Balázs Gaszner

Invasive validation of a new oscillometric device (Arteriograph) for measuring augmentation index, central blood pressure and aortic pulse wave velocity

Background The importance of measuring aortic pulse wave velocity (PWVao), aortic augmentation index (Aix) and central systolic blood pressure (SBPao) has been shown under different clinical conditions; however, information on these parameters is hard to obtain. The aim of this study was to evaluate the accuracy of a new, easily applicable oscillometric device (Arteriograph), determining these parameters simultaneously, against invasive measurements. Methods Aortic Aix, SBPao and PWVao were measured invasively during cardiac catheterization in 16, 55 and 22 cases, respectively, and compared with the values measured by the Arteriograph. Results We found strong correlation between the invasively measured aortic Aix and the oscillometrically measured brachial Aix on either beat-to-beat or mean value per patient basis (r = 0.9, P < 0.001; r = 0.94, P < 0.001), which allowed the noninvasive calculation of the aortic Aix without using generalized transfer function. Similarly strong correlation (r = 0.95, P < 0.001) was found between the invasively measured and the noninvasively calculated central SBPao; furthermore, the BHS assessment of the paired differences fulfilled the ‘B’ grading. The PWVao values measured invasively and by Arteriograph were 9.41 ± 1.8 m/s and 9.46 ± 1.8 m/s, respectively (mean ± SD); furthermore, the Pearsons correlation was 0.91 (P < 0.001). The limits of agreement were 11.4% for aortic Aix and 1.59 m/s for PWVao. Conclusion Aix, SBPao and PWVao, measured oscillometrically, showed strong correlation with the invasively obtained values. The observed limits of agreement are encouragingly low for accepting the method for clinical use. Our results suggest that the PWVao values, measured by Arteriograph, are close to the true aortic PWV, determined invasively.

Urocortinergic neurons respond in a differentiated manner to various acute stressors in the Edinger-Westphal nucleus in the rat

Corticotropin‐releasing factor (CRF) was implicated as being a major contributor to the neurochemically mediated central regulation of stress response; however, an increasing body of evidence suggests that, besides CRF, other members of this neuropeptide family, such as urocortin (Ucn), may also play a role in modifying the efferent components of immune, endocrine, and behavioral responses to stress. Ucns distribution in the rat brain has been demonstrated, with the most abundant Ucn‐immunoreactive perikarya present in the Edinger‐Westphal nucleus (E‐WN). Acute pain and immobilization stresses recruit E‐WN neurons, however, the activation pattern of E‐WN Ucn neurons in response to various acute systemic and neurogenic challenges has not been compared in a single study. We therefore combined quantitative Fos imaging as a marker for neuronal activation with urocortin immunohistochemistry to visualize neurons induced by intravenous lipopolysaccharide (LPS; 100 μg/kg), ether inhalation, restraint, hyperosmotic (1.5 M NaCl i.p.), and hypotensive hemorrhage challenges. Neurons in the E‐WN responded with the strongest Fos induction to LPS, but ether and restraint stress also resulted in massive Fos immunoreactivity 2 hours after stress. Unexpectedly, hyperosmotic and hypotensive hemorrhage stresses did not induce urocortinergic perikarya in this brain area 2 hours poststress. This challenge‐specific recruitment of E‐WN neurons was independent of stress‐induced adrenal response. The biological significance and the stress‐specific activation of E‐WN urocortinergic neurons will be discussed. J. Comp. Neurol. 480:170–179, 2004.

Chronic stress induces sex-specific alterations in methylation and expression of corticotropin-releasing factor gene in the rat

