CRH mRNA Research Articles

Targeted knockout of a chemokine-like gene increases anxiety and fear responses

Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Short-term exposure to repeated chasing stress does not induce habituation in Senegalese sole, Solea senegalensis

Animals can habituate to certain repeated stressors and reduce the physiological response that such stressor evoked initially. Studies related to stress habituation in fish are scarce and the available data differ depending on the species and on the type, duration and severity of the stressor. The main objective of this study was to investigate the stress response of juvenile Senegalese sole (Solea senegalensis) submitted to repeated chasing stress for 3days previous to the experiment in order to evaluate the occurrence of habituation to those stress conditions in this fish species. Thus, five different experimental groups were evaluated: not stressed fish (control, C), fish stressed only on the experimental day (ST/naïve), and fish stressed on the experimental day and on the 3 previous days: during the day (ST/Dt), at night (ST/Nt) or both (ST/Dt+Nt). Parameters related to primary and secondary responses to chasing were evaluated in plasma, liver and brain. Chasing in ST/naïve group induced incremented values of plasma cortisol, glucose and lactate but no changes in catecholamine levels compared to controls. In trained fish, higher cortisol but decreased glucose, lactate and catecholamine levels were observed after stress compared to controls and to ST/naïve groups. In the liver, stress did not induce any changes with respect to controls whereas ST/Dt and ST/Dt+Nt showed lower values of glucose and glycogen than stressed naïve fish. In the brain, ST/naïve group presented no significant changes in serotonergic activity. However, incremented serotonergic activity was detected in fish previously trained. Furthermore, dopaminergic activity decreased in diurnal trained and nocturnal trained groups with respect to ST/naïve fish. Crh expression in hypothalamus was higher in ST/naïve fish but not in fish submitted to repeated stress compared to controls. In summary, it seems that there was no habituation to the repeated acute stress protocol in Solea senegalensis in terms of serotonergic activity and cortisol release during the physiological stress response. However, the decreased levels of plasma catecholamines and energy metabolites, and of the hypothalamic crh mRNA abundance and dopaminergic activity, indicate a modulation of the stress response in trained fish. Altogether, the results suggest that either the chasing stressor was too strong or the training period too short for the animals to habituate, indicating that repeated chasing within short periods should be avoided when manipulating fish in order to keep proper welfare conditions in this species.

CART neurons in the arcuate nucleus and lateral hypothalamic area exert differential controls on energy homeostasis

ObjectiveThe cocaine- and amphetamine-regulated transcript (CART) codes for a pivotal neuropeptide important in the control of appetite and energy homeostasis. However, limited understanding exists for the defined effector sites underlying CART function, as discrepant effects of central CART administration have been reported. MethodsBy combining Cart-cre knock-in mice with a Cart adeno-associated viral vector designed using the flip-excision switch (AAV-FLEX) technology, specific reintroduction or overexpression of CART selectively in CART neurons in the arcuate nucleus (Arc) and lateral hypothalamic area (LHA), respectively, was achieved. The effects on energy homeostasis control were investigated. ResultsHere we show that CART neuron-specific reintroduction of CART into the Arc and LHA leads to distinct effects on energy homeostasis control. Specifically, CART reintroduction into the Arc of otherwise CART-deficient Cartcre/cre mice markedly decreased fat mass and body weight, whereas CART reintroduction into the LHA caused significant fat mass gain and lean mass loss, but overall unaltered body weight. The reduced adiposity in ArcCART;Cartcre/cre mice was associated with an increase in both energy expenditure and physical activity, along with significantly decreased Npy mRNA levels in the Arc but with no change in food consumption. Distinctively, the elevated fat mass in LHACART;Cartcre/cre mice was accompanied by diminished insulin responsiveness and glucose tolerance, greater spontaneous food intake, and reduced energy expenditure, which is consistent with the observed decrease of brown adipose tissue temperature. This is also in line with significantly reduced tyrosine hydroxylase (Th) and notably increased corticotropin-releasing hormone (Crh) mRNA expressions in the paraventricular nucleus (PVN). ConclusionsTaken together, these results identify catabolic and anabolic effects of CART in the Arc and LHA, respectively, demonstrating for the first time the distinct and region-specific functions of CART in controlling feeding and energy homeostasis.

The role of prolactin in the suppression of Crh mRNA expression during pregnancy and lactation in the mouse.

