RESUMO
AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas do Tecido Nervoso/genética , ATPase Trocadora de Sódio-Potássio/genética , Adenosina Trifosfatases , Sequência de Aminoácidos , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Sequência de Bases , Western Blotting , Encéfalo/enzimologia , Encéfalo/metabolismo , Proteínas de Transporte de Cátions , Adesão Celular , Células Cultivadas , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Imunofluorescência , Biblioteca Gênica , Cinética , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Rubídio/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
The capacity for long-distance migration of the oligodendrocyte precursor cell, oligodendrocyte-type 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-methyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit. We show that NMDA evokes an increase in cytosolic Ca2+ that can be blocked by the NMDA receptor antagonist AP-5 and by Mg2+. Blocking the activity of these receptors dramatically diminished O-2A cell migration from explants. We also show that NMDA receptor activity is necessary for the expression by O-2A cells of the highly sialylated polysialic acid-neural cell adhesion molecule (PSA-NCAM) that is required for their migration. Thus, glutamate or glutamate receptor ligands may regulate O-2A cell migration by modulating expression of PSA-NCAM. These studies demonstrate how interactions between ionotropic receptors, intracellular signaling, and cell adhesion molecule expression influence cell surface properties, which in turn are critical determinants of cell migration.
Assuntos
Moléculas de Adesão de Célula Nervosa/metabolismo , Oligodendroglia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Ácidos Siálicos/metabolismo , Cálcio/metabolismo , Movimento Celular , Células Cultivadas , Humanos , Oligodendroglia/citologia , Técnicas de Patch-Clamp , Neuro-Hipófise/citologia , RNA/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/metabolismo , Células-Tronco/citologiaRESUMO
Regional brain activation was assessed by mapping of Fos-related protein expression in rats trained to self-administration of intravenous nicotine and cocaine. Both drugs produced specific overlapping patterns of activation in the shell and the core of the nucleus accumbens, medial prefrontal cortex, and medial caudate areas, but not in the amygdala. Thus, the reinforcing properties of cocaine and nicotine map on selected structures of the terminal fields of the mesocorticolimbic dopamine system, supporting the idea that common substrates for these addictive drugs exist.
Assuntos
Encéfalo/efeitos dos fármacos , Cocaína/farmacologia , Nicotina/farmacologia , Transtornos Relacionados ao Uso de Opioides/etiologia , Transtornos Relacionados ao Uso de Substâncias/etiologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Encéfalo/metabolismo , Mapeamento Encefálico , Cocaína/administração & dosagem , DNA/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nicotina/administração & dosagem , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Reforço Psicológico , Autoadministração , Fator de Transcrição AP-1/metabolismoRESUMO
Adhesion molecule on glia (AMOG) is a 45 - 50 kD cell surface glycoprotein structurally similar to the Na, K-ATPase beta-subunit and associated with the catalytic subunit of this enzyme. Previous immunofluorescence results had suggested that AMOG is transiently expressed on Bergmann glia during mouse cerebellar development, and antibody-inhibition results have implicated it in the migration of granule neurons. We report that, while AMOG mRNA is detected in Bergmann glia during the migratory period, this astrocyte derivative continues to express AMOG mRNA at similar levels in adult mice suggesting a functional role for AMOG in adulthood. Evidence from RNA and protein blot analyses that AMOG is present before birth, increasing about ten fold in adult mouse brain and cerebellum is also provided. RNA blot analysis of astrocyte-enriched cell populations and in situ hybridization results show that astrocytes synthesize AMOG mRNA in all regions of the developing and adult brain. In the adult, AMOG mRNA is more abundant in grey than white matter and, among grey matter regions, highest in cerebellar cortex. These results indicate a relationship between density of neuronal elements and AMOG expression. It is further speculated that AMOG is part of a Na,K-ATPase complex expressed preferentially by astrocytes in mouse brain.