Background Although the higher prevalence of depression in women than in men is well known, the neuronal basis of this sex difference is largely elusive. Methods Male and female rats were exposed to chronic variable mild stress (CVMS) after which immediate early gene products, corticotropin-releasing factor (CRF) mRNA and peptide, various epigenetic-associated enzymes and DNA methylation of the Crf gene were determined in the hypothalamic paraventricular nucleus (PVN), oval (BSTov) and fusiform (BSTfu) parts of the bed nucleus of the stria terminalis, and central amygdala (CeA). Results CVMS induced site-specific changes in Crf gene methylation in all brain centers studied in female rats and in the male BST and CeA, whereas the histone acetyltransferase, CREB-binding protein was increased in the female BST and the histone-deacetylase-5 decreased in the male CeA. These changes were accompanied by an increased amount of c-Fos in the PVN, BSTfu and CeA in males, and of FosB in the PVN of both sexes and in the male BSTov and BSTfu. In the PVN, CVMS increased CRF mRNA in males and CRF peptide decreased in females. Conclusions The data confirm our hypothesis that chronic stress affects gene expression and CRF transcriptional, translational and secretory activities in the PVN, BSTov, BSTfu and CeA, in a brain center-specific and sex-specific manner. Brain region-specific and sex-specific changes in epigenetic activity and neuronal activation may play, too, an important role in the sex specificity of the stress response and the susceptibility to depression.

Pituitary adenylate cyclase-activating polypeptide plays a key role in nitroglycerol-induced trigeminovascular activation in mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (PAC1, VPAC) are present in sensory neurons and vascular smooth muscle. PACAP infusion was found to trigger migraine-like headache in humans and we showed its central pro-nociceptive function in several mouse pain models. Nitroglycerol (NTG)-induced pathophysiological changes were investigated in this study in PACAP gene-deleted (PACAP(-/-)) and wildtype (PACAP(+/+)) mice. Chemical activation of the trigeminovascular system was induced by 10 mg/kg i.p. NTG. Light-aversive behavior was determined in a light-dark box, meningeal microcirculation by laser Doppler blood perfusion scanning and the early neuronal activation marker c-Fos with immunohistochemistry. NTG-induced photophobia both in the early (0-30 min) and late phases (90-120 min) due to direct vasodilation and trigeminal sensitization, respectively, was significantly reduced in PACAP(-/-) mice. Meningeal blood flow increased by 30-35% during 4 h in PACAP(+/+) mice, but only a 5-10% elevation occurred from the second hour in PACAP(-/-) ones. The number of c-Fos expressing cells referring to neuronal activation in the trigeminal ganglia and nucleus caudalis significantly increased 4h after NTG in PACAP(+/+), but not in PACAP(-/-) animals. Similar PAC1 receptor immunostaining was detected in both groups, which did not change 4 h after NTG treatment. PACAP-38 (300 μg/kg, i.p.) produced photophobia similarly to NTG and 30% meningeal vasodilatation for 30 min in PACAP(+/+), but not in PACAP(-/-) mice. It significantly increased neural activation 4h later in the trigeminal ganglia of both groups, but in the nucleus caudalis of only the PACAP(+/+) mice. We provide the first experimental results that PACAP is a pivotal mediator of trigeminovascular activation/sensitization and meningeal vasodilation related to migraine.

HOUSEKEEPING GENES REVISITED : DIFFERENT EXPRESSIONS DEPENDING ON GENDER, BRAIN AREA AND STRESSOR

Housekeeping gene (HKG) mRNAs are used to normalize expression data of genes of interest in quantitative reverse transcriptase polymerase chain reaction studies. Such normalization assumes constant HKG gene expression under all circumstances. Although sporadic evidence suggests that HKG expression may not always fulfill this requirement and, therefore, such normalization may lead readily to erroneous results, this fact is generally not sufficiently appreciated by investigators. Here, we have systematically analyzed the expression of three common HKGs, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunit 18S and beta-actin, in two different stress paradigms, in various brain areas, in male and in female rats. HKG expressions differed considerably with respect to brain area, type of stressor and gender, in an HKG-specific manner. Therefore, we conclude that before final experimentation, pilot expression studies are necessary to select an HKG which expression is unaffected by the experimental factor(s), allowing reliable interpretation of expression data of genes of interest.

