RESUMEN
We recently demonstrated that the hypoxic ventilatory response (HVR) is facilitated by the AMP-activated protein kinase (AMPK) in catecholaminergic neural networks that likely lie downstream of the carotid bodies within the caudal brainstem. Here, we further subcategorise the neurons involved, by cross-comparison of mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic (TH-Cre) or adrenergic (PNMT-Cre) neurons. As expected, the HVR was markedly attenuated in mice with AMPK-α1/α2 deletion in catecholaminergic neurons, but surprisingly was modestly augmented in mice with AMPK-α1/α2 deletion in adrenergic neurons when compared against a variety of controls (TH-Cre, PNMT-Cre, AMPK-α1/α2 floxed). Moreover, AMPK-α1/α2 deletion in catecholaminergic neurons precipitated marked hypoventilation and apnoea during poikilocapnic hypoxia, relative to controls, while mice with AMPK-α1/α2 deletion in adrenergic neurons entered relative hyperventilation with reduced apnoea frequency and duration. We conclude, therefore, that AMPK-dependent modulation of non-adrenergic networks may facilitate increases in ventilatory drive that shape the classical HVR, whereas AMPK-dependent modulation of adrenergic networks may provide some form of negative feedback or inhibitory input to moderate HVR, which could, for example, protect against hyperventilation-induced hypocapnia and respiratory alkalosis.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Neuronas Adrenérgicas , Tronco Encefálico , Hiperventilación , Hipocapnia , Animales , Ratones , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Apnea/etiología , Apnea/genética , Tronco Encefálico/enzimología , Hiperventilación/complicaciones , Hipoxia/metabolismo , Neuronas Adrenérgicas/enzimología , Hipocapnia/etnología , Hipocapnia/genética , Eliminación de GenRESUMEN
Many studies have shown that a maternal low-protein diet increases the susceptibility of offspring to cardiovascular disease in later-life. Moreover, a lower incidence of cardiovascular disease in females than in males is understood to be largely due to the protective effect of high levels of estrogens throughout a woman's reproductive life. However, to our knowledge, the role of estradiol in moderating the later-life susceptibility of offspring of nutrient-deprived mothers to cardiovascular disease is not fully understood. The present study is aimed at investigating whether oxidative stress in the brainstem caused by a maternal low-protein diet administered during a critical period of fetal/neonatal brain development (i.e during gestation and lactation) is affected by estradiol levels. Female Wistar rat offspring were divided into four groups according to their mothers' diets and to the serum estradiol levels of the offspring at the time of testing: (1) 22 days of age/control diet: (2) 22 days of age/low-protein diet; (3) 122 days of age/control diet: (4) 122 days of age/low-protein diet. Undernutrition in the context of low serum estradiol compared to undernutrition in a higher estradiol context resulted in increased levels of oxidative stress biomarkers and a reduction in enzymatic and non-enzymatic antioxidant defenses. Total global oxy-score showed oxidative damage in 22-day-old rats whose mothers had received a low-protein diet. In the 122-day-old group, we observed a decrease in oxidative stress biomarkers, increased enzymatic antioxidant activity, and a positive oxy-score when compared to control. We conclude from these results that following a protein deficiency in the maternal diet during early development of the offspring, estrogens present at high levels at reproductive age may confer resistance to the oxidative damage in the brainstem that is very apparent in pre-pubertal rats.
