RESUMO
Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.(AU)
Assuntos
Humanos , Animais , Foto-Oxidação , Actinas/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/ultraestrutura , Amarelo de Eosina-(YS)/metabolismo , Amarelo de Eosina-(YS)/farmacologia , Corantes Fluorescentes/farmacologia , Imageamento Tridimensional/métodos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Microscopia de Fluorescência/métodos , Modelos Moleculares , Oxirredução , Faloidina/farmacologia , Fótons , Coloração e Rotulagem/métodosRESUMO
Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.
Assuntos
Humanos , Animais , Actinas/metabolismo , Amarelo de Eosina-(YS)/farmacologia , Amarelo de Eosina-(YS)/metabolismo , Foto-Oxidação , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/ultraestrutura , Coloração e Rotulagem/métodos , Corantes Fluorescentes/farmacologia , Faloidina/farmacologia , Imageamento Tridimensional/métodos , Modelos Moleculares , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Microscopia de Fluorescência/métodos , Oxirredução , FótonsRESUMO
Specific ligand binding to rat hippocampal adenosine A1 receptor after administration of the convulsant drug 3-mercaptopropionic acid (MP) was studied by means of a quantitative autoradiographic method. 2-Chloro-N6-[cyclopentyl-2,3,4,5-3H adenosine] ([3H]CCPA), a potent and selective A1 receptor ligand, was selected for binding studies. MP administration (150 mg/kg, i.p.), at seizure, caused significant increases in the following CA1 layers: pyramidal (45%), radiatum (18%) and lacunosum molecular (35%); in CA2 area, a significant decrease in stratum oriens (36%) and an increase in stratum radiatum (14%) and lacunosum molecular (33%) layers was observed. In CA3 area a rise in pyramidal (40%) and radiatum layers (26%), as well as in hillus (97%) was found. At postseizure, changes were restricted to CA1, CA2 and CA3 pyramidal layers and to CA1 lacunosum molecular layer, with increases ranging from 22 to 50%. These results show that [3H]CCPA binding is modified diversely in intrahippocampal layers and areas, thus indicating their dissimilar role in seizure activity.
Assuntos
Adenosina/análogos & derivados , Hipocampo/fisiopatologia , Receptores Purinérgicos P1/metabolismo , Convulsões/metabolismo , Adenosina/metabolismo , Animais , Autorradiografia , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Ensaio Radioligante , Ratos , Ratos Wistar , TrítioRESUMO
Specific [3H]-MK801 binding to rat NMDA receptors following the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied in striatal membrane fractions. MP administration (150 mg/kg, i.p.) caused an increase of 53% and 82% in [3H]-MK801 binding during seizure and the postseizure period respectively. Administration of CPA (2 mg/kg, i.p.) raised [3H]-MK801 binding by 72%. When CPA was administered 30 min before MP and rats sacrificed at seizure (CPA + MPc), an increase of 64%, was observed. Saturation results indicate that receptor sites increased their maximal binding capacity (Bmax) in all treatments while the apparent dissociation constant (Kd) remained unchanged. MP administration brought about an increase of 52% and 42% in [3H]-MK801 binding sites during seizure and postseizure respectively. Administration of CPA raised receptor density by 75%. When CPA was administered 30 min before MP and rats sacrificed at seizure (CPA + MPc), an increase of 62%, was observed. These results show that striatal NMDA receptors have a selective role in seizure activity in the basal ganglia and that the adenosine analogue administration may modify [3H]-MK801 binding in a way similar to that of the convulsant drug.
Assuntos
Ácido 3-Mercaptopropiônico , Adenosina/análogos & derivados , Convulsivantes , Corpo Estriado/metabolismo , Epilepsia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Adenosina/farmacologia , Animais , Membrana Celular/metabolismo , Corpo Estriado/efeitos dos fármacos , Maleato de Dizocilpina/metabolismo , Epilepsia/induzido quimicamente , Masculino , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Trítio , Regulação para CimaRESUMO
Rat CNS adenosine A2A receptors were studied after administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) by means of a quantitative autoradiographic method. Specific binding was quantified in striatum only. The highest density was found in caudate-putamen (2.50 fmol/mm2), followed by nuclei accumbens (1.85 fmol/mm2) and the lowest values in the olfactory tubercle (1.26 fmol/mm2). These differences were statistically significant. MP administration (150 mg/kg) caused significant increases (12-18%) in caudate-putamen and nuclei accumbens in both stages: seizure and postseizure and no changes in the olfactory tubercle. CPA administration (2 mg/kg) originated a rise of 16% in nuclei accumbens but no change in the other two regions. When CPA was injected 30 minutes before MP, an increase (18 to 45%) in caudate-putamen and nuclei accumbens at seizure and postseizure stages was observed. Saturation results, in striatal membrane fraction, indicate that receptor sites increased their maximal binding capacity (Bmax) while the apparent dissociation constant (Kd) remained unchanged. These results suggest the involvement of the adenosine A2A receptors in convulsant activity and that CPA administration at the dose selected brings about a rise in neuronal excitability in this area.
