RESUMO
Obstructive sleep apnea (OSA), a chronic sleep disorder characterized by repetitive reduction or cessation of airflow during sleep, is widely prevalent and is associated with adverse neurocognitive sequelae including increased risk of Alzheimer's disease (AD). In humans, OSA is more common in elderly males. OSA is characterized by sleep fragmentation and chronic intermittent hypoxia (CIH), and recent epidemiological studies point to CIH as the best predictor of neurocognitive sequelae associated with OSA. The sex- and age- specific effects of OSA-associated CIH on specific cell populations such as γ-aminobutyric acid (GABA)-ergic neurons in the hippocampus and the medial prefrontal cortex (mPFC), regions important for cognitive function, remain largely unknown. The present study examined the effect of 35 days of either moderate (10% oxygen) or severe (5% oxygen) CIH on GABAergic neurons in the mPFC and hippocampus of young and aged male and female mice as well as post-accelerated ovarian failure (AOF) female mice. In the mPFC and hippocampus, the number of GABA-labeled neurons increased in aged and young severe CIH males compared to controls but not in young moderate CIH males. This change was not representative of the individual GABAergic cell subpopulations, as the number of parvalbumin-labeled neurons decreased while the number of somatostatin-labeled neurons increased in the hippocampus of severe CIH young males only. In all female groups, the number of GABA-labeled cells was not different between CIH and controls. However, in the mPFC, CIH increased the number of parvalbumin-labeled neurons in young females and the number of somatostatin-labeled cells in AOF females but decreased the number of somatostatin-labeled cells in aged females. In the hippocampus, CIH decreased the number of somatostatin-labeled neurons in young females. CIH decreased the density of vesicular GABA transporter in the mPFC of AOF females only. These findings suggest sex-specific changes in GABAergic neurons in the hippocampus and mPFC with males showing an increase of this cell population as compared to their female counterparts following CIH. Age at exposure and severity of CIH also differentially affect the GABAergic cell population in mice.
Assuntos
Neurônios GABAérgicos/patologia , Hipocampo/patologia , Hipóxia Encefálica/patologia , Córtex Pré-Frontal/patologia , Fatores Etários , Animais , Contagem de Células , Feminino , Hipocampo/metabolismo , Hipóxia Encefálica/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/metabolismo , Caracteres SexuaisRESUMO
Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulation of the hypothalamic paraventricular nucleus (PVN). Many forms of glutamate-dependent plasticity are mediated by NMDA receptor GluN1 subunit expression and the distribution of functional receptor to the plasma membrane of dendrites. Here, we use a combined ultrastructural and functional analysis to examine the relationship between PVN NMDA receptors and the blood pressure increase induced by chronic infusion of a low dose of Ang II. We report that the increase in blood pressure produced by a 2 week administration of a subpressor dose of Ang II results in an elevation in plasma membrane GluN1 in dendrites of PVN neurons in adult male mice. The functional implications of these observations are further demonstrated by the finding that GluN1 deletion in PVN neurons attenuated the Ang II-induced increases in blood pressure. These results indicate that NMDA receptor plasticity in PVN neurons significantly contributes to the elevated blood pressure mediated by Ang II.