Asunto(s)
Tronco Encefálico/metabolismo , Dieta con Restricción de Proteínas/efectos adversos , Desnutrición/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Neuronas/metabolismo , Neuroprotección , Estrés Oxidativo , Animales , Animales Recién Nacidos , Biomarcadores/sangre , Biomarcadores/metabolismo , Tronco Encefálico/enzimología , Estradiol/sangre , Femenino , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Lactancia , Peroxidación de Lípido , Desnutrición/sangre , Desnutrición/etiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Oxidación-Reducción , Oxidorreductasas/metabolismo , Embarazo , Carbonilación Proteica , Ratas WistarRESUMEN
Mutations within the lysosomal enzyme ß-glucocerebrosidase (GC) result in Gaucher disease and represent a major risk factor for developing Parkinson disease (PD). Loss of GC activity leads to accumulation of its substrate glucosylceramide and α-synuclein. Since lysosomal activity of GC is tightly linked to expression of its trafficking receptor, the lysosomal integral membrane protein type-2 (LIMP-2), we studied α-synuclein metabolism in LIMP-2-deficient mice. These mice showed an α-synuclein dosage-dependent phenotype, including severe neurological impairments and premature death. In LIMP-2-deficient brains a significant reduction in GC activity led to lipid storage, disturbed autophagic/lysosomal function, and α-synuclein accumulation mediating neurotoxicity of dopaminergic (DA) neurons, apoptotic cell death, and inflammation. Heterologous expression of LIMP-2 accelerated clearance of overexpressed α-synuclein, possibly through increasing lysosomal GC activity. In surviving DA neurons of human PD midbrain, LIMP-2 levels were increased, probably to compensate for lysosomal GC deficiency. Therefore, we suggest that manipulating LIMP-2 expression to increase lysosomal GC activity is a promising strategy for the treatment of synucleinopathies.
Asunto(s)
Glucosilceramidasa/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/enzimología , Tronco Encefálico/patología , Tronco Encefálico/ultraestructura , Gliosis/complicaciones , Gliosis/patología , Humanos , Lípidos/química , Proteínas de Membrana de los Lisosomas/deficiencia , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/patología , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Neurotoxinas/toxicidadRESUMEN
BACKGROUND AND OBJECTIVES: Studies in humans and animal models have established a close relationship between early environment insult and subsequent risk of development of non-communicable diseases, including the cardiovascular. Whereas experimental evidences highlight the early undernutrition and the late cardiovascular disease relation, the central mechanisms linking the two remain unknown. Owing to the oxidative balance influence in several pathologies, the aim of the present study was to evaluate the effects of maternal undernutrition (i.e. a low-protein (LP) diet) on oxidative balance in the brainstem. METHODS AND RESULTS: Male rats from mothers fed with an LP diet (8% casein) throughout the perinatal period (i.e. gestation and lactation) showed 10× higher lipid peroxidation levels than animals treated with normoprotein (17% casein) at 100 days of age. In addition, we observed the following reductions in enzymatic activities: superoxide dismutase, 16%; catalase, 30%; glutathione peroxidase, 34%; glutathione-S-transferase, 51%; glutathione reductase, 23%; glucose-6-phosphate dehydrogenase, 31%; and in non-enzymatic glutathione system, 46%. DISCUSSION: This study is the first to focus on the role of maternal LP nutrition in oxidative balance in a central nervous system structure responsible for cardiovascular control in adult rats. Our data observed changes in oxidative balance in the offspring, therefore, bring a new concept related to early undernutrition and can help in the development of a new clinical strategy to combat the effects of nutritional insult. Wherein the central oxidative imbalance is a feasible mechanism underlying the hypertension risk in adulthood triggered by maternal LP diet.
Asunto(s)
Antioxidantes/metabolismo , Tronco Encefálico/metabolismo , Dieta con Restricción de Proteínas/efectos adversos , Lactancia , Fenómenos Fisiologicos Nutricionales Maternos , Neuronas/metabolismo , Estrés Oxidativo , Animales , Tronco Encefálico/enzimología , Femenino , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Peroxidación de Lípido , Masculino , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Oxidación-Reducción , Oxidorreductasas/metabolismo , Embarazo , Complicaciones del Embarazo/etiología , Complicaciones del Embarazo/metabolismo , Complicaciones del Embarazo/fisiopatología , Deficiencia de Proteína/etiología , Deficiencia de Proteína/metabolismo , Deficiencia de Proteína/fisiopatología , Ratas WistarRESUMEN
Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.