Sex-dependent and differential responses to acute restraint stress of corticotropin-releasing factor–producing neurons in the rat paraventricular nucleus, central amygdala, and bed nucleus of the stria terminalis

Male and female rodents respond differently to acute stress. We tested our hypothesis that this sex difference is based on differences in stress sensitivity of forebrain areas, by determining possible effects of a single acute psychogenic stressor (1‐hr restraint stress) on neuronal gene expression (c‐Fos and FosB immunoreactivities), storage of corticotropin‐releasing factor (CRF) immunoreactivity, and CRF production (CRF mRNA in situ hybridization) as well as the expression of genes associated with epigenetic processes (quantitative RT‐PCR) in the rat paraventricular nucleus (PVN), the oval and fusiform subdivisions of the bed nucleus of the stria terminalis (BSTov and BSTfu, respectively), and the central amygdala (CeA), in both males and females. Compared with females, male rats responded to the stressor with a stronger rise in corticosterone titer and a stronger increase in neuronal contents of c‐Fos, CRF mRNA, and CREB‐binding protein mRNA in the PVN. In the BSTov, females but not males showed an increase in c‐Fos, whereas the CRF mRNA content was increased in males only. In the BSTfu, males and females showed similar stress‐induced increases in c‐Fos and FosB, whereas in the CeA, both sexes revealed similar increases in c‐Fos and in CRF mRNA. We conclude that male and female rats differ in their reactivity to acute stress with respect to possibly epigenetically mediated (particularly in the PVN) neuronal gene expression and neuropeptide dynamics (PVN and BSTov) and that this difference may contribute to the sex dependence of the animals physiological and behavioral responses to an acute stressor.

The behavioral phenotype of pituitary adenylate-cyclase activating polypeptide-deficient mice in anxiety and depression tests is accompanied by blunted c-Fos expression in the bed nucleus of the stria terminalis, central projecting Edinger–Westphal nucleus, ventral lateral septum, and dorsal raphe nucleus

Pituitary adenylate-cyclase activating polypeptide (PACAP) has been implicated in the (patho)physiology of stress-adaptation. PACAP deficient (PACAP(-/-)) mice show altered anxiety levels and depression-like behavior, but little is known about the underlying mechanisms in stress-related brain areas. Therefore, we aimed at investigating PACAP(-/-) mice in light-dark box, marble burying, open field, and forced swim paradigms. We also analyzed whether the forced swim test-induced c-Fos expression would be affected by PACAP deficiency in the following stress-related brain areas: magno- and parvocellular paraventricular nucleus of the hypothalamus (PVN); basolateral (BLA), medial (MeA), and central (CeA) amygdaloid nuclei; ventral (BSTv), dorsolateral (BSTdl), dorsomedial (BSTdm), and oval (BSTov) nuclei of the bed nucleus of stria terminalis; dorsal (dLS) and ventral parts (vLS) of lateral septal nucleus, central projecting Edinger-Westphal nucleus (EWcp), dorsal (dPAG) and lateral (lPAG) periaqueductal gray matter, dorsal raphe nucleus (DR). Our results revealed that PACAP(-/-) mice showed greatly reduced anxiety and increased locomotor activity compared with wildtypes. In forced swim test PACAP(-/-) mice showed increased depression-like behavior. Forced swim exposure increased c-Fos expression in all examined brain areas in wildtypes, whereas this was markedly blunted in the DR, EWcp, BSTov, BSTdl, BSTv, PVN, vLS, dPAG, and in the lPAG of PACAP(-/-) mice vs. wildtypes, strongly suggesting their involvement in the behavioral phenotype of PACAP(-/-) mice. PACAP deficiency did not influence the c-Fos response in the CeA, MeA, BSTdm, and dLS. Therefore, we propose that PACAP exerts a brain area-specific effect on stress-induced neuronal activation and it might contribute to stress-related mood disorders.

Neuropeptide Y activates urocortin 1 neurons in the nonpreganglionic Edinger-Westphal nucleus.