Assuntos
Angiotensina II/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Proteínas do Tecido Nervoso/metabolismo , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Análise de Variância , Animais , Lateralidade Funcional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Imunoeletrônica , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/ultraestrutura , Óxido Nítrico Sintase Tipo I/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Hipotalâmico Paraventricular/ultraestrutura , Pletismografia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de N-Metil-D-Aspartato/genética , VasoconstritoresRESUMO
Adaptive changes in glutamatergic signaling within the hypothalamic paraventricular nucleus (PVN) may play a role in the neurohumoral dysfunction underlying the hypertension induced by "slow-pressor" ANG II infusion. We hypothesized that these adaptive changes alter production of gp91phox NADPH oxidase (NOX)-derived reactive oxygen species (ROS) or nitric oxide (NO), resulting in enhanced glutamatergic signaling in the PVN. Electron microscopic immunolabeling showed colocalization of NOX2 and N-methyl-D-aspartate receptor (NMDAR) NR1 subunits in PVN dendrites, an effect enhanced (+48%, P < 0.05 vs. saline) in mice receiving ANG II (600 ng·kg⻹·min⻹ sc). Isolated PVN cells or spinally projecting PVN neurons from ANG II-infused mice had increased levels of ROS at baseline (+40 ± 5% and +57.6 ± 7.7%, P < 0.01 vs. saline) and after NMDA (+24 ± 7% and +17 ± 5.5%, P < 0.01 and P < 0.05 vs. saline). In contrast, ANG II infusion suppressed NO production in PVN cells at baseline (-29.1 ± 5.2%, P < 0.05 vs. saline) and after NMDA (-18.9 ± 2%, P < 0.01 vs. saline), an effect counteracted by NOX inhibition. In whole cell recording of unlabeled and spinally labeled PVN neurons in slices, NMDA induced a larger inward current in ANG II than in saline groups (+79 ± 24% and +82.9 ± 6.6%, P < 0.01 vs. saline), which was reversed by the ROS scavenger MnTBAP and the NO donor S-nitroso-N-acetylpenicillamine (P > 0.05 vs. control). These findings suggest that slow-pressor ANG II increases the association of NR1 with NOX2 in dendrites of PVN neurons, resulting in enhanced NOX-derived ROS and reduced NO during glutamatergic activity. The resulting enhancement of NMDAR activity may contribute to the neurohumoral dysfunction underlying the development of slow-pressor ANG II hypertension.
Assuntos
Angiotensina II/efeitos adversos , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Glicoproteínas de Membrana/metabolismo , N-Metilaspartato/metabolismo , NADPH Oxidases/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Superóxidos/metabolismo , Angiotensina II/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Modelos Animais , NADPH Oxidase 2 , Neurônios/citologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Óxido Nítrico/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/ultraestrutura , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
NADPH oxidase-generated reactive oxygen species (ROS) are highly implicated in the development of angiotensin II (AngII)-dependent hypertension mediated in part through the hypothalamic paraventricular nucleus (PVN). This region contains vasopressin and non-vasopressin neurons that are responsive to cardiovascular dysregulation, but it is not known whether ROS is generated by one or both cell types in response to "slow-pressor" infusion of AngII. We addressed this question using ROS imaging and electron microscopic dual labeling for vasopressin and p47(phox), a cytoplasmic NADPH oxidase subunit requiring mobilization to membranes for the initiation of ROS production. C57BL/6 mice or vasopressin-enhanced green fluorescent protein (VP-eGFP) mice were infused systemically with saline or AngII (600 ng · kg(-1) · min(-1), s.c.) for 2 weeks, during which they slowly developed hypertension. Ultrastructural analysis of the PVN demonstrated p47(phox) immunolabeling in many glial and neuronal profiles, most of which were postsynaptic dendrites. Compared with saline, AngII recipient mice had a significant increase in p47(phox) immunolabeling on endomembranes just beneath the plasmalemmal surface (+42.1 ± 11.3%; p < 0.05) in non-vasopressin dendrites. In contrast, AngII infusion decreased p47(phox) immunolabeling on the plasma membrane (-35.5 ± 16.5%; p < 0.05) in vasopressin dendrites. Isolated non-VP-eGFP neurons from the PVN of AngII-infused mice also showed an increase in baseline ROS production not seen in VP-eGFP neurons. Our results suggest that chronic low-dose AngII may offset the homeostatic control of blood pressure by differentially affecting membrane assembly of NADPH oxidase and ROS production in vasopressin and non-vasopressin neurons located within the PVN.