Asunto(s)
Tronco Encefálico/enzimología , Endocitosis/fisiología , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/metabolismo , Sinapsis/enzimología , Vesículas Sinápticas/enzimología , Animales , Tronco Encefálico/efectos de los fármacos , Endocitosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacosRESUMEN
The locus coeruleus (LC) noradrenergic system regulates arousal and modulates attention through its extensive projections across the brain. LC dysfunction has been implicated in a broad range of neurodevelopmental, neurodegenerative and psychiatric disorders, as well as in the cognitive changes observed during normal aging. Magnetic resonance imaging (MRI) has been used to characterize the human LC (elevated contrast relative to surrounding structures), but there is limited understanding of the factors underlying putative LC contrast that are critical to successful biomarker development and confidence in localizing nucleus LC. We used ultra-high-field 7 T magnetic resonance imaging (MRI) to acquire T1-weighted microscopy resolution images (78 µm in-plane resolution) of the LC from post-mortem tissue samples. Histological analyses were performed to characterize the distribution of tyrosine hydroxylase (TH) and neuromelanin in the scanned tissue, which allowed for direct comparison with MR microscopy images. Our results indicate that LC-MRI contrast corresponds to the location of neuromelanin cells in LC; these also correspond to norepinephrine neurons. Thus, neuromelanin appears to serve as a natural contrast agent for nucleus LC that can be used to localize nucleus LC and may have the potential to characterize neurodegenerative disease.
Asunto(s)
Locus Coeruleus/anatomía & histología , Anciano , Anciano de 80 o más Años , Biomarcadores , Tronco Encefálico/anatomía & histología , Tronco Encefálico/enzimología , Cadáver , Colorantes , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Locus Coeruleus/enzimología , Imagen por Resonancia Magnética , Masculino , Melaninas/metabolismo , Persona de Mediana Edad , Cambios Post Mortem , Reproducibilidad de los Resultados , Tirosina 3-Monooxigenasa/análisisRESUMEN
Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010). In individual mice, the clinical phenotype evolves progressively and rapidly (3-6weeks) from clasping, over general motor impairment to severe reduction in body-weight into the terminal phase that announces imminent death (<3days). Surprisingly, co-expression of GSK3ß with Tau.P301L significantly prolonged survival of bigenic biGT mice (Terwel et al., 2008), which we here assign to delayed development of brainstem tauopathy. Eventually, brainstem tauopathy became as prominent in old biGT mice in the specified brainstem nuclei as in the parental Tau.P301L mice, resulting in similar clinical deterioration and terminal phase preceding death, although at later age. Biochemically, in both genotypes the pathway to neurofibrillary tangles and neuropil threads was similar: phosphorylation of protein Tau and formation of soluble oligomers and insoluble aggregates, ending in the typical tangles and threads of tauopathy. The extra GSK3ß activity led to expected increased phosphorylation of protein Tau, particularly at residues S262 and S396, which we must conclude to delay the aggregation of protein Tau in the brainstem of aging biGT mice. The unexpected, paradoxical alleviation of the brainstem problems in biGT mice allowed them to grow older and thereby develop more severe tauopathy in forebrain than Tau.P301L mice, which succumb at younger age.
Asunto(s)
Tronco Encefálico/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Tauopatías/enzimología , Proteínas tau/química , Proteínas tau/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Tronco Encefálico/metabolismo , Femenino , Glucógeno Sintasa Quinasa 3 beta , Masculino , Ratones , Ratones Transgénicos , Fosforilación , Análisis de Supervivencia , Tauopatías/metabolismoRESUMEN
Prednisolone is a member of synthetic glucocorticoids which are widely used to treat chronic inflammatory diseases. In this study, neuronal degeneration and cell death, and glial reaction were investigated in the rat trigeminal ganglion (TG) and brainstem after subcutaneous injection of prednisolone for 7 days. Expression of c-Jun activating transcription factor 3 and caspase-3 was absent or infrequent in the TG, and cranial sensory and motor nuclei of saline- and prednisolone-treated animals. In these animals, distribution of calcitonin gene-related peptide-immunoreactive (-IR) neurons and nerve fibers was similar in the brainstem. In addition, the number of Iba1- and glial fibrillary acidic protein (GFAP)-IR cells with some processes in the brainstem was barely affected by prednisolone treatment. However, the treatment increased ramification of Iba1-IR processes in the subnucleus caudalis of the trigeminal sensory complex. Prednisolone scarcely influenced the morphology of GFAP-IR cells in the brainstem. Expression of p38 mitogen-activated protein kinase was very rare in the brainstem of saline- and prednisolone-treated animals. The present study suggests that microglia are activated by prednisolone in the subnucleus caudalis of the trigeminal sensory complex. The glucocorticoid may affect nociceptive transmission in the brainstem.
