressed in NK-92MI. CRH can decrease NK cell migration in a time-/dose-dependent manner. Conclusion: These data suggest CRH can inhibit NK cell migration to target cells.

Enhancement of Cell Migration by Corticotropin-Releasing Hormone (CRH) in Human Gastric Cancer Cell Line, MKN-28

Background: Corticotropin-Releasing Hormone (CRH), an important regulator of stress response, has a potent immunoregulatory effect with the ability to promote the growth of various cancer through CRH receptor type 1 under stress. Although the metastasized cancers through cell migration are more aggressive than the primary cancers, little is known about the effect of CRH on cell migration. Gastric cancer is prone to metastasize to other tissues and it is reported that gastric cancer is response to various stresses such as oxidative stress. Herein, we studied the relationship between CRH and gastric cancer cell migration. Methods: We used gastric cancer cell line, MKN-28 and tested the CRH receptor type 1 expression on MKN-28 by RT-PCR. To examine the change in the ability of migration by CRH in MKN-28, cells were incubated with CRH and then migration ability was measured using a cell migration assay. Results: We confirmed that CRH receptor type 1 was expressed in MKN-28 and HaCaT cells. The migration ability of MKN-28 cells was increased by CRH in a time-, dosedependent manner. Conclusion: These data suggest that CRH increases migration ability in gastric cancer cell line and that CRH may be a critical regulator in the metastasis of gastric cancer cell. (Immune Network 2004;4(4):244-249)