Maternal stress is associated with negative health consequences for both the mother and her offspring. To prevent these adverse outcomes, activity of the hypothalamic-pituitary-adrenal (HPA) axis is attenuated during pregnancy and lactation. Although the mechanisms generating this adaptive change have not been defined fully, the anterior pituitary hormone prolactin may play a significant role. The present study investigated the role of prolactin in regulating the basal activity of the HPA axis during pregnancy and lactation in the mouse, focussing upon the corticotrophin-releasing hormone (CRH) neurones. Using in situ hybridisation, a decrease in Crh mRNA-expressing cell number in pregnant (55.6±9.0 cells per section) and lactating (97.4±4.9) mice compared to virgin controls was characterised (186.8±18.7, P<.01 Tukey-Kramer test; n=6-7 per group). Removal of the pups (24hours) and thus the associated suckling-induced prolactin secretion, restored CRH neurone number (180.1±19.7). To specifically test the role of prolactin in suppressing Crh mRNA expression in lactation, prolactin levels were selectively manipulated in lactating mice. Lactating mice were treated with ovine prolactin (1500μgday-1 , osmotic minipump, s.c.; n=7) or vehicle (n=6) for 24hours following pup removal. This was sufficient to suppress Crh mRNA expression from 108.0±13.5 to 53.7±16.7 cells per section (P<.05 Student's t-test). Additional cohorts of lactating mice were treated with bromocriptine (300μg over 24hours, s.c.; n=7) or vehicle (n=5) to suppress endogenous prolactin secretion; however, no change in Crh mRNA expression was detected. Thus, although prolactin was sufficient to suppress Crh mRNA expression in the paraventricular nucleus, it does not appear to be required for the ongoing regulation of the CRH neurones in lactation.

Methamphetamine Induces TET1- and TET3-Dependent DNA Hydroxymethylation of Crh and Avp Genes in the Rat Nucleus Accumbens

Methamphetamine (METH) addiction is a biopsychosocial disorder that is accompanied by multiple relapses even after prolonged abstinence, suggesting the possibilities of long-lasting maladaptive epigenetic changes in the brain. Here, we show that METH administration produced time-dependent increases in the expression of corticotropin-releasing hormone (Crh/Crf), arginine vasopressin (Avp), and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) mRNAs in the rat nucleus accumbens (NAc). Chromatin immunoprecipitation (ChIP) assays revealed that METH increased the abundance of phosphorylated CREB (pCREB) at the promoter of Cartpt but not at Avp or Crh DNA sequences. In contrast, METH produced DNA hypomethylation at sites near the Crh transcription start site (TSS) and at intragenic Avp sequences. METH also increased DNA hydroxymethylation at the Crh TSS and at intragenic Avp sites. In addition, METH increased the protein expression of ten-eleven-translocation enzymes that catalyze DNA hydroxymethylation. Importantly, METH increased TET1 binding at the Crh promoter and increased TET3 binding at Avp intragenic regions. We further tested the role of TET enzymes in METH-induced changes in gene expression by using the TET inhibitor, 1,5-isoquinolinediol (IQD), and found that IQD blocked METH-induced increases in Crh and Avp mRNA expression. Together, these results indicate that METH produced changes in neuropeptide transcription by both activation of the cAMP/CREB pathway and stimulation of TET-dependent DNA hydroxymethylation. These results provide molecular evidence for epigenetic controls of METH-induced changes in the expression of neuropeptides.

The effects of continuously acoustical stress on cortisol in milkfish (Chanos chanos)

Strong underwater acoustic noise has been known that may cause hearing loss and actual stress in teleost. However, the long-term physiological effects of relatively quiet but continuously noise on fish were less understood. In present study, milkfish, Chanos chanos, were exposed to the simulated-wind farm noise either quiet (109dB re 1μPa/125.4Hz; approx. 10–100m distant from the wind farm) or noisy (138dB re 1μPa/125.4Hz; nearby the wind farm) conditions for 24h, 3days and 1week. Comparing to the control group (80dB re 1μPa/125.4Hz), the fish exposed to noisy conditions had higher plasma cortisol levels in the first 24h. However, the cortisol levels of 24h spot returned to the resting levels quickly. The fish exposed under noisy condition had significantly higher head kidney star (steroidogenic acute regulatory) and hsd11b2 (11-β-hydroxysteroid dehydrogenase 2) mRNA levels at the following treatment time points. In addition, noise exposure did not change hypothalamus crh (Corticotropin-releasing hormone) mRNA levels in this experiment. The results implied that the weak but continuously noise was a potential stressor to fish, but the impacts may be various depending on the sound levels and exposure time. Furthermore, this study showed that the continuous noise may up-regulate the genes that are related to cortisol synthesis and possibly make the fish more sensitive to ambient stressors, which may influence the energy allocation appearance in long-term exposures.

