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
Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. Here, we hypothesize that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induce ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identify a critical period of vulnerability to GALC ablation between P4-6 in mice. Early Galc-iKO induction causes a worse KD phenotype, higher psychosine levels in the rodent brainstem and spinal cord, and a significantly shorter life-span of the mice. Intriguingly, GALC expression peaks during this critical developmental period in mice. Further analysis of this mouse model reveals a cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis.
Asunto(s)
Tronco Encefálico/enzimología , Tronco Encefálico/crecimiento & desarrollo , Tronco Encefálico/metabolismo , Galactosilceramidasa/genética , Galactosilceramidasa/metabolismo , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Animales , Tronco Encefálico/embriología , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Fenotipo , Psicosina/metabolismo , Tamoxifeno , TranscriptomaRESUMEN
The development of knockin mice with Cre recombinase expressed under the control of the promoter for choline acetyltransferase (ChAT) has allowed experimental manipulation of cholinergic circuits. However, currently available ChATCre mouse lines are on the C57BL/6J strain background, which shows early onset age-related hearing loss attributed to the Cdh23753A mutation (a.k.a., the ahl mutation). To develop ChATCre mice without accelerated hearing loss, we backcrossed ChATIRES-Cre mice with CBA/CaJ mice that have normal hearing. We used genotyping to obtain mice homozygous for ChATIRES-Cre and the wild-type allele at the Cdh23 locus (ChATCre,Cdh23WT). In the new line, auditory brainstem response thresholds were â¼20 dB lower than those in 9 month old ChATIRES-Cre mice at all frequencies tested (4-31.5 kHz). These thresholds were stable throughout the period of testing (3-12 months of age). We then bred ChATCre,Cdh23WT animals with Ai14 reporter mice to confirm the expression pattern of ChATCre. In these mice, tdTomato-labeled cells were observed in all brainstem regions known to contain cholinergic cells. We then stained the tissue with a neuron-specific marker, NeuN, to determine whether Cre expression was limited to neurons. Across several brainstem nuclei (pontomesencephalic tegmentum, motor trigeminal and facial nuclei), 100% of the tdTomato-labeled cells were double-labeled with anti-NeuN (n = 1896 cells), indicating Cre-recombinase was limited to neurons. Almost all of these cells (1867/1896 = 98.5%) also stained with antibodies against ChAT, indicating that reporter label was expressed almost exclusively in cholinergic neurons. Finally, an average 88.7% of the ChAT+ cells in these nuclei were labeled with tdTomato, indicating that the Cre is expressed in a large proportion of the cholinergic cells in these nuclei. We conclude that the backcrossed ChATCre,Cdh23WT mouse line has normal hearing and expresses Cre recombinase almost exclusively in cholinergic neurons. This ChATCre,Cdh23WT mouse line may provide an opportunity to manipulate cholinergic circuits without the confound of accelerated hearing loss associated with the C57BL/6J background. Furthermore, comparison with lines that do show early hearing loss may provide insight into possible cholinergic roles in age-related hearing loss.
Asunto(s)
Tronco Encefálico/enzimología , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/enzimología , Pérdida Auditiva/prevención & control , Audición , Integrasas/metabolismo , Animales , Umbral Auditivo , Tronco Encefálico/fisiopatología , Cadherinas/genética , Colina O-Acetiltransferasa/genética , Cruzamientos Genéticos , Proteínas de Unión al ADN/metabolismo , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Técnicas de Sustitución del Gen , Pérdida Auditiva/enzimología , Pérdida Auditiva/genética , Pérdida Auditiva/fisiopatología , Integrasas/genética , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Especificidad de la EspecieRESUMEN
Previous studies showed that chlorpyrifos (CPF) acute exposure impaired cardiorespiratory reflexes. Evidence also indicates that continuous exposure to organophosphorus compounds impairs cardiovascular function. However, the effect of intermittent exposure to CPF, as may be experienced in the real world, on tonic and reflex cardiorespiratory function remains unexplored. Wistar rats were injected with saline or CPF for 4 weeks (3 times/week) or 12 weeks (once/week) at the doses of 7 mg/kg and 10 mg/kg. After exposure, blood pressure (BP), heart rate (HR), respiratory rate (fR), tidal volume (VT), and minute volume (VE) were recorded. Systolic BP and pulse interval (PI) variability, HR spectrum, spontaneous baroreflex and chemoreflex function were also evaluated. Plasma butyrylcholinesterase and brainstem acetylcholinesterase activities were quantified. Enzymatic activity of the CPF animals was reduced after both treatment periods. Baseline BP, HR, and fR, as well as systolic BP and PI variability indices, did not change, after CPF treatment. VT and VE were elevated in CPF animals. CPF exposure increased the very low-frequency component of the HR spectrum. Baroreflex gain was reduced after CPF 4-week exposure. Chemoreflex bradycardia was reduced in the CPF-treated rats. These data show that intermittent exposure to CPF impairs cardiorespiratory function in rats. These results may have important clinical implications for workers seasonally exposed to these compounds.