Assuntos
Hipertensão/patologia , NADPH Oxidases/metabolismo , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/patologia , Espécies Reativas de Oxigênio/metabolismo , Angiotensina II/administração & dosagem , Angiotensina II/efeitos adversos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Esquema de Medicação , Sistemas de Liberação de Medicamentos , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Hipertensão/induzido quimicamente , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Imunoeletrônica , N-Metilaspartato/farmacologia , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Transfecção , Vasoconstritores/administração & dosagem , Vasoconstritores/efeitos adversos , Vasopressinas/genética , Vasopressinas/metabolismoRESUMO
The medial region of the nucleus tractus solitarius (mNTS) is a key brain stem site controlling cardiovascular function, wherein ANG II modulates neuronal L-type Ca(2+) currents via activation of ANG II type 1 receptors (AT(1)R) and production of reactive oxygen species (ROS). ANG II type 2 receptors (AT(2)R) induce production of nitric oxide (NO), which may interact with ROS and modulate AT(1)R signaling. We sought to determine whether AT(2)R-mediated NO production occurs in mNTS neurons and, if so, to elucidate the NO source and the functional interaction with AT(1)R-induced ROS or Ca(2+) influx. Electron microscopic (EM) immunolabeling showed that AT(2)R and neuronal NO synthase (nNOS) are coexpressed in neuronal somata and dendrites receiving synapses in the mNTS. In the presence of the AT(1)R antagonist losartan, ANG II increased NO production in isolated mNTS neurons, an effect blocked by the AT(2)R antagonist PD123319, but not the angiotensin (1-7) antagonist D-Ala. Studies in mNTS neurons of nNOS-null or endothelial NOS (eNOS)-null mice established nNOS as the source of NO. ANG II-induced ROS production was enhanced by PD123319, the NOS inhibitor N(G)-nitro-l-arginine (LNNA), or in nNOS-null mice. Moreover, in the presence of losartan, ANG II reduced voltage-gated L-type Ca(2+) current, an effect blocked by PD123319 or LNNA. We conclude that AT(2)R are closely associated and functionally coupled with nNOS in mNTS neurons. The resulting NO production antagonizes AT(1)R-mediated ROS and dampens L-type Ca(2+) currents. The ensuing signaling changes in the NTS may counteract the deleterious effects of AT(1)R on cardiovascular function.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Óxido Nítrico/biossíntese , Receptores de Angiotensina/metabolismo , Núcleo Solitário/metabolismo , Animais , Células Cultivadas , Radicais Livres/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-DawleyRESUMO
Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly developing chemosensory-dependent blood pressure elevation ascribed in part to NMDA receptor-dependent plasticity and reduced nitric oxide (NO) signaling in the carotid body. The hypothalamic paraventricular nucleus (PVN) is responsive to hypoxic stress and also contains neurons that express NMDA receptors and neuronal nitric oxide synthase (nNOS). We tested the hypothesis that extended (35 d) CIH results in a decrease in the surface/synaptic availability of the essential NMDA NR1 subunit in nNOS-containing neurons and NMDA-induced NO production in the PVN of mice. As compared with controls, the 35 d CIH-exposed mice showed a significant increase in blood pressure and an increased density of NR1 immunogold particles located in the cytoplasm of nNOS-containing dendrites. Neither of these between-group differences was seen after 14 d, even though there was already a reduction in the NR1 plasmalemmal density at this time point. Patch-clamp recording of PVN neurons in slices showed a significant reduction in NMDA currents after either 14 or 35 d exposure to CIH compared with sham controls. In contrast, NO production, as measured by the NO-sensitive fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein, was suppressed only in the 35 d CIH group. We conclude that CIH produces a reduction in the surface/synaptic targeting of NR1 in nNOS neurons and decreases NMDA receptor-mediated currents in the PVN before the emergence of hypertension, the development of which may be enabled by suppression of NO signaling in this brain region.
Assuntos
Hipóxia/patologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Óxido Nítrico/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Arginina/farmacologia , Gasometria/métodos , Pressão Sanguínea/fisiologia , Óxidos N-Cíclicos/farmacologia , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Sequestradores de Radicais Livres/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hipóxia/fisiopatologia , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão/métodos , N-Metilaspartato/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo I/ultraestrutura , Núcleo Hipotalâmico Paraventricular/patologia , Núcleo Hipotalâmico Paraventricular/ultraestrutura , Receptores de N-Metil-D-Aspartato/ultraestrutura , S-Nitroso-N-Acetilpenicilamina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vasopressinas/metabolismoRESUMO
Chronic intermittent hypoxia (CIH) is a frequent concomitant of sleep apnea, which can increase sympathetic nerve activity through mechanisms involving chemoreceptor inputs to the commissural nucleus of the solitary tract (cNTS). These chemosensory inputs co-store glutamate and substance P (SP), an endogenous ligand for neurokinin-1 (NK(1)) receptors. Acute hypoxia results in internalization of NK(1) receptors, suggesting that CIH also may affect the subcellular distribution of NK(1) receptors in subpopulations of cNTS neurons, some of which may express tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis (TH). To test this hypothesis, we examined dual immunolabeling for the NK(1) receptor and TH in the cNTS of male mice subjected to 10days or 35days of CIH or intermittent air. Electron microscopy revealed that NK(1) receptors and TH were almost exclusively localized within separate somatodendritic profiles in cNTS of control mice. In dendrites, immunogold particles identifying NK(1) receptors were prevalent in the cytoplasm and on the plasmalemmal surface. Compared with controls, CIH produced a significant region-specific decrease in the cytoplasmic (10 and 35days, P<0.05, unpaired Student t-test) and extrasynaptic plasmalemmal (35days, P<0.01, unpaired Student t-test) density of NK(1) immunogold particles exclusively in small (<0.1microm) dendrites without TH immunoreactivity. These results suggest that CIH produces a duration-dependent reduction in the availability of NK(1) receptors preferentially in small dendrites of non-catecholaminergic neurons in the cNTS. The implications of our findings are discussed with respect to their potential involvement in the slowly developing hypertension seen in sleep apnea patients.