Anatomical and functional implications of corticotrophin-releasing hormone neurones in a septal nucleus of the avian brain: an emphasis on glial-neuronal interaction via V1a receptors in vitro.

Previously, we showed that corticotrophin-releasing hormone immunoreactive (CRH-IR) neurones in a septal structure are associated with stress and the hypothalamic-pituitary-adrenal axis in birds. In the present study, we focused upon CRH-IR neurones located within the septal structure called the nucleus of the hippocampal commissure (NHpC). Immunocytochemical and gene expression analyses were used to identify the anatomical and functional characteristics of cells within the NHpC. A comparative morphometry analysis showed that CRH-IR neurones in the NHpC were significantly larger than CRH-IR parvocellular neurones in the paraventricular nucleus of the hypothalamus (PVN) and lateral bed nucleus of the stria terminalis. Furthermore, these large neurones in the NHpC usually have more than two processes, showing characteristics of multipolar neurones. Utilisation of an organotypic slice culture method enabled testing of how CRH-IR neurones could be regulated within the NHpC. Similar to the PVN, CRH mRNA levels in the NHpC were increased following forskolin treatment. However, dexamethasone decreased forskolin-induced CRH gene expression only in the PVN and not in the NHpC, indicating differential inhibitory mechanisms in the PVN and the NHpC of the avian brain. Moreover, immunocytochemical evidence also showed that CRH-IR neurones reside in the NHpC along with the vasotocinergic system, comprising arginine vasotocin (AVT) nerve terminals and immunoreactive vasotocin V1a receptors (V1aR) in glia. Hence, we hypothesised that AVT acts as a neuromodulator within the NHpC to modulate activity of CRH neurones via glial V1aR. Gene expression analysis of cultured slices revealed that AVT treatment increased CRH mRNA levels, whereas a combination of AVT and a V1aR antagonist treatment decreased CRH mRNA expression. Furthermore, an attempt to identify an intercellular mechanism in glial-neuronal communication in the NHpC revealed that brain-derived neurotrophic factor (BDNF) and its receptor (TrkB) could be involved in the signalling mechanism. Immunocytochemical results further showed that both BDNF and TrkB receptors were found in glia of the NHpC. Interestingly, in cultured brain slices containing the NHpC, the use of a selective TrkB antagonist decreased the AVT-induced increase in CRH gene expression levels. The results from the present study collectively suggest that CRH neuronal activity is modulated by AVT via V1aR involving BDNF and TrkB glia in the NHpC.

Molecular cloning and tissue distribution of Crh and Pomc mRNA in the fat-tailed dunnart (Sminthopsis crassicaudata), an Australian marsupial

Like all vertebrates, marsupials respond to stressors with the activation of the hypothalamo-pituitary-adrenal axis. However, peptides operating at the higher regulatory levels of this hormonal system, i.e. corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH), have not been investigated in marsupials. Here we report the molecular cloning of the precursor cDNAs of CRH (prepro-CRH) and of ACTH (proopiomelanocortin; POMC) in an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). Dunnart POMC and prepro-CRH are predicted to be peptides of 399 and 200 amino acids, respectively. While the ACTH and β-endorphin sequences within the POMC sequence are highly conserved, the POMC sequence shows some unique features in this species, and perhaps all Australian marsupials, including the loss of a γ-melanotropin sequence and duplications of the ACTH sequence. Mature dunnart CRH is identical to CRH in human, mouse, rat and chicken. Pomc and Crh mRNA is mainly expressed in dunnart pituitary gland and brain, respectively, but both are also present in a range of peripheral tissues.

Prolactin regulation of the HPA axis is not mediated by a direct action upon CRH neurons: evidence from the rat and mouse.