Asunto(s)
Barorreflejo/efectos de los fármacos , Tronco Encefálico/efectos de los fármacos , Cloropirifos/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Corazón/inervación , Insecticidas/toxicidad , Pulmón/inervación , Acetilcolinesterasa/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Tronco Encefálico/enzimología , Tronco Encefálico/fisiopatología , Butirilcolinesterasa/sangre , Cardiotoxicidad , Células Quimiorreceptoras/efectos de los fármacos , Células Quimiorreceptoras/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratas Wistar , Frecuencia Respiratoria/efectos de los fármacos , Volumen de Ventilación Pulmonar/efectos de los fármacos , Factores de TiempoRESUMEN
Sodium deficiency elevates aldosterone, which in addition to epithelial tissues acts on the brain to promote dysphoric symptoms and salt intake. Aldosterone boosts the activity of neurons that express 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone-sensitive cells. To better characterize these neurons, we combine immunolabeling and in situ hybridization with fate mapping and Cre-conditional axon tracing in mice. Many cells throughout the brain have a developmental history of Hsd11b2 expression, but in the adult brain one small brainstem region with a leaky blood-brain barrier contains HSD2 neurons. These neurons express Hsd11b2, Nr3c2 (mineralocorticoid receptor), Agtr1a (angiotensin receptor), Slc17a6 (vesicular glutamate transporter 2), Phox2b, and Nxph4; many also express Cartpt or Lmx1b. No HSD2 neurons express cholinergic, monoaminergic, or several other neuropeptidergic markers. Their axons project to the parabrachial complex (PB), where they intermingle with AgRP-immunoreactive axons to form dense terminal fields overlapping FoxP2 neurons in the central lateral subnucleus (PBcL) and pre-locus coeruleus (pLC). Their axons also extend to the forebrain, intermingling with AgRP- and CGRP-immunoreactive axons to form dense terminals surrounding GABAergic neurons in the ventrolateral bed nucleus of the stria terminalis (BSTvL). Sparse axons target the periaqueductal gray, ventral tegmental area, lateral hypothalamic area, paraventricular hypothalamic nucleus, and central nucleus of the amygdala. Dual retrograde tracing revealed that largely separate HSD2 neurons project to pLC/PB or BSTvL. This projection pattern raises the possibility that a subset of HSD2 neurons promotes the dysphoric, anorexic, and anhedonic symptoms of hyperaldosteronism via AgRP-inhibited relay neurons in PB.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Aldosterona/farmacología , Tronco Encefálico/efectos de los fármacos , Neuronas/efectos de los fármacos , Prosencéfalo/efectos de los fármacos , Núcleo Solitario/efectos de los fármacos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Animales , Regulación del Apetito , Axones/efectos de los fármacos , Axones/enzimología , Tronco Encefálico/citología , Tronco Encefálico/enzimología , Encefalinas/genética , Encefalinas/metabolismo , Conducta Alimentaria , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Genes Reporteros , Hibridación Fluorescente in Situ , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/enzimología , Técnicas de Trazados de Vías Neuroanatómicas , Neuronas/enzimología , Prosencéfalo/citología , Prosencéfalo/enzimología , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Núcleo Solitario/citología , Núcleo Solitario/enzimologíaRESUMEN
Serotonin (5-HT) acts as both a morphogenetic factor during early embryonic development and a neuromodulator of circuit plasticity in the mature brain. Dysregulation of serotonin signaling during critical periods is involved in developmental neurological disorders, such as schizophrenia and autism. In this study we focused on the consequences of defect reelin signaling for the development of the brainstem serotonergic raphe system. We observed that reelin signaling components are expressed by serotonergic neurons during the critical period of their lateral migration. Further, we found that reelin signaling is important for the normal migration of rostral, but not caudal hindbrain raphe nuclei and that reelin deficiency results in the malformation of the paramedian raphe nucleus and the lateral wings of the dorsal raphe nuclei. Additionally, we showed that serotonergic neurons projections to laminated brain structures were severely altered. With this study, we propose that the perturbation of canonical reelin signaling interferes with the orientation of tangentially, but not radially, migrating brainstem 5-HT neurons. Our results open the window for further studies on the interaction of reelin and serotonin and the pathogenesis of neurodevelopmental disorders.