Assuntos
Dendritos/metabolismo , Hipóxia Encefálica/metabolismo , Receptores da Neurocinina-1/metabolismo , Síndromes da Apneia do Sono/metabolismo , Núcleo Solitário/metabolismo , Animais , Catecolaminas/metabolismo , Doença Crônica , Dendritos/patologia , Dendritos/ultraestrutura , Imunofluorescência , Hipóxia Encefálica/patologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Reflexo/fisiologia , Mecânica Respiratória/fisiologia , Síndromes da Apneia do Sono/patologia , Núcleo Solitário/patologia , Substância P/metabolismo , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologia , Núcleo Espinal do Trigêmeo/metabolismo , Núcleo Espinal do Trigêmeo/patologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Dandruff and seborrheic dermatitis (D/SD) are common hyperproliferative scalp disorders with a similar etiology. Both result, in part, from metabolic activity of Malassezia globosa and Malassezia restricta, commensal basidiomycete yeasts commonly found on human scalps. Current hypotheses about the mechanism of D/SD include Malassezia-induced fatty acid metabolism, particularly lipase-mediated breakdown of sebaceous lipids and release of irritating free fatty acids. We report that lipase activity was detected in four species of Malassezia, including M. globosa. We isolated lipase activity by washing M. globosa cells. The isolated lipase was active against diolein, but not triolein. In contrast, intact cells showed lipase activity against both substrates, suggesting the presence of at least another lipase. The diglyceride-hydrolyzing lipase was purified from the extract, and much of its sequence was determined by peptide sequencing. The corresponding lipase gene (LIP1) was cloned and sequenced. Confirmation that LIP1 encoded a functional lipase was obtained using a covalent lipase inhibitor. LIP1 was differentially expressed in vitro. Expression was detected on three out of five human scalps, as indicated by reverse transcription-PCR. This is the first step in a molecular description of lipid metabolism on the scalp, ultimately leading toward a test of its role in D/SD etiology.
Assuntos
Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lipase/genética , Lipase/metabolismo , Malassezia/enzimologia , Couro Cabeludo/microbiologia , Clonagem Molecular , Diglicerídeos/química , Regulação Fúngica da Expressão Gênica , Glicerídeos/química , Humanos , Lipídeos/química , Modelos Biológicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trioleína/químicaRESUMO
In vivo microdialysis in C57BL/6J (B6) mouse was used to test the hypothesis that activating adenosine A(2A) receptors in the pontine reticular formation (PRF) increases acetylcholine (ACh) release and rapid eye movement (REM) sleep. Eight concentrations of the adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; CGS) were delivered to the PRF and ACh in the PRF was quantified. ACh release was significantly increased by dialysis with 3 mum CGS and significantly decreased by dialysis with 10 and 100 microm CGS. Co-administration of the adenosine A(2A) receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM) blocked the CGS-induced increase in ACh release. In a second series of experiments, CGS (3 microm) was delivered by dialysis to the PRF for 2 h while recording sleep and wakefulness. CGS significantly decreased time in wakefulness (-51% in h 1; -54% in h 2), increased time in non-rapid eye movement (NREM) sleep (90% in h 1; 151% in h 2), and increased both time in REM sleep (331% in h 2) and the number of REM sleep episodes (488% in h 2). The enhancement of REM sleep is consistent with the interpretation that adenosine A(2A) receptors in the PRF of the B6 mouse contribute to REM sleep regulation, in part, by increasing ACh release in the PRF. A(2A) receptor activation may promote NREM sleep via GABAergic inhibition of arousal promoting neurons in the PRF.