RESUMO
The effect of L-5-hydroxytryptophan (5-HTP) on the threshold for maximal electroconvulsions was compared with concomitant changes in levels of 5-HTP, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in plasma and brain of rats. A single injection of 5-HTP (100 mg/kg, i.p.) caused significant elevation in seizure threshold which was markedly intensified by pretreatment with the decarboxylase inhibitor carbidopa (10 mg/kg, i.p., 0.5 hr previously). Pretreatment with carbidopa also resulted in behavioural changes, i.e. the characteristic "wet-dog shake" behaviour became much more prominent. Biochemically, administration of 5-HTP gave rise to significant elevation of levels of 5-HTP, 5-HT and 5-HIAA in plasma and brain. Carbidopa increased levels of 5-HTP in the brain, decreased 5-HIAA in the periphery but did not alter the elimination rate of 5-HTP in plasma. In both naive rats and rats pretreated with carbidopa, a significant correlation was found between levels of 5-HTP and 5-HIAA in plasma and brain following injection of 5-HTP. Furthermore, in the absence of carbidopa, the increases of levels of 5-HT in plasma and brain induced by 5-HTP were correlated in a significant fashion. When the changes in the electroconvulsive threshold were compared with respective changes in levels of 5-hydroxyindoles, a significant correlation was obtained between threshold elevations and increases of 5-HTP and 5-HT in the brain. In rats treated with 5-HTP, without decarboxylase inhibitor, a significant correlation was found between increases in 5-HT in plasma and the seizure threshold. The results suggest that analysis of 5-hydroxyindoles in plasma may represent a useful tool for the estimation of 5-HT metabolism in brain.
Assuntos
5-Hidroxitriptofano/farmacologia , Anticonvulsivantes , Química Encefálica/efeitos dos fármacos , Ácido Hidroxi-Indolacético/metabolismo , Serotonina/metabolismo , 5-Hidroxitriptofano/metabolismo , Animais , Carbidopa/farmacologia , Eletrochoque , Feminino , Ácido Hidroxi-Indolacético/sangue , Ratos , Ratos Endogâmicos , Serotonina/sangue , Fatores de TempoRESUMO
Age-related decline of cognition and memory, in humans and other animals, appears to be associated with neuronal loss. Experimental and clinical evidence has shown that the hippocampal formation is one of the brain regions most vulnerable to the ageing process. Because excess of glutamate is neurotoxic to hippocampal neurons, abnormalities in glutamate neurotransmitter function may play a crucial role in neurodegenerative disorders, especially in conjunction with brain ageing. We have used in situ hybridization to study the expression of the two major alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor subunits, involved in the control of calcium permeability in the young adult and aged rat hippocampus. We show that the levels of messenger RNA encoding the AMPA-selective glutamate receptor subunit-1 (GluR1 or GluRA) and AMPA-selective glutamate receptor subunit-2 (GluR2 or GluRB) are highest in the dentate gyrus, followed by the CA1 and CA3 hippocampal subfields. We also show that the levels of both messenger RNAs decrease differentially with age in all subfields of the hippocampus. Finally, the GluR1/GluR2 messenger RNA ratios increase in the aged hippocampus, particularly in the CA3 subfield, suggesting that altered calcium homeostasis may contribute to age-related neuronal death.
Assuntos
Envelhecimento/metabolismo , Cálcio/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de AMPA/biossíntese , Animais , Autorradiografia , Morte Celular/fisiologia , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hibridização In Situ , Masculino , Sondas de Oligonucleotídeos , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
The process of degeneration and dendritic reorganization of cholinergic neurons was investigated in the rat basal forebrain under the conditions of chronic neurotoxic injury induced by long-term consumption of ethanol. After 28 weeks of ethanol treatment (20% v/v), both the number of choline acetyltransferase-immunoreactive basal forebrain neurons and levels of biochemical measures of cholinergic neurons, such as the activity of choline acetyltransferase and the synthesis and content of acetylcholine, were decreased by about 60-80%. The number of cholinergic neurons showing a positive hybridization signal to choline acetyltransferase messenger RNA was decreased to a similar extent. On the contrary, the reduction in the number of neurons immunoreactive for nerve growth factor receptor p75, which in control brains is highly co-localized with the expression of choline acetyltransferase, was much less pronounced and reached only 20-30%. The loss of choline acetyltransferase expression was associated with a cellular hypertrophy. Neurons which had survived the neurotoxic damage, furthermore, showed a remodelling of the dendritic organization which was quantitatively investigated after Golgi impregnation. This process of dendritic reorganization was mainly characterized by an increase in number and length of terminal dendritic segments. The results indicate that under the conditions of the present paradigm of chronic neurodegeneration, a certain number of cholinergic neurons persists in a form where they lost their ability to express detectable amounts of choline acetyltransferase messenger RNA and the enzyme protein. Persisting neurons, however, show both expression of nerve growth factor receptor p75 and signs of perikaryal and dendritic growth. It might, therefore, be hypothesized that chronic degeneration of cholinergic basal forebrain neurons triggers reactive attempts of repair which involve the action of trophic factors such as nerve growth factor.