Centrally acting prolactin has been shown to have anti-stress effects by modulating the activity of the hypothalamic-pituitary-adrenal axis. We tested the hypothesis that prolactin directly targets hypothalamic corticotropin-releasing hormone (CRH) neurons. In situ hybridisation confirmed expression of mRNA encoding the long, but not the short, isoform of the prolactin receptor (PRLR) within the paraventricular nucleus (PVN) of the virgin rat; however, only 6% of CRH neurons expressed long-form Prlr mRNA. Examination of the functional response of CRH neurons to intracerebroventricular prolactin (500ng) showed that these neurons did not respond with activation of phosphorylated signal transducer and activator of transcription 5 (pSTAT5), a marker of long-form PRLR activation. However, as only a subset of neurons expressing Crh mRNA could be detected using immunohistochemistry, we utilised a transgenic mouse model to label CRH neurons with a fluorescent reporter (CRH-Cre-tdTomato). In lactating animals, chronically elevated prolactin levels resulted in significantly increased pSTAT5 expression in the PVN. Overall, few tdTomato-labelled CRH neurons were double-labelled, although a small subset of CRH neurons in the caudal PVN were pSTAT5 positive (approximately 10% of tdTomato neurons at this level, compared to 1% in the rostral PVN). These data suggest that most CRH neurons do not respond directly to prolactin. To confirm that prolactin was not activating another signalling pathway, we used a transgenic mouse line to label PRLR-expressing neurons with Cre-dependent green fluorescent protein (GFP) expression (CRH-Cre-Prlr lox/lox ). No GFP-expressing cells were evident in the PVN, indicating that in the mouse, as in the rat, the CRH neurons do not express either PRLR isoform. Together these data showed that the anti-stress effects of prolactin are not the result of prolactin directly regulating CRH neurons.

Postnatal Light Effects on Pup Stress Axis Development Are Independent of Maternal Behavior.

Postnatal environment shapes brain development during key critical periods. We have recently found that postnatal light environment has long-term effects on the stress and circadian systems, which can lead to altered stress responses, circadian behavior and a depressive phenotype in adulthood. However, it is still unclear how light experience affects the postnatal development of specific stress markers in the pup brain and the role played by maternal behavior and stress. To test this, we raised mice under either light-dark cycles (LD), constant light (LL) or constant darkness (DD) during the suckling stage. After weaning, all mice were exposed to LD until adulthood. Results show that postnatal light environment does not have any significant effects on dam stress levels (plasma corticosterone concentration, Arginine-vasopressin and Glucocorticoid receptor (GR) protein expression in the brain) or maternal behavior, including licking and grooming. Light environment does not have a major effect on litter characteristics or pup growth either. Interestingly, light environment during the suckling stage significantly impacted Corticotrophin-releasing hormone (CRH) and Gr mRNA expression in pup brain during development. Furthermore, a difference in Crh mRNA expression between LL- and DD-raised mice was still observed in adulthood, long after the exposure to abnormal light environments had stopped. Taken together, these data suggest that the long-term effects of postnatal light environment on the pups' stress system cannot be attributed to alterations in either maternal behavior and/or stress axis function. Instead, postnatal light experience may act directly on the pup stress axis and/or indirectly via circadian system alterations.

Repetitive transcranial magnetic stimulation for treating depressive behavior in response to chronic but unpredictable mild stress

Objective To observe the effect of repetitive transcranial magnetic stimulation (rTMS) on behavior in response to chronic but unpredictable mild stress and explore potential neuroendocrine mechanisms. Methods Forty adult SD male rats were randomly divided into a control group (n=8) and a model preparation group (n=32). The control group was given normal care while a model of depression was induced in the model preparation group through giving an unpredictable mild stimulus (CUMS). The depressive rats were randomly divided into a model group, an rTMS group and a sham rTMS group (8 cases in each group). The rTMS group and sham rTMS groups accepted the rTMS or sham stimulation for 3 weeks. The changes in behavior in each group were quantified using body weight, sucrose consumption and an open field test before and after stimulation. Enzyme-linked immunosorbent assays (Elisas) were conducted to detect plasma adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels. Reverse-transcription polymerase chain reactions (RT-PCRs) were carried out to allow the detection of mRNA expression in hypothalamus related to levels of adrenocorticotropic hormone releasing hormone (CRH). Results After the modeling there were significant differences between the model preparation group and the control group in terms of weight increase, sucrose consumption and open field test results. After rTMS the rate of weight increase, sucrose consumption and the scores in the open field test of the rTMS group had increased significantly more than in the control group. Elisas showed significantly higher plasma ACTH and CORT levels in the model group as well. The average expression of CRH mRNA in the model group was significantly higher than in either of the other two groups. Conclusions rTMS can relieve depression-like behavior induced by chronic stress, at least in rats. This may be related to a downgrading of the hyperactive functioning of the hypothalamus-pituitary-adrenal axis. Key words: Depression; Transcranial magnetic stimulation; Hypothalamus-pituitary-adrenal axis; Stress

Loss of hypothalamic corticotropin-releasing hormone markedly reduces anxiety behaviors in mice.