Central regulatory pathways promoting stress adaptation utilize various neurotransmitters/neuropeptides, such as urocortin 1 (Ucn1) and neuropeptide Y (NPY). Ucn1 is abundantly expressed in the nonpreganglionic Edinger‐Westphal nucleus (npEW), where it is codistributed with NPY‐immunoreactive (ir) terminals. A special role for both neuropeptides has been postulated in stress adaptation. Using double‐labeling immunohistochemistry, we observed close appositions between NPY‐ir terminals and neurons immunoreactive for Ucn1 in the rat, as well as in the human npEW. Therefore, we hypothesized that NPY might control the activity of Ucn1‐positive neurons in the npEW. To test this hypothesis, NPY was injected into the lateral cerebral ventricle of rats, resulting in a strong activation of npEW Ucn1 neurons as revealed by Fos immunohistochemistry. Ucn1 mRNA was also upregulated in the npEW 2 hours after the injection of NPY. In a search for the type of NPY receptor that mediates this NPY‐induced recruitment of npEW‐Ucn1 cells, we found that the great majority of Ucn1 cells exhibited NPY Y5 receptor immunoreactivity, and only a few of the Ucn1 cells coexpressed the Y1 receptor. We concluded that NPY, via NPY Y5 and to a lesser extent via the Y1 receptors, exerts a stimulatory action on Ucn1 cells in the npEW. Further studies are currently in progress to elucidate the significance of this NPY–Ucn1 interaction in the npEW. J. Comp. Neurol. 500:708–719, 2007.

Sex-specific effects of fasting on urocortin 1, cocaine- and amphetamine-regulated transcript peptide and nesfatin-1 expression in the rat Edinger–Westphal nucleus

Leptin is critical for normal food intake and energy metabolism. While leptin receptor (ObR) function has been well studied in hypothalamic feeding circuitries, the functional relevance of ObR in extrahypothalamic areas is largely unknown. Central regulatory pathways involved in food intake utilize various neuropeptides, such as urocortin 1 (Ucn1), cocaine- and amphetamine-regulated transcript peptide (CART) and nesfatin-1. Ucn1 is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition to Ucn1, other satiety signals, such as CART and nesfatin-1, are highly expressed in neurons of the npEW. Using immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), we here show the presence of short and long forms of ObR in the rat npEW. Then, we tested our hypothesis that a change in plasma leptin will modulate the activity of npEW neurons containing Ucn1, CART and nesfatin-1. First, by double-labeling immunocytochemistry, we observed that almost all npEW neurons colocalizing Ucn1, CART and nesfatin-1 also contain ObR. Fasting rats for two days caused a marked body weight loss and reduced leptin plasma level in both genders. Ucn1 mRNA and CART mRNA were upregulated after fasting in males (3.3 and 2.4 times, respectively; P<0.05) but not in females. However, their peptide levels were not significantly changed. The peptide level and mRNA of nesfatin-1 were unaffected by fasting. We conclude that npEW-neurons containing Ucn1, CART and nesfatin-1 co-express ObR, and may be involved in leptin-mediated feeding control in male rats only.

Stress-related changes in the activity of cocaine- and amphetamine-regulated transcript and nesfatin neurons in the midbrain non-preganglionic Edinger-Westphal nucleus in the rat.

Cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1/nucleobindin 2 (NUCB2) are assumed to play a role in feeding and adaptation to stress. Both peptides are highly expressed in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW), a center implicated in the regulation of stress adaptation and in the pathogenesis of stress-induced brain disorders, in a sex-specific manner. The present study was undertaken to test whether CART and nesfatin are involved in these actions of the npEW in the rat. Acute restraint and chronic variable mild stress were used. Following stress, physiological parameters (serum corticosterone levels, body, adrenal and thymus weights) were determined, CART and nesfatin-like immunoreactivity (LI) as well as mRNA expression were analyzed in the npEW nucleus. Our results depict the following changes: (1) Acute stress resulted in an increase in serum corticosterone levels that was higher in females; (2) In males, data on corticosterone and body weight gain and in females, data on body weight gain revealed an effect of chronic stress; (3) Both acute and chronic stress activated npEW neurons expressing CART and nesfatin-LI, as shown by increased cFos immunoreactivity; (4) Chronic, but not acute stress increased the amount of CART and nesfatin-LI in both males and females; (5) Neither acute nor chronic stress had an effect on CART and NUCB2 mRNA contents of npEW neurons in either sex. Taken together, our data suggest that CART and nesfatin are involved in the response of npEW neurons to chronic stress.