RESUMEN
The astroglial marker, glial fibrillary acidic protein (GFAP) was investigated by immunohistochemistry in various brain areas in order to see its fluctuations in various functional states. Different neuronal states were either experimentally induced or studied under physiological conditions. To produce experimental alterations the visual system was chosen as a model. Upon lesioning of the lateral geniculate body with the stereotaxic injection of ibotenic acid an increase in GFAP immunoreactivity could be induced in layers III and IV of the ipsilateral visual cortex where geniculo-cortical fibres terminate. Electron microscopy has revealed a synchronous degeneration of synaptic terminals and the hypertrophy of perisynaptic astrocyte processes. To study changes in the intact animal the effect of illumination was observed. In the lateral geniculate body the dorsal subnucleus was found immunonegative when studied at day and positive at night. Similar changes were observed in the suprachiasmatic nucleus. As to more generalized influences, the effect of gonadal steroids on the GFAP-reaction interpeduncular nucleus, an area not involved in hormonal regulatory mechanisms was studied. In males only castration could reduce constantly high GFAP immunoreactivity, whereas in females GFAP showed wide-range sexual cycle-related fluctuations. It was concluded that changes in GFAP immunoreactivity can indicate synaptic events within a circumscribed area of the brain.
Asunto(s)
Encéfalo , Proteína Ácida Fibrilar de la Glía/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/anatomía & histología , Encéfalo/metabolismo , Femenino , Masculino , Neuronas/citología , Ratas , Ratas Wistar , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
The expression of the two members of the dystrobrevin (DB) family in the adult brain was thought to be highly specific for the two main cell types: alpha-dystrobrevin (alpha-DB) and beta-dystrobrevin (beta-DB) has been identified as glial and neuronal proteins, respectively. In the present work we show that a subset of neurons in the hypothalamus contains alpha-DB. Comparative immunohistochemical studies with two alpha-DB antibodies of different specificity indicate that the neurons contain short alpha-DB isoform(s) alpha-DB-2 and/or alpha-DB-4. Immunoreactive multipolar or spindle-shaped neurons form clusters with bilateral symmetry, localized predominantly in the lateral hypothalamic area, with extensions into the zona incerta and the dorso-medial and ventro-medial hypothalamic region. alpha-DB immunoreactivity was localized in cell processes and at postsynaptic densities, furthermore in the endoplasmic reticulum within the perikarya. alpha-DB-positive neurons are beta-dystrobrevin immunoreactive, but alpha- and beta-DB do not co-localize with their usual molecular anchors like dystrophins or high molecular weight forms of utrophin. Colocalization with nNOS was also not observed. The pattern of alpha-DB immunoreactive neurons gave a perfect colocalization with melanin-concentrating hormone (MCH) neurons throughout the whole region studied. We propose that alpha-DB plays a role in a structure or regulation mechanism unique to MCH-expressing neurons.
Asunto(s)
Proteínas Asociadas a la Distrofina/metabolismo , Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Melaninas/metabolismo , Neuronas/metabolismo , Hormonas Hipofisarias/metabolismo , Sinapsis/metabolismo , Animales , Mapeo Encefálico , Proteínas Asociadas a la Distrofina/química , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Técnica del Anticuerpo Fluorescente , Área Hipotalámica Lateral/citología , Área Hipotalámica Lateral/metabolismo , Hipotálamo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peso Molecular , Neuronas/citología , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Succinate (SUC), a citrate (CIT) cycle intermediate, and carbenoxolone (CBX), a gap junction inhibitor, were shown to displace [3H]gamma-hydroxybutyrate ([3H]GHB), which is specifically bound to sites present in synaptic membrane subcellular fractions of the rat forebrain and the human nucleus accumbens. Elaboration on previous work revealed that acidic pH-induced specific binding of [3H]SUC occurs, and it has been shown to have a biphasic displacement profile distinguishing high-affinity (K(i,SUC) = 9.1 +/- 1.7 microM) and low-affinity (K(i,SUC) = 15 +/- 7 mM) binding. Both high- and low- affinity sites were characterized by the binding of GHB (K(i,GHB) = 3.9 +/- 0.5 microM and K(i,GHB) = 5.0 +/- 2.0 mM) and lactate (LAC; K(i,LAC) = 3.9 +/- 0.5 microM and K(i,LAC) = 7.7 +/- 0.9 mM). Ligands, including the hemiester ethyl-hemi-SUC, and the gap junction inhibitors flufenamate, CBX, and the GHB binding site-selective NCS-382 interacted with the high-affinity site (in microM: K(i,EHS) = 17 +/- 5, K(i,FFA) = 24 +/- 13, K(i,CBX) = 28 +/- 9, K(i,NCS-382) = 0.8 +/- 0.1 microM). Binding of the Na+,K+-ATPase inhibitor ouabain, the proton-coupled monocarboxylate transporter (MCT)-specific alpha-cyano-hydroxycinnamic acid (CHC), and CIT characterized the low-affinity SUC binding site (in mM: K(i,ouabain) = 0.13 +/- 0.05, K(i,CHC) = 0.32 +/- 0.07, K(i,CIT) = 0.79 +/- 0.20). All tested compounds inhibited [3H]SUC binding in the human nucleus accumbens and had K(i) values similar to those observed in the rat forebrain. The binding process can clearly be recognized as different from synaptic and mitochondrial uptake or astrocytic release of SUC, GHB, and/or CIT by its unique GHB selectivity. The transient decrease of extracellular SUC observed during epileptiform activity suggested that the function of the synaptic target recognizing protonated succinate monocarboxylate may vary under different (patho)physiological conditions. Furthermore, we put forward a hypothesis on the synaptic activity-regulated signaling between astrocytes and neurons via SUC protonation.