Asunto(s)
Tronco Encefálico/fisiología , Moléculas de Adhesión Celular Neuronal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Proteínas del Tejido Nervioso/fisiología , Núcleos del Rafe/fisiología , Serina Endopeptidasas/fisiología , Neuronas Serotoninérgicas/fisiología , Animales , Western Blotting , Tronco Encefálico/anatomía & histología , Tronco Encefálico/enzimología , Ratones , Ratones Noqueados , Plasticidad Neuronal/fisiología , Núcleos del Rafe/anatomía & histología , Núcleos del Rafe/embriología , Proteína Reelina , Serotonina/fisiología , Transducción de Señal/fisiologíaRESUMEN
Although it is well-established that severe poisoning by organophosphorus (OP) compounds strongly affects the cardiorespiratory system, the effects of sub-lethal exposure to these compounds on the neural control of cardiovascular function are poorly explored. The aim of this study was to evaluate the effects of acute sub-lethal exposure to chlorpyrifos (CPF), a commonly used OP insecticide, on three basic reflex mechanisms involved in blood pressure regulation, the peripheral chemoreflex, the baroreflex and the Bezold-Jarisch reflex. Adult male Wistar rats were injected intraperitoneally with a single dose of CPF (30â¯mg/kg) or saline (0.9%). 24â¯h after injections, cardiovascular reflexes were tested in awake rats. Potassium cyanide (KCN) and phenylbiguanide (PBG) were injected intravenously to activate the chemoreflex and the Bezold-Jarisch reflex, respectively. The baroreflex was activated by phenylephrine and sodium nitroprusside infusions. Blood samples were taken for measurements of butyrylcholinesterase (BChE) activity while acetylcholinesterase (AChE) activity was measured in brainstem samples. Animals treated with CPF presented signs of intoxication such as ataxia, tremor, lacrimation, salivation, tetany, urination and defecation. The hypertensive and the bradycardic responses of the chemoreflex as well as the hypotensive and bradycardic responses of the Bezold-Jarisch reflex were attenuated in CPF treated animals (Pâ¯<â¯0.05). Concerning the baroreflex responses, CPF treatment reduced the bradycardia plateau, the range and the gain of the reflex (Pâ¯<â¯0.05). Plasma BChE and brainstem AChE were both reduced significantly after CPF treatment (Pâ¯<â¯0.05). Our results showed that acute sub-lethal exposure to CPF impairs the cardiovascular responses of homeostatic and defensive cardiovascular reflexes. These effects are associated with a marked inhibition of plasma BChE and brainstem AChE.
Asunto(s)
Barorreflejo/efectos de los fármacos , Tronco Encefálico/efectos de los fármacos , Cloropirifos/toxicidad , Acetilcolinesterasa/sangre , Acetilcolinesterasa/metabolismo , Animales , Tronco Encefálico/enzimología , Butirilcolinesterasa/sangre , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/toxicidad , Proteínas Ligadas a GPI/sangre , Proteínas Ligadas a GPI/metabolismo , Insecticidas/toxicidad , Masculino , Proyectos Piloto , Ratas , Ratas Wistar , Pruebas de Toxicidad AgudaRESUMEN
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
The spinocerebellar projection has an essential role in sensorimotor coordination of limbs and the trunk. Multiple groups of spinocerebellar projections have been identified in retrograde labeling studies. In this study, we aimed at characterizing projection patterns of these groups using a combination of anterograde labeling of the thoracic spinal cord and aldolase C immunostaining of longitudinal stripes of the cerebellar cortex in the mouse. We reconstructed 22 single spinocerebellar axons, wholly in the cerebellum and brain stem and partly, in the spinal cord. They were classified into three groups, (a) non-crossed axons of Clarke's column neurons (NCC, 8 axons), (b) non-crossed axons of marginal Clarke's column neurons (NMCC, 7 axons), and (c) crossed axons of neurons in the medial ventral horn (CMVH, 7 axons), based on previous retrograde labeling studies. While NCC axons projected mainly to multiple bilateral stripes in vermal lobules II-IV and VIII-IX, and the ipsilateral medial cerebellar nucleus, NMCC axons projected mainly to ipsilateral stripes in paravermal lobules II-V and copula pyramidis, and the anterior interposed nucleus. CMVH axons projected bilaterally to multiple stripes in lobules II-V with a small number of terminals but had abundant collaterals in the spinal cord and medullary reticular nuclei as well as in the vestibular and cerebellar nuclei. The results indicate that, while CMVH axons overlap with propriospinal and spinoreticular projections, NCC and NMCC axons are primarily spinocerebellar axons, which seem to be involved in relatively more proximal and distal sensorimotor controls, respectively.