Assuntos
Colina O-Acetiltransferase/genética , Degeneração Neural , Fatores de Crescimento Neural/genética , Prosencéfalo/patologia , Consumo de Bebidas Alcoólicas , Intoxicação Alcoólica , Animais , Autorradiografia , Contagem de Células , Imuno-Histoquímica , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Substância Negra , Fatores de TempoRESUMO
Amongst different intrinsic and extrinsic inputs, cholinergic striatal interneurons receive afferents from the dopaminergic nigrostriatal projection and from local collaterals of striatonigral cells containing substance P. The following study demonstrates that both dopamine D2 and substance P (neurokinin-1) receptors are expressed by a large proportion of cholinergic interneurons. Using in situ hybridization on triplet adjacent sections with radioactive probes specific for choline acetyltransferase, substance P receptor, and D2 receptor long-splicing form messenger RNAs, we show that these interneurons can be divided into four subpopulations in terms of substance P and D2 receptor expression. A majority of these neurons coexpress both receptors (76%), while other minor subpopulations express either one (respectively, 16% and 2%) or none of them (6%). Our results also show that substance P receptor is expressed by striatal neurons that are not cholinergic. These findings are in agreement with the concept that striatal cholinergic interneurons are heterogeneous in terms of input-output connections and the type of receptors expressed. Moreover, the presence of substance P and D2 receptors on a majority of these neurons is relevant to a putative role of cholinergic interneurons in several conditions such as various neurodegenerative disorders or antipsychotic drug administration, where substance P and dopamine inputs are modified.
Assuntos
Corpo Estriado/metabolismo , Neurônios/metabolismo , Sistema Nervoso Parassimpático/metabolismo , RNA Mensageiro/biossíntese , Receptores de Dopamina D2/biossíntese , Receptores de Neurotransmissores/biossíntese , Animais , Southern Blotting , Colina O-Acetiltransferase/biossíntese , Corpo Estriado/citologia , Hibridização In Situ , Técnicas In Vitro , Masculino , Oligonucleotídeos/metabolismo , Sistema Nervoso Parassimpático/citologia , Sondas RNA , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-1RESUMO
The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed by several different subunits and the overall subunit composition of the receptor appears to determine its physiological and pharmacological properties. Although glutamatergic mechanisms have been implicated in various forms of hippocampal stress responses, the impact of stress on glutamate receptor subunit composition has not yet been elucidated. We have used cell-by-cell quantitative in situ hybridization to assess stress-induced changes in transcript levels of N-methyl-D-aspartate and AMPA receptor subunit genes in subdivisions of the rat hippocampus and hypothalamus that are implicated in the stress response. We found that 24 h after a single immobilization stress there was a significant increase in the cellular level of NR1 subunit messenger RNA (about 35-45% above control values) in hippocampal CA3 and CA1 pyramidal cells as well as in neurons of the hypothalamic supraoptic and paraventricular nuclei. Moreover, in the CA3 area we have detected a concomitant increase (50% above controls) in the level of NR2B subunit messenger RNA, while the expression of NR2A subunit gene did not change after stress. Stress induced a selective decrease in the level of AMPA receptor subunit glutamate receptor A messenger RNA in neurons of both the CA3 and CA1 areas (18 and 24%, respectively, below control values). These results suggest that the regulation of specific subunit messenger RNAs of the N-methyl-D-aspartate and AMPA receptors may be involved in altered hippocampal and hypothalamic responsiveness to glutamate and thus could play a critical role in stress-induced changes in their function.