Assuntos
Adenosina/análogos & derivados , Corpo Estriado/efeitos dos fármacos , Convulsões/metabolismo , Ácido 3-Mercaptopropiônico/farmacologia , Adenosina/farmacologia , Animais , Autorradiografia , Convulsivantes/farmacologia , Corpo Estriado/metabolismo , Corpo Estriado/fisiopatologia , Masculino , Ratos , Ratos Wistar , Convulsões/induzido quimicamenteRESUMO
Specific [3H]MK801 binding to rat brain NMDA receptors after the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied by means of a quantitative autoradiographic method. MP administration (150 mg/kg, i.p.) caused significant decreases in [3H]MK801 binding in several hippocampus subareas and layers, mainly in CA1 and CA3 at seizure (11-27%) and postseizure (8-16%) and in cerebral occipital cortex at seizure (18-22%). In nucleus accumbens, a rise was observed at postseizure (44%) and a tendency to increase at seizure (24%). CPA (2mg/kg, i.p.) decreased ligand binding in hippocampus (CAI, CA2, CA3) (17-22%) and in occipital cerebral cortex (18-24%). When CPA was administered 30 minutes before MP (which delayed seizure onset) and rats were sacrificed at seizure, decreases in [3H]MK801 binding in several layers of CA1 and CA3 of hippocampus (11-27%) and in CA1, CA2, CA3 (24-35%) after CPA+MP postseizure, and an increase in CA2 after CPA and CPA+MP postseizure (20-34%), were observed. A drop was found in the occipital subarea (18-24%) after CPA and in the frontal and occipital subarea after CPA+MP postseizure (24-34%) while no changes were observed in any treatment involving the other cerebral cortex regions, thalamic nuclei, caudate putamen and olfactory tubercle. These results show that [3H]MK801 binding changes according to drug treatment and the area being studied, thus indicating a different role in seizure activity.
Assuntos
Adenosina/análogos & derivados , Encéfalo/efeitos dos fármacos , Convulsivantes/administração & dosagem , Maleato de Dizocilpina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Adenosina/farmacologia , Animais , Autorradiografia , Encéfalo/metabolismo , Convulsivantes/farmacologia , Masculino , Ligação Proteica , Ratos , Ratos Wistar , TrítioRESUMO
Rat CNS adenosine A1 receptors were studied by quantitative autoradiography after the administration of convulsant 3-mercaptopropionic acid (MP) and an adenosine analogue cyclopentyladenosine (CPA), using 2-chloro-N6-[cyclopentyl-2,3,4,5-3H adenosine]-([3H]CCPA) as radioactive ligand. Specific binding was quantified in hippocampus, cerebellum, cerebral cortex, thalamic nuclei, superior colliculus and striatum, and the highest densities were found in CA1, CA2, and CA3 hippocampus subareas and the lowest levels in superior colliculus and striatum. MP administration (150 mg/kg, i.p.) produced significant increases in [3H]CCPA binding in CA1 subarea at seizure (15%) and postseizure (21%) and in CA2 at seizure (15%) but a tendency to decrease in dentate gyrus. There was an increase in cerebellum at seizure (18%) but no significant changes in the other studied regions. CPA injection (2 mg/kg, i.p.) enhanced [3H]CCPA binding in CA1 and CA2 areas (17-18%) but not in CA3 area of the hippocampus. When CPA was administered before MP, which delayed seizure onset, an increase in [3H]CCPA binding in CA1 hippocampus subarea (19%) and cerebellum (28%) was also observed. Results showed that the administration of convulsant MP and adenosine analogue CPA exerts differential effects on adenosine A1 receptors in CNS areas; hippocampus is the most affected area with all treatments, specially CA1 subarea, supporting an essential role in convulsant activity as well as in seizure prevention.