Asunto(s)
Axones , Tronco Encefálico/citología , Cerebelo/citología , Fructosa-Bifosfato Aldolasa/metabolismo , Médula Espinal/citología , Animales , Axones/enzimología , Biotina/análogos & derivados , Tronco Encefálico/enzimología , Cerebelo/enzimología , Dextranos , Femenino , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Vías Nerviosas/citología , Vías Nerviosas/enzimología , Técnicas de Trazados de Vías Neuroanatómicas , Trazadores del Tracto Neuronal , Médula Espinal/enzimología , Vértebras TorácicasRESUMEN
The Wistar Audiogenic Rat (WAR) is a well-characterized seizure-prone, inbred rodent strain that, when acutely stimulated with high-intensity sounds, develops brainstem-dependent tonic-clonic seizures that can evolve to limbic-like, myoclonic (forebrain) seizures when the acoustic stimuli are presented chronically (audiogenic kindling). In order to investigate possible mechanisms underlying WAR susceptibility to seizures, we evaluated Na,K-ATPase activity, Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and oxidative stress markers in whole forebrain and whole brainstem samples of naïve WAR, as compared to samples from control Wistar rats. We also evaluated the expression levels of α1 and α3 isoforms of Na,K-ATPase in forebrain samples. We observed increased Na,K-ATPase activity in forebrain samples and increased oxidative stress markers (lipid peroxidation, glutathione peroxidase and superoxide dismutase) in brainstem samples of WAR. The Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and expression levels of α1 and α3 isoforms of Na,K-ATPase were unaltered. In view of previous data showing that the membrane potentials from naïve WAR's neurons are less negative than that from neurons from Wistar rats, we suggest that Na,K-ATPase increased activity might be involved in a compensatory mechanism necessary to maintain WAR's brains normal activity. Additionally, ongoing oxidative stress in the brainstem could bring Na,K-ATPase activity back to normal levels, which may explain why WAR's present increased susceptibility to seizures triggered by high-intensity sound stimulation.
Asunto(s)
Tronco Encefálico/enzimología , Estrés Oxidativo/fisiología , Prosencéfalo/enzimología , Convulsiones , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estimulación Acústica/efectos adversos , Adenosina Trifosfatasas/metabolismo , Animales , Tronco Encefálico/patología , Modelos Animales de Enfermedad , Glutatión Peroxidasa/metabolismo , Excitación Neurológica/fisiología , Peroxidación de Lípido , Neuronas/enzimología , Prosencéfalo/patología , Isoformas de Proteínas/metabolismo , Ratas , Ratas Wistar , Convulsiones/etiología , Convulsiones/metabolismo , Convulsiones/patologíaRESUMEN
Objective To explore the changes of cytochrome oxidase (COX) activity in the pre-Botzinger complex (pre-BotC) of the brainstem. Methods The double labeling of COX histochemistry and pre-BotC marker neurokinin-1 receptor (NK1R) nanogold-silver immunohistochemical staining was conducted to determine COX activity in the pre-BotC, especially within different subcellular structures of this nucleus. COX activity was semi-quantitatively analyzed. Results Under the light microscope, NK1R-immunoreactive (NK1R-ir) product was mainly distributed along the neuronal membrane, clearly outlining pre-BotC neurons. COX histochemical staining in brown was extensively expressed in the somata and processes of NK1R-ir neurons. Under the electron microscope, NK1R-ir gold particles were mainly distributed along the inner surface of the membrane of the somata and dendrites. The cytoplasm was also found labeled with NK1R-ir gold particles. The mitochondrial shape and distribution were different in different subcellular structures (somata, axon terminals, dendrites) of the pre-BotC. They were usually round or oval in the somata and axon terminals, whereas in the dendrites, slender elongated mitochondria were the most common. Tubular and vesicular cristae were more commonly visualized in the somata, but lamellar-oriented cristae were frequently encountered in the dendrites and axon terminals. The mitochondria appeared clustered together in the axon terminals, but in scattered distribution and close to the membrane in the dendrites except at synapses, where they were densely distributed and enlarged locally close to the postsynaptic membrane. The close link of the mitochondria with synapses indicated functional requirement that postsynaptic signal neurotransmission needs a large amount of ATP consumption. COX active product was expressed in the mitochondrial cristae, where different densities of the cristae represented different level of COX activity. The higher level of COX activity was evident in the axon terminals and dendrites than that in the somata, being significantly different. Conclusion Subcellular different regions in the pre-BotC function differently and need different energy metabolisms, thereby axon terminals and dendrites require higher COX activity than somata. In particular at synapses, mitochondria are densely localized with high COX activity. The present study provides a new approach by combination of COX histochemistry with immuno-electron microscopic techniques to detect regional COX activity in different subcellular structures of neurons.