A long-standing paradigm posits that hypothalamic corticotropin-releasing hormone (CRH) regulates neuroendocrine functions such as adrenal glucocorticoid release, while extra-hypothalamic CRH plays a key role in stressor-triggered behaviors. Here we report that hypothalamus-specific Crh knockout mice (Sim1CrhKO mice, created by crossing Crhflox with Sim1Cre mice) have absent Crh mRNA and peptide mainly in the paraventricular nucleus of the hypothalamus (PVH) but preserved Crh expression in other brain regions including amygdala and cerebral cortex. As expected, Sim1CrhKO mice exhibit adrenal atrophy as well as decreased basal, diurnal and stressor-stimulated plasma corticosterone secretion and basal plasma ACTH, but surprisingly, have a profound anxiolytic phenotype when evaluated using multiple stressors including open field, elevated plus maze, holeboard, light-dark box, and novel object recognition task. Restoring plasma corticosterone did not reverse the anxiolytic phenotype of Sim1CrhKO mice. Crh-Cre driver mice revealed that PVHCrh fibers project abundantly to cingulate cortex and the nucleus accumbens shell, and moderately to medial amygdala, locus coeruleus, and solitary tract, consistent with the existence of PVHCrh-dependent behavioral pathways. Although previous, nonselective attenuation of CRH production or action, genetically in mice and pharmacologically in humans, respectively, has not produced the anticipated anxiolytic effects, our data show that targeted interference specifically with hypothalamic Crh expression results in anxiolysis. Our data identify neurons that express both Sim1 and Crh as a cellular entry point into the study of CRH-mediated, anxiety-like behaviors and their therapeutic attenuation.

Environmental enrichment imparts disease-modifying and transgenerational effects on genetically-determined epilepsy and anxiety

IntroductionThe absence epilepsies are presumed to be caused by genetic factors, but the influence of environmental exposures on epilepsy development and severity, and whether this influence is transmitted to subsequent generations, is not well known. We assessed the effects of environmental enrichment on epilepsy and anxiety outcomes in multiple generations of GAERS – a genetic rat model of absence epilepsy that manifests comorbid elevated anxiety-like behaviour. MethodsGAERS were exposed to environmental enrichment or standard housing beginning either prior to, or after epilepsy onset, and underwent EEG recordings and anxiety testing. Then, we exposed male GAERS to early enrichment or standard housing and generated F1 progeny, which also underwent EEG recordings. Hippocampal CRH mRNA expression and DNA methylation were assessed using RT-PCR and pyrosequencing, respectively. ResultsEarly environmental enrichment delayed the onset of epilepsy in GAERS, and resulted in fewer seizures in adulthood, compared with standard housed GAERS. Enrichment also reduced the frequency of seizures when initiated in adulthood. Anxiety levels were reduced by enrichment, and these anti-epileptogenic and anxiolytic effects were heritable into the next generation. We also found reduced expression of CRH mRNA in GAERS exposed to enrichment, but this was not due to changes in DNA methylation. ConclusionsEnvironmental enrichment produces disease-modifying effects on genetically determined absence epilepsy and anxiety, and these beneficial effects are transferable to the subsequent generation. Reduced CRH expression was associated with these phenotypic improvements. Environmental stimulation holds promise as a naturalistic therapy for genetically determined epilepsy which may benefit subsequent generations.

Isoform switching of steroid receptor co-activator-1 attenuates glucocorticoid-induced anxiogenic amygdala CRH expression.