Asunto(s)
Microglía/metabolismo , Prednisolona/farmacología , Núcleo Caudal del Trigémino/metabolismo , Factor de Transcripción Activador 3/metabolismo , Animales , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/enzimología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Caspasa 3/metabolismo , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Núcleo Caudal del Trigémino/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The present investigation was carried out to evaluate alterations in oxidative stress parameter [lipid peroxidation (LPx)] and antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] in rat brainstem in response to neonatal hypothyroidism during development (from birth to 7, 15 and 30 days old) and adulthood (90 days old). Hypothyroidism in rats was induced by feeding the lactating mothers (from the day of parturition till weaning, 25 days old) or directly to the pups with 0.05 % [6-n-propyl 2-thiouracil (PTU)] in drinking water. Increased level of LPx was observed in brainstem of 7 days old hypothyroid rats, accompanied by augmented activities of SOD and GPx. In 15 and 30 days old hypothyroid rat brainstem, a significant decline in LPx was observed. Significantly increased activities of CAT and GPx were observed in 15 and 30 days PTU-treated rats. Decreased level of LPx was observed in brainstem of rats treated with PTU from birth to 30 days followed by withdrawal up to 90 days of age (transient hypothyroidism) as compared to control and persistent treatment of PTU up to 90 days of age. Activities of CAT and GPx were decreased in persistent hypothyroid rats of 90 days old with respect to control and transient hypothyroid rats. On the other hand, SOD activity was decreased in both persistent and transient hypothyroid rats with respect to control rats. These results suggest that the PTU-induced neonatal hypothyroidism modulates the antioxidant defence system during postnatal development and adulthood in brainstem of rats.
Asunto(s)
Antioxidantes/metabolismo , Tronco Encefálico/enzimología , Hipotiroidismo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Animales Lactantes , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Hipotiroidismo/inducido químicamente , Peroxidación de Lípido , Masculino , Oxidación-Reducción , Estrés Oxidativo , Propiltiouracilo/toxicidad , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
Phospholipases A(2) (PLA(2)) are enzymes that cleave the sn-2 bond of membrane phospholipids to yield free fatty acids and lysophospholipids. Secretory PLA2-III (sPLA(2)-III) has been suggested to be important for neuronal differentiation, growth and survival, and is highly expressed in the spinal cord. The aim of this study is to elucidate its expression and distribution in different regions of the adult rat CNS. Quantitative RT-PCR analyses showed high levels of sPLA(2)-III mRNA expression in the brainstem and spinal cord and low expression in the olfactory bulb. Western blot analyses showed high level of expression in the brainstem, spinal cord and cerebral neocortex. A dense band corresponding to the catalytically active, mature/cleaved form, and a faint band corresponding to the full length sPLA(2)-III were detected in post-mitochondrial supernatants, from different parts of the CNS. Subcellular fractionation of spinal cord homogenates showed that sPLA(2)-III protein is present in the 'light membrane/cytosol' fraction, but not the nucleus, synaptosomal membrane or synaptic vesicle-enriched fractions. sPLA(2)-III was immunolocalized to neurons in the cerebral neocortex, Purkinje neurons in the cerebellar cortex, periaqueductal gray, red nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Electron microscopy of the spinal cord and cerebral neocortex showed that sPLA(2)-III was localized in dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled, putatively glutamatergic, axon terminals. The localization of mature/cleaved form of sPLA(2)-III in postsynaptic structures suggest a physiological role of the enzyme in neurotransmission or synaptic plasticity.