Asunto(s)
Tronco Encefálico/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Animales , Tronco Encefálico/ultraestructura , Dendritas/enzimología , Inmunohistoquímica , Microscopía Inmunoelectrónica , Terminales Presinápticos/enzimología , Ratas , Ratas Sprague-Dawley , Receptores de Neuroquinina-1/análisisRESUMEN
Purinergic signaling is involved in pain generation and modulation in the nociceptive sensory nervous system. Adenosine triphosphate (ATP) induces pain via activation of ionotropic P2X receptors while adenosine mediates analgesia via activation of metabotropic P1 receptors. These purinergic signaling are determined by ecto-nucleotidases that control ATP degradation and adenosine generation. Using enzymatic histochemistry, we detected ecto-AMPase activity in dental pulp, trigeminal ganglia (TG) neurons, and their nerve fibers. Using immunofluorescence staining, we confirmed the expression of ecto-5'-nucleotidase (CD73) in trigeminal nociceptive neurons and their axonal fibers, including the nociceptive nerve fibers projecting into the brainstem. In addition, we detected the existence of CD73 and ecto-AMPase activity in the nociceptive lamina of the trigeminal subnucleus caudalis (TSNC) in the brainstem. Furthermore, we demonstrated that incubation with specific anti-CD73 serum significantly reduced the ecto-AMPase activity in the nociceptive lamina in the brainstem. Our results indicate that CD73 might participate in nociceptive modulation by affecting extracellular adenosine generation in the trigeminal nociceptive pathway. Disruption of TG neuronal ecto-nucleotidase expression and axonal terminal localization under certain circumstances such as chronic inflammation, oxidant stress, local constriction, and injury in trigeminal nerves may contribute to the pathogenesis of orofacial neuropathic pain.
Asunto(s)
5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/fisiología , Adenosina Trifosfatasas/metabolismo , Axones/enzimología , Tronco Encefálico/enzimología , Pulpa Dental/enzimología , Fibras Nerviosas/enzimología , Núcleo Caudal del Trigémino/enzimología , Nervio Trigémino/enzimología , Adolescente , Animales , Pulpa Dental/citología , Dolor Facial/enzimología , Técnica del Anticuerpo Fluorescente , Humanos , Hidrólisis , Tercer Molar , Transducción de Señal , Coloración y Etiquetado , Adulto JovenRESUMEN
Microglia are ubiquitously distributed throughout the central nervous system (CNS) and play a critical role in the maintenance of neuronal homeostasis. Recent advances have shown that microglia, never resting cells of the CNS, continuously monitor and influence neuronal/synaptic activity levels, by communicating with neurons with the aid of their dynamic processes. The brainstem contains many catecholaminergic nuclei that are key to many aspects of brain function. This includes C1 neurons of the ventrolateral medulla that are thought to play a critical role in control of the circulation. Despite the role of catecholaminergic brainstem neurons in normal physiology, the presence of microglia that surrounds them is poorly understood. Here, we investigate the spatial distribution and morphology of microglia in catecholaminergic nuclei of the brainstem in 3 strains of rat: Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Our data reveal that microglia are heterogeneously distributed within and across different strains of rats. Interestingly, intra-strain comparison of tyrosine hydroxylase-immunoreactive (TH-ir) neuronal and microglial number reveals that microglial number varies with the TH-ir neuronal number in the brainstem. Even though microglial spatial distribution varies across brainstem nuclei, microglial morphology (% area covered, number of end point processes and branch length) does not differ significantly. This work provides the first evidence that even though microglia, in their surveilling state, do not vary appreciably in their morphology across brainstem areas, they do have a heterogeneous pattern of distribution that may be influenced by their local environment.