Maladaptive glucocorticoid effects contribute to stress-related psychopathology. The glucocorticoid receptor (GR) that mediates many of these effects uses multiple signaling pathways. We have tested the hypothesis that manipulation of downstream factors ('coregulators') can abrogate potentially maladaptive GR-mediated effects on fear-motivated behavior that are linked to corticotropin releasing hormone (CRH). For this purpose the expression ratio of two splice variants of steroid receptor coactivator-1 (SRC-1) was altered via antisense-mediated 'exon-skipping' in the central amygdala of the mouse brain. We observed that a change in splicing towards the repressive isoform SRC-1a strongly reduced glucocorticoid-induced responsiveness of Crh mRNA expression and increased methylation of the Crh promoter. The transcriptional GR target gene Fkbp5 remained responsive to glucocorticoids, indicating gene specificity of the effect. The shift of the SRC-1 splice variants altered glucocorticoid-dependent exploratory behavior and attenuated consolidation of contextual fear memory. In conclusion, our findings demonstrate that manipulation of GR signaling pathways related to the Crh gene can selectively diminish potentially maladaptive effects of glucocorticoids.

Adolescent chronic stress causes hypothalamo–pituitary–adrenocortical hypo-responsiveness and depression-like behavior in adult female rats

Adolescence is a period of substantial neuroplasticity in stress regulatory neurocircuits. Chronic stress exposure during this period leads to long-lasting changes in neuroendocrine function and emotional behaviors, suggesting adolescence may be a critical period for development of stress vulnerability. This study investigated the effects of exposure to 14 days of chronic variable stress (CVS) in late-adolescent (pnd 45–58) female rats on neuroendocrine function, neuropeptide mRNA expression and depressive-like behavior in adolescence (pnd 59) and in adulthood (pnd 101). Adult females exposed to CVS in adolescence have a blunted hypothalamo-pituitary-adrenocortical (HPA) axis in response to a novel stressor and increased immobility in the forced swim test. Blunted HPA axis responses were accompanied by reduced vasopressin mRNA expression in the paraventricular nucleus of the hypothalamus (PVN), suggesting decreased central drive. Adolescent females tested immediately after CVS did not exhibit differences in stress reactivity or immobility in the forced swim test, despite evidence for enhanced central HPA axis drive (increased CRH mRNA expression in PVN). Overall, our study demonstrates that exposure to chronic stress in adolescence is sufficient to induce lasting changes in neuroendocrine drive and behavior, potentially altering the developmental trajectory of stress circuits as female rats age into adulthood.

Exposure to Chronic Mild Stress Differentially Alters Corticotropin-Releasing Hormone and Arginine Vasopressin mRNA Expression in the Stress-Responsive Neurocircuitry of Male and Female Rats Prenatally Exposed to Alcohol.

Prenatal alcohol exposure (PAE) results in dysregulation of the offspring hypothalamic-pituitary-adrenal (HPA) axis, increasing sensitivity to stressors and vulnerability to stress-related disorders. We have previously shown that exposure to chronic mild stress (CMS) in adulthood significantly increases anxiety-like behaviors (elevated plus maze) in PAE males and females compared to controls. To explore neurobiological mechanisms linking HPA dysregulation and altered anxiety-like behavior, we investigated neuropeptide (corticotropin-releasing hormone [CRH] and arginine vasopressin [AVP]) expression in brain areas involved in the stress neurocircuitry of animals from this previous behavioral study. Adult PAE, pair-fed (PF), and ad libitum fed control (C) male and female offspring exposed to CMS or remaining undisturbed (non-CMS) were terminated 30 minutes following behavioral testing. In the paraventricular nucleus, CMS increased CRH mRNA levels in PAE compared to PF and C males and increased AVP mRNA levels in PAE compared to C males, with no differential effects for CRH or AVP in females. In the central nucleus of the amygdala, there was an increase in CRH mRNA expression overall, regardless of CMS condition or sex, in PAE compared to C animals. Moreover, in PF males, CMS increased AVP mRNA levels in the paraventricular nucleus, resulting in a decreased CRH/AVP ratio compared to PAE males, and decreased amygdala CRH mRNA compared to that in the non-CMS condition. CMS differentially altered central HPA peptide expression in PAE and PF animals compared to their control counterparts, with a possible shift toward greater CRH mediation of HPA regulation in PAE males, and greater AVP mediation of HPA regulation in PF males. However, changes in CRH and AVP expression do not align fully with the anxiogenic profile observed in our previous behavior study, suggesting that other neuronal substrates and limbic forebrain regions also contribute to increased anxiety-like behavior following CMS.