Asunto(s)
Sistema Nervioso Central/enzimología , Fosfolipasas A2 Grupo III/biosíntesis , Animales , Tronco Encefálico/enzimología , Tronco Encefálico/ultraestructura , Corteza Cerebral/enzimología , Corteza Cerebral/ultraestructura , Dendritas/enzimología , Dendritas/ultraestructura , Masculino , Terminales Presinápticos/ultraestructura , Ratas , Médula Espinal/enzimología , Médula Espinal/ultraestructuraRESUMEN
The aims of the present study were to determine the mechanism underlying enhanced neuronal nitric oxide synthase (nNOS) activity in the brain of hypertensive Dahl salt-sensitive (DSS) rats and the consequences of enhanced nNOS activity. Male DSS rats were fed either a regular (0.4% NaCl) or high-salt (8% NaCl) diet, with or without 0.25% nifedipine, for 4 weeks. The effects of nifedipine, which lowers blood pressure peripherally, on central nNOS were determined by measuring nNOS activity, as well as the number of nNOS-positive neurons in the brain stem and diencephalon. The effects of chronic (12 days) infusion of 7 µg (0.5 µL/h, i.c.v.) S-methyl-L-thiocitrulline (SMTC; a stereoselective competitive nNOS inhibitor) on mean arterial pressure were assessed in conscious DSS rats using a radiotelemetry system. In addition, the number of central nNOS-positive neurons was compared between DSS and salt-insensitive Sprague-Dawley rats. Normalization of blood pressure by nifedipine attenuated the increase in nNOS activity in the brain stem of DSS rats. Chronic i.c.v. infusion of SMTC further enhanced hypertension in DSS rats. Feeding of a high-salt diet increased nNOS-positive neurons in the lateral parabrachial nucleus, rostral ventrolateral medulla and nucleus tractus solitarius of DSS compared with Sprague-Dawley rats, whereas nNOS-positive neurons in the paraventricular nucleus remained downregulated in DSS rats. The results of the present study suggest that hypertension, rather than a high-salt diet, increases central nNOS activity in hypertensive DSS rats to buffer high blood pressure. However, this compensatory response may be insufficient to relieve salt-induced hypertension.
Asunto(s)
Tronco Encefálico/enzimología , Hipertensión/enzimología , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Presión Arterial/efectos de los fármacos , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/patología , Citrulina/análogos & derivados , Citrulina/farmacología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Hipertensión/patología , Inmunohistoquímica , Masculino , Neuronas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Ratas , Ratas Endogámicas Dahl , Ratas Sprague-Dawley , Cloruro de Sodio Dietético/efectos adversos , Telemetría , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
The distribution of nitroxide ergic neurons in the medulla oblongata nuclei in Wistar rats (n = 8) was studied histochemically (NADPH-diaphorase) and using immunohistochemistry with an antiserum against neuronal form of nitric oxide synthase (nNOS). NADPH-diaphorase activity was found in large and small neurons of the sensory, autonomic and motor nuclei. The latter were especially rich in the cells demonstrating the activity of the enzyme. Unlike NADPH-diaphorase, nNOS in the corresponding nuclei was always detected in the fewer number of neurons, predominantly of small sizes. The sensory nuclei (nucleus of solitary tract, reticular parvocellular and lateral nuclei, spinal nucleus of the trigeminal nerve) contained 1.5-3 times more nNOS neurons than in motor nuclei. In some nuclei (nucleus ambiguus, hypoglossal nerve nucleus), containing numerous NADPH-diaphorase-positive neurons, immunoreactive cells were particularly rare.