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Microglía/enzimología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Presión Sanguínea/fisiología , Tronco Encefálico/enzimología , Catecolaminas/metabolismo , Masculino , Bulbo Raquídeo/enzimología , Microglía/citología , Neuronas/enzimología , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-DawleyRESUMEN
Cytochrome b 5 reductase (Cb 5R) and cytochrome b 5 (Cb 5) form an enzymatic redox system that plays many roles in mammalian cells. In the last 15 years, it has been proposed that this system is involved in the recycling of ascorbate, a vital antioxidant molecule in the brain and that its deregulation can lead to the production of reactive oxygen species that play a major role in oxidative-induced neuronal death. In this work, we have performed a regional and cellular distribution study of the expression of this redox system in adult rat brain by anti-Cb 5R isoform 3 and anti-Cb 5 antibodies. We found high expression levels in cerebellar cortex, labeling heavily granule neurons and Purkinje cells, and in structures such as the fastigial, interposed and dentate cerebellar nuclei. A large part of Cb 5R isoform 3 in the cerebellum cortex was regionalized in close proximity to the lipid raft-like nanodomains, labeled with cholera toxin B, as we have shown by fluorescence resonance energy transfer imaging. In addition, vestibular, reticular and motor nuclei located at the brain stem level and pyramidal neurons of somatomotor areas of the brain cortex and of the hippocampus have been also found to display high expression levels of these proteins. All these results point out the enrichment of Cb 5R isoform 3/Cb 5 system in neuronal cells and structures of the cerebellum and brain stem whose functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia, as well as in brain areas highly prone to undergo oxidative stress-induced neurodegeneration.
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Encéfalo/enzimología , Cerebelo/enzimología , Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/metabolismo , Células Piramidales/enzimología , Animales , Tronco Encefálico/enzimología , Hipocampo/enzimología , Isoenzimas/metabolismo , Masculino , Microdominios de Membrana/enzimología , Neocórtex/enzimología , Neuroglía/enzimología , Ratas , Ratas WistarRESUMEN
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.
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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
Obesity is a major risk factor for asthma, which is characterized by airway hyperreactivity (AHR). In obesity-associated asthma, AHR may be regulated by non-TH2 mechanisms. We hypothesized that airway reactivity is regulated by insulin in the CNS, and that the high levels of insulin associated with obesity contribute to AHR. We found that intracerebroventricular (ICV)-injected insulin increases airway reactivity in wild-type, but not in vesicle acetylcholine transporter knockdown (VAChT KD(HOM-/-)), mice. Either neutralization of central insulin or inhibition of extracellular signal-regulated kinases (ERK) normalized airway reactivity in hyperinsulinemic obese mice. These effects were mediated by insulin in cholinergic nerves located at the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA), which convey parasympathetic outflow to the lungs. We propose that increased insulin-induced activation of ERK in parasympathetic pre-ganglionic nerves contributes to AHR in obese mice, suggesting a drug-treatable link between obesity and asthma.
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Tronco Encefálico/enzimología , Hiperreactividad Bronquial/complicaciones , Neuronas Colinérgicas/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hiperinsulinismo/complicaciones , Sistema de Señalización de MAP Quinasas , Animales , Hiperreactividad Bronquial/enzimología , Hiperreactividad Bronquial/fisiopatología , Broncoconstricción , Neuronas Colinérgicas/patología , Dieta Alta en Grasa , Activación Enzimática , Hiperinsulinismo/enzimología , Hiperinsulinismo/fisiopatología , Inflamación/patología , Inyecciones Intraventriculares , Insulina/metabolismo , Cloruro de Metacolina , Ratones Endogámicos C57BL , Ratones Obesos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Receptor de Insulina/metabolismoRESUMEN
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.
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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
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.
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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
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.
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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
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.