Prenatal stress programs neuroendocrine stress responses and affective behaviors in second generation rats in a sex-dependent manner

An adverse environment in early life is often associated with dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis and higher rates of mood disorders in adulthood. In rats, exposure to social stress during pregnancy results in hyperactive HPA axis responses to stress in the adult offspring and heightened anxiety behavior in the males, but not the females. Here we tested whether, without further intervention, the effects of prenatal stress (PNS) in the first filial generation (F1) are transmitted to the F2 generation via the maternal line. F1 control and PNS female rats were mated with control males and housed under non-stress conditions throughout pregnancy. HPA axis responses to acute stress, anxiety- and depressive-like behavior were assessed in the adult F2 offspring.ACTH and corticosterone responses to an acute stressor were markedly enhanced in F2 PNS females compared with controls. This was associated with greater corticotropin releasing hormone (Crh) mRNA expression in the paraventricular nucleus and reduced hippocampal glucocorticoid (Gr) and mineralocorticoid receptor (Mr) mRNA expression. Conversely, in the F2 PNS males, HPA axis responses to acute stress were attenuated and hippocampal Gr mRNA expression was greater compared with controls.F2 PNS males exhibited heightened anxiety-like behavior (light-dark box and elevated plus maze) compared with F2 control males. Anxiety-like behavior did not differ between F2 control and PNS females during metestrus/diestrus, however at proestrus/estrus, F2 control females displayed a reduction in anxiety-like behavior, but this effect was not observed in the F2 PNS females. Heightened anxiety in the F2 PNS males was associated with greater Crh mRNA expression in the central nucleus of the amygdala compared with controls. Moreover, Crh receptor-1 (Crhr1) mRNA expression was significantly increased, whereas Crhr2 mRNA was significantly decreased in discrete regions of the amygdala in F2 PNS males compared with controls, with no differences in the F2 females. No differences in depressive-like behavior (sucrose preference or forced swim test) were observed in either sex. In conclusion, the effects of maternal stress during pregnancy on HPA axis regulation and anxiety-like behavior can be transmitted to future generations in a sex-dependent manner. These data have implications for human neuropsychiatric disorders with developmental origins.

PII-LBA8 USE OF LINEAR DISCRIMINANT ANALYSIS IN A URINE-BASED TEST FOR BLADDER CANCER DIAGNOSIS

You have accessJournal of UrologyPlenary Session II – Late Breaking Abstracts1 Apr 2015PII-LBA8 USE OF LINEAR DISCRIMINANT ANALYSIS IN A URINE-BASED TEST FOR BLADDER CANCER DIAGNOSIS Ellen Wallace, Kathleen E. Mach, Leena McCann, Lai Yi Mandy Sin, Ruchika Mohan, Malini Satya, Huilin Wei, Jun Zhang, Chris Lykke, Russell Higuchi, and Joseph C. Liao Ellen WallaceEllen Wallace More articles by this author , Kathleen E. MachKathleen E. Mach More articles by this author , Leena McCannLeena McCann More articles by this author , Lai Yi Mandy SinLai Yi Mandy Sin More articles by this author , Ruchika MohanRuchika Mohan More articles by this author , Malini SatyaMalini Satya More articles by this author , Huilin WeiHuilin Wei More articles by this author , Jun ZhangJun Zhang More articles by this author , Chris LykkeChris Lykke More articles by this author , Russell HiguchiRussell Higuchi More articles by this author , and Joseph C. LiaoJoseph C. Liao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.03.088AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We aim to develop a rapid, accurate and non-invasive molecular assay for bladder cancer diagnosis for use on the GeneXpert® (Cepheid, Sunnyvale CA) platform with sample-in-answer-out capability. Linear discriminant analysis (LDA) was used both to help choose the best markers for the assay, and to optimize the assay's ability to discriminate negative and positive samples. METHODS Appropriate local IRB approvals were obtained. The GeneXpert® Instrument Systems automate and integrate sample purification and multiplex RT-qPCR detection. The systems utilize single-use cartridges that hold the reagents and conduct the RT-qPCR process. The assay requires less than two minutes of hands-on time and results are available in approximately 90 minutes. A training set of 497 urine samples collected from 18 sites was tested for the expression levels of ABL1, CRH, IGF2, ANXA10, KRT20, AR, PIK3CA, UPK1B, UPK2 and MGEA5 mRNA. The ten markers were analyzed using stepwise logistic regression to choose the best 5 marker signature and develop an LDA equation to determine test outcome. An assay was developed for the mRNA detection of a 5 marker signature that includes a sample adequacy control and an internal RT-qPCR control. The assay with integrated LDA equation was tested with a set of 243 urine samples from 7 sites to validate the signature. RESULTS Backwards stepwise regression was used with the training set of samples and data with 10 markers to reduce the model and obtain the best 5-marker multivariable equation to determine test results. The resulting LDA equation using Ct values for ABL1, CRH, IGF2, ANXA10 and UPK1B was shown to yield sensitivities of 98% (79/81) for high grade cancer (papillary and CIS combined), 78% (31/40) for low grade and specificity of 83% (78/94) with patients undergoing hematuria workup. An assay for detection of ABL1, CRH, IGF2, ANXA10 and UPK1B mRNA was formulated and optimized for use on the GeneXpert®. A validation set of 243 urine samples was tested with the new assay to evaluate equivalence relative to the prior training set results. The new assay's sensitivity was 92% (12/13) for high-grade cancer (papillary and CIS combined) and 86% (12/14) for low-grade. The specificity was 89% (24/27) for patients undergoing workup for hematuria and 96% (144/150) with healthy volunteers. A prospective study with the newly developed test is underway. CONCLUSIONS We have developed a simple to use and accurate test for the detection of bladder cancer. The assay utilizes a proven diagnostic platform of GeneXpert® with automated sample preparation providing test results within 90 minutes. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e498 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ellen Wallace More articles by this author Kathleen E. Mach More articles by this author Leena McCann More articles by this author Lai Yi Mandy Sin More articles by this author Ruchika Mohan More articles by this author Malini Satya More articles by this author Huilin Wei More articles by this author Jun Zhang More articles by this author Chris Lykke More articles by this author Russell Higuchi More articles by this author Joseph C. Liao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Bisphenol A disrupts gene expression in human placental trophoblast cells.