Asunto(s)
Bulbo Raquídeo/enzimología , NADPH Deshidrogenasa/metabolismo , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Tronco Encefálico/enzimología , Tronco Encefálico/metabolismo , Tronco Encefálico/ultraestructura , Inmunohistoquímica , Bulbo Raquídeo/metabolismo , Bulbo Raquídeo/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Óxidos de Nitrógeno/metabolismo , Ratas , Ratas Wistar , Distribución TisularRESUMEN
Our recent study demonstrated that central cannabinoid receptor 1 (CB1R) activation caused dose-related pressor response in conscious rats, and reported studies implicated the brainstem phosphatidylinositol 3-kinase (PI3K)/Akt-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in blood pressure control. Therefore, in this study, we tested the hypothesis that the modulation of brainstem PI3K/Akt-ERK1/2 signaling plays a critical role in the central CB(1)R-mediated pressor response. In conscious freely moving rats, the pressor response elicited by intracisternal (i.c.) (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt (WIN55,212-2) (15 µg) was associated with significant increases in ERK1/2 phosphorylation in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS). In contrast, Akt phosphorylation was significantly reduced in the same neuronal pools. Pretreatment with the selective CB1R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (30 µg i.c.) attenuated the neurochemical responses elicited by central CB1R activation. Furthermore, pretreatment with the ERK/mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) (5 µg i.c.) abrogated WIN55,212-2-evoked increases in blood pressure and neuronal ERK1/2 phosphorylation but not the reduction in Akt phosphorylation. On the other hand, prior PI3K inhibition with wortmannin (0.4 µg i.c.) exacerbated the WIN55,212-2 (7.5 and 15 µg i.c.) dose-related increases in blood pressure and ERK1/2 phosphorylation in the RVLM. The present neurochemical and integrative studies yield new insight into the critical role of two brainstem kinases, PI3K and ERK1/2, in the pressor response elicited by central CB1R activation in conscious rats.
Asunto(s)
Benzoxazinas/farmacología , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Tronco Encefálico/enzimología , Tronco Encefálico/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Morfolinas/farmacología , Naftalenos/farmacología , Proteína Oncogénica v-akt/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Androstadienos/farmacología , Animales , Western Blotting , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Bulbo Raquídeo/fisiología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Fosforilación , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/fisiología , Núcleo Solitario/fisiología , WortmaninaRESUMEN
Brain noradrenergic system has been implicated in early-life stress effects on adult physiology and behavior; however, the mechanisms for this relationship are not clear. Here we tested the hypothesis that stress hormones, glucocorticoids, may affect noradrenergic system activity by modulating gene expression and function of tyrosine hydroxylase (TH), the key enzyme for catecholamine synthesis, in the rat brain during perinatal life. We have shown that TH mRNA levels and enzyme activity increase in the fetal rat brainstem during the last days of pregnancy. Administration of hydrocortisone or dexamethasone to female rats on day 20 of pregnancy significantly increased TH mRNA levels (real-time PCR) and enzyme activity (DOPA accumulation after inhibition of aromatic L: -amino acid decarboxylase with NSD-1015) as well as noradrenaline concentrations in the brainstem of fetuses 6 h after the treatment. Similar glucocorticoid effects on fetal TH and noradrenaline were observed 72 h after the treatment with hydrocortisone on days 16 and 18 of pregnancy. In contrast to fetuses, no effects on the TH were revealed in the brainstem of neonatal pups after single or repeated injections of hydrocortisone or dexamethasone. TH gene expression remains at a relatively constant level in the early neonatal rat brain. The results suggest that glucocorticoids are capable of inducing TH at both transcriptional and enzyme activity levels in the brainstem of near-term fetuses.