Asunto(s)
Astrocitos/metabolismo , Neuronas/metabolismo , Núcleo Accumbens/citología , Oxibato de Sodio/metabolismo , Succinatos/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Interacciones Farmacológicas , Humanos , Técnicas In Vitro , Neuronas/citología , Núcleo Accumbens/efectos de los fármacos , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Oxibato de Sodio/farmacocinética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Succinatos/farmacocinética , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructura , Sinaptosomas/efectos de los fármacos , Tritio/farmacocinéticaRESUMEN
The relationship between mitochondrial Ca2+ accumulation and reactive oxygen species formation is studied by means of fast kinetics with fluorescence detection. Succinate or elevated [Na+] and [Ca2+] provided alternative conditions for induction of reactive oxygen species in isolated rat cerebrocortical mitochondria. Depending on Na+/Ca2+ exchanger and superoxide dismutase activities, formation of reactive oxygen species in mitochondria exposed to elevated [Na+] and [Ca2+] levels was characterized by t(1/2) = 12.3+/-2.9 ms. Production of reactive oxygen species by xanthine oxidase followed a similar time course. Both the processes were blocked by para-benzoquinone, indicating the primary formation of O2-*. Substantial increase in mitochondrial O2-*. was observed at [Ca2+] > or = 2.5 microM. Mitochondrial Ca2+ accumulation obeyed similar [Ca2+] dependence but shorter t(1/2) (2.5+/-0.3 ms), providing kinetic evidence for Ca2+ accumulation-dependent primary formation of O2-* in brain mitochondria.
Asunto(s)
Encéfalo/ultraestructura , Calcio/metabolismo , Mitocondrias/metabolismo , Oxígeno/metabolismo , Animales , Microscopía Electrónica de Transmisión/métodos , Mitocondrias/ultraestructura , Ratas , Especies Reactivas de Oxígeno/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Superóxido Dismutasa/metabolismo , Factores de TiempoRESUMEN
The pacemaker of the biological clock, the suprachiasmatic nucleus (SCN) of the hypothalamus, was studied in intact male rats to determine its immunoreactivity to glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Animals were kept under 12-h light-dark cycles in synchrony with day-night periods. Immunohistochemical reactions were carried out at midday and late at night in both winter (January) and summer (July). In winter, GFAP immunoreactivity was found to be low during the day and high at night. The findings were reversed in summer, when GFAP immunoreactivity was high during the day and low at night. Increased GFAP immunoreactivity appeared in the form of an abundance of thick immunopositive fibres rather than of cell bodies. This was interpreted as a hypertrophy of pre-existing astrocytes due to alternating photic stimulation conveyed by retinofugal fibres to the SCN. The observed seasonal reversal in the direction of GFAP oscillations raises the possibility that a circannual timer exists outside the SCN.
Asunto(s)
Ritmo Circadiano/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Núcleo Supraquiasmático/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Astrocitos/patología , Relojes Biológicos/fisiología , Hipertrofia , Masculino , Estimulación Luminosa , Ratas , Ratas Wistar , Estaciones del Año , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/patologíaRESUMEN
We previously demonstrated that in rat, astrocytic glial fibrillary acidic protein- (GFAP) expression in the interpeduncular nucleus (IPN) was responsive to testosterone and in females the intensity of GFAP-immunoreactivity (IR) followed the periodic hormonal changes of the estrous cycle. The aim of this study was to test whether 17beta-estradiol (E(2)), in the absence of other ovarian hormones, can influence GFAP-expression within individual subnuclei of the IPN and to determine the cellular distribution of estrogen receptor beta (ERbeta) in the IPN. Quantitative surface-density analysis was used to compare the intensity of GFAP-IR at different anterio-posterior (AP) levels of the IPN in ovariectomized female rats 24 h after treatment with E(2) or vehicle. Estrogen-treatment resulted in a significant increase in GFAP-IR in the rostrolateral subnucleus of the IPN at AP: -5.60, in the lateral-, dorsolateral-, dorsomedial- and central subnuclei at -6.04 and in the lateral subnucleus at -6.72. No significant differences were observed at -5.80 and -6.30. These results indicate that E(2), in the absence of other ovarian hormones, modulates GFAP-expression within select IPN subnuclei and that these affects are dependent on position along the AP axis. To determine whether ERbeta was a possible mediator of the observed estrogenic effects, adjacent section pairs of the IPN were immunostained for ERbeta or GFAP. Using the 'mirror' method, ERbeta-IR was detected in the cytoplasm of GFAP-immunopositive astroglia and in the nuclei of GFAP-immunonegative neurons. These findings suggest that in the IPN, E(2) may directly modulate GFAP-expression through ERbeta-mediated mechanisms.