This study examined the effect of bisphenol A (BPA) on human placental gene expression using primary trophoblast cells as an in vitro model system. Trophoblast cells were isolated from human placentas at term, cultured and then exposed to environmentally relevant concentrations of BPA (0.1-2 μg/ml) for up to 24h, after which levels of 11β-HSD2 mRNA, protein and activity were determined by standard radiometric conversion assay, western blotting, and qRT-PCR, respectively. The mRNA levels of several other prominent placental hormones/factors were also assessed by qRT-PCR. BPA dramatically increased levels of 11β-HSD2 activity, protein and mRNA in a time- and concentration-dependent manner (> 4-fold). BPA also augmented aromatase, glucose transporter-1, CRH, and hCG mRNA levels while reducing the level of leptin mRNA. These findings demonstrate that BPA severely disrupts human placental gene expression in vitro, which suggests that exposure to BPA may contribute to altered placental function and consequent pregnancy complications.

The balance between stress resilience and vulnerability is regulated by corticotropin-releasing hormone during the critical postnatal period for sensory development.

Determining whether a stressful event will lead to stress-resilience or vulnerability depends probably on an adjustable stress response set point, which is most likely effective during postnatal sensory development and involves the regulation of corticotrophin-releasing hormone (CRH) expression. During the critical period of thermal-control establishment in 3-day-old chicks, heat stress was found to render resilient or sensitized response, depending on the ambient temperature. These two different responses were correlated with the amount of activation of the hypothalamic-pituitary-adrenal (HPA) axis. The expression of CRH mRNA in the hypothalamic paraventricular nucleus was augmented during heat challenge a week after heat conditioning in chicks which were trained to be vulnerable to heat, while it declined in chicks that were trained to be resilient. To study the role of CRH in HPA-axis plasticity, CRH or Crh-antisense were intracranially injected into the third ventricle. CRH caused an elevation of both body temperature and plasma corticosterone level, while Crh-antisense caused an opposite response. Moreover, these effects had long term implications by reversing a week later, heat resilience into vulnerability and vice versa. Chicks that had been injected with CRH followed by exposure to mild heat stress, normally inducing resilience, demonstrated, a week later, an elevation in body temperature, and Crh mRNA level similar to heat vulnerability, while Crh-antisense injected chicks, which were exposed to harsh temperature, responded in heat resilience. These results demonstrate a potential role for CRH in determining the stress resilience/vulnerability balance.