Asunto(s)
Encéfalo/enzimología , Regulación Enzimológica de la Expresión Génica , Glucocorticoides/fisiología , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/genética , Factores de Edad , Animales , Animales Recién Nacidos , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/enzimología , Inducción Enzimática/fisiología , Femenino , Embarazo , ARN Mensajero/biosíntesis , Ratas , Ratas WistarRESUMEN
RATIONALE: Angiotensin converting enzyme type 2 (ACE2) is a new member of the brain renin-angiotensin system, that might be activated by an overactive renin-angiotensin system. OBJECTIVE: To clarify the role of central ACE2 using a new transgenic mouse model with human (h)ACE2 under the control of a synapsin promoter, allowing neuron-targeted expression in the central nervous system. METHODS AND RESULTS: Syn-hACE2 (SA) transgenic mice exhibit high hACE2 protein expression and activity throughout the brain. Baseline hemodynamic parameters (telemetry), autonomic function, and spontaneous baroreflex sensitivity (SBRS) were not significantly different between SA mice and nontransgenic littermates. Brain-targeted ACE2 overexpression attenuated the development of neurogenic hypertension (Ang II infusion: 600 ng/kg per minute for 14 days) and the associated reduction of both SBRS and parasympathetic tone. This prevention of hypertension by ACE2 overexpression was reversed by blockade of the Ang-(1-7) receptor (d-Ala7-Ang-[1-7]; 600 ng/kg per minute). Brain angiotensin II type 2 (AT(2))/AT(1) and Mas/AT(1) receptor ratios were significantly increased in SA mice. They remained higher following Ang II infusion but were dramatically reduced after Ang-(1-7) receptor blockade. ACE2 overexpression resulted in increased NOS and NO levels in the brain, and prevented the Ang II-mediated decrease in NOS expression in regions modulating blood pressure regulation. CONCLUSIONS: ACE2 overexpression attenuates the development of neurogenic hypertension partially by preventing the decrease in both SBRS and parasympathetic tone. These protective effects might be mediated by enhanced NO release in the brain resulting from Mas and AT(2) receptor upregulation. Taken together, our data highlight the compensatory role of central ACE2 and its potential benefits as a therapeutic target for neurogenic hypertension.
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Encéfalo/metabolismo , Regulación Enzimológica de la Expresión Génica , Hipertensión/genética , Peptidil-Dipeptidasa A/genética , Angiotensina II/sangre , Angiotensina II/metabolismo , Angiotensina II/farmacología , Enzima Convertidora de Angiotensina 2 , Animales , Barorreflejo/fisiología , Presión Sanguínea/efectos de los fármacos , Western Blotting , Encéfalo/enzimología , Tronco Encefálico/enzimología , Tronco Encefálico/metabolismo , Femenino , Humanos , Hipertensión/enzimología , Hipertensión/fisiopatología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Sistema Nervioso Parasimpático/fisiología , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Peptidil-Dipeptidasa A/metabolismo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Immunocytochemical method was used to determine the distribution of neurons expressing heme oxygenase-2 (HO-2-positive neurons) in the nuclei of various parts of the brainstem of 16 male Wistar rats. The sizes of neurons and the optical density of the product of histochemical reaction in their cytoplasm were determined in the nuclei studied. HO-2-positive neurons, differing in shape, size and numbers, were identified in the nuclei of the medulla oblongata, pons and midbrain. HO-2-positive cells were found 3-5 times more frequently in the sensory nuclei as compared to the motor ones. At the same time, relatively large number of nuclei was detected, which contained either no or a few HO-2-positive neurons.
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Tronco Encefálico/enzimología , Hemo Oxigenasa (Desciclizante)/análisis , Animales , Masculino , Neuronas/enzimología , Ratas , Ratas WistarRESUMEN
Amyotrophic lateral sclerosis (ALS) is a paralyzing disorder characterized by the progressive degeneration and death of motor neurons and occurs both as a sporadic and familial disease. Mutant SOD1 (mtSOD1) in motor neurons induces vulnerability to the disease through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities, defective axonal transport- and growth factor signaling, excitotoxicity, and neuro-inflammation.Melittin is a 26 amino acid protein and is one of the components of bee venom which is used in traditional Chinese medicine to inhibit of cancer cell proliferation and is known to have anti-inflammatory and anti-arthritic effects.The purpose of the present study was to determine if melittin could suppress motor neuron loss and protein misfolding in the hSOD1G93A mouse, which is commonly used as a model for inherited ALS. Meltittin was injected at the 'ZuSanLi' (ST36) acupuncture point in the hSOD1G93A animal model. Melittin-treated animals showed a decrease in the number of microglia and in the expression level of phospho-p38 in the spinal cord and brainstem. Interestingly, melittin treatment in symptomatic ALS animals improved motor function and reduced the level of neuron death in the spinal cord when compared to the control group. Furthermore, we found increased of α-synuclein modifications, such as phosphorylation or nitration, in both the brainstem and spinal cord in hSOD1G93A mice. However, melittin treatment reduced α-synuclein misfolding and restored the proteasomal activity in the brainstem and spinal cord of symptomatic hSOD1G93A transgenic mice.Our research suggests a potential functional link between melittin and the inhibition of neuroinflammation in an ALS animal model.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/enzimología , Meliteno/farmacología , Meliteno/uso terapéutico , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Conducta Animal/efectos de los fármacos , Tronco Encefálico/citología , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/enzimología , Tronco Encefálico/patología , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Pliegue de Proteína/efectos de los fármacos , Prueba de Desempeño de Rotación con Aceleración Constante , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/enzimología , Médula Espinal/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Tasa de Supervivencia , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Neck muscle nociception mediated by nitric oxide may play a role in the pathophysiology of tension-type headache. OBJECTIVE: The present study addresses the involvement of neuronal nitric oxide synthase (nNOS) in the facilitation of neck muscle nociception after local application of nerve growth factor (NGF). METHODS: After administration of NGF into semispinal neck muscles, the impact of neck muscle noxious input on brainstem processing was monitored by the jaw-opening reflex in anesthetized mice. The modulatory effect of preceding and subsequent administration of an inhibitor of neuronal nitric oxide synthase on central facilitation was addressed in a controlled study. RESULTS: With preceding i.p. application of saline or 0.096 mg/kg of the specific nNOS inhibitor Nω-propyl-L-arginine (NPLA), NGF induced a sustained reflex facilitation within 60 minutes. Preceding injection of 0.96 mg/kg or 1.92 mg/kg NPLA completely prevented the potentially facilitatory effect of NGF. Subsequent administration of 0.96 mg/kg NPLA did not affect established NGF-evoked reflex facilitation. Thus, NPLA prevents facilitation of brainstem processing by noxious myofascial input from neck muscles in a dose-dependent manner. CONCLUSION: These findings suggest that nNOS is involved in the induction but not the maintenance of NGF-evoked facilitation of nociception in the brainstem. These results from an experimental animal model may support the idea of NOS and nNOS as potential targets for pharmacological treatment of tension-type headache.
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Músculos del Cuello/inervación , Óxido Nítrico Sintasa de Tipo I/metabolismo , Dolor/enzimología , Cefalea de Tipo Tensional/enzimología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Tronco Encefálico/enzimología , Modelos Animales de Enfermedad , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos del Cuello/efectos de los fármacos , Músculos del Cuello/fisiopatología , Factor de Crecimiento Nervioso/toxicidad , Dolor/fisiopatología , Reflejo/efectos de los fármacos , Cefalea de Tipo Tensional/inducido químicamente , Cefalea de Tipo Tensional/fisiopatologíaRESUMEN
Cyclic changes in activity of Ca2+-dependent neutral protease occur during preparation for hibernation, with an increase in September and November and decrease in October and December. During hibernation proteolytic enzyme activity decreased, while during self-warming after induced awakening, the role of Ca2+-dependent processes in the tissues of ground squirrels increased according to the body temperature.
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Tronco Encefálico/enzimología , Calpaína/metabolismo , Corteza Cerebral/enzimología , Hibernación , Miocardio/enzimología , Sciuridae/fisiología , Animales , Especificidad de Órganos , Sciuridae/metabolismoRESUMEN
Investigated by immunohistochemistry the presence and distribution of CBS in the nuclei of the spinal cord and brainstem in 8 mens of 18-44 years old, died from mechanical injury is not related to the central nervous system damage. Established that the CBS-neurons are revealed in all parts of stem and spinal cord, but their content in the studied cores, ranging from 0.9 to 17 %. Observed a definite relationship between the activity of CBS and the quantity of neurons. Large cells of motor nuclei often have a high and very high intensity of the reaction. In the sensory nuclei of a high proportion of small neurons with low activity of the enzyme.