Assuntos
Hipocampo/ultraestrutura , Hipotálamo Anterior/ultraestrutura , RNA Mensageiro/metabolismo , Receptores de AMPA/genética , Receptores de N-Metil-D-Aspartato/genética , Estresse Fisiológico/fisiopatologia , Animais , Expressão Gênica/fisiologia , Hipocampo/química , Hipotálamo Anterior/química , Hibridização In Situ , Masculino , Núcleo Hipotalâmico Paraventricular/química , Núcleo Hipotalâmico Paraventricular/ultraestrutura , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Receptores de AMPA/ultraestrutura , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/ultraestrutura , Restrição Física , Estresse Fisiológico/genética , Núcleo Supraóptico/química , Núcleo Supraóptico/ultraestrutura , Transcrição Gênica/fisiologiaRESUMO
Long-term consumption of ethanol both in human and rodent induces a process of chronic degeneration of cholinergic basal forebrain neurons which results in a cholinergic deafferentation of the cortical mantle. We have used quantitative northern blot analysis and in situ hybridization to demonstrate that these degenerative events in rat evoke an increase in the expression of the nerve growth factor gene in a number of brain areas, including the cholinergic basal forebrain nuclei and their cortical target regions. By combining non-radioactive in situ hybridization and immunohistochemistry activated astrocytes were identified as the major source of altered nerve growth factor gene expression. This increased nerve growth factor expression is paralleled by a dendritic remodelling of basal forebrain neurons, while the expression of choline acetyltransferase in surviving neurons remains the same. This failure of nerve growth factor to rescue the expression of choline acetyltransferase differs from the effects of exogenously administered nerve growth factor in acutely lesioned systems. The results indicate that under certain conditions of chronic neurodegeneration, the utilization of nerve growth factor might be impaired, which could be due to a defective nerve growth factor signalling mechanism.
Assuntos
Astrócitos/patologia , Degeneração Neural , Fatores de Crescimento Neural/genética , Consumo de Bebidas Alcoólicas , Animais , Autorradiografia , Northern Blotting , Colina O-Acetiltransferase/genética , Hipocampo , Hibridização In Situ , Masculino , Prosencéfalo/patologia , Ratos , Ratos Sprague-DawleyRESUMO
The time course of different pharmacological effects of L-5-hydroxytryptophan (5-HTP) during continued treatment was studied in rats. 5-HTP was administered three times daily at 100 mg/kg IP in combination with the peripheral decarboxylase inhibitor carbidopa (10 mg/kg) for 14 days. 5-HTP induced a pronounced increase of the threshold for maximal electroconvulsions, decreased body temperature and body weight and induced characteristic "wet-dog" shake behaviour. Whereas the anticonvulsant effect increased during the 14 days of treatment, tolerance developed to the excitatory and, less rapidly, to the hypothermic and anorexigenic effects of 5-HTP. Biochemical determinations showed marked increases in 5-HTP and its metabolites, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid, in both plasma and brain throughout the period of treatment. The mechanisms underlying the different time-courses of the functional effects of 5-HTP during continued treatment are not clear, but effects on catecholaminergic systems as well as regional differences in 5-HT increases in the brain might be involved.
Assuntos
5-Hidroxitriptofano/farmacologia , Transtornos dos Movimentos/induzido quimicamente , Convulsões/induzido quimicamente , 5-Hidroxitriptofano/análise , Animais , Temperatura Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Carbidopa/farmacologia , Interações Medicamentosas , Tolerância a Medicamentos , Feminino , Ácido Hidroxi-Indolacético/análise , Ratos , Ratos Endogâmicos , Serotonina/análiseRESUMO
To determine whether paraventricular corticotropin-releasing factor (CRF) neurons express NMDA, AMPA or kainate-preferring glutamate receptors, we have colocalized, by in situ hybridization (ISH), transcripts of various glutamate receptor subunit genes with the CRF messenger RNA on doublet adjacent sections of the rat hypothalamus. We found that more than 70% of CRF-positive neurons contain the NMDA receptor subunit NR1 mRNA whereas NR2A and NR2B subunit mRNAs were not detectable in CRF cells. A significant proportion of identified CRF cells express AMPA receptor subunit GluRA (46%), GluRB (21%) as well as the kainate-preferring receptor subunit KA2 (31%) mRNAs. These results support the hypothesis that the excitatory transmitter glutamate may directly influence CRF neurons through NMDA as well as non-NMDA receptors.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Receptores de Glutamato/genética , Animais , Expressão Gênica/fisiologia , Hibridização In Situ , Masculino , Neurônios/fisiologia , Neurônios/ultraestrutura , Sondas de Oligonucleotídeos , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos WKY , Coloração pela PrataRESUMO
The effect of aminooxyacetic acid (AOAA, 90 mg/kg i.v.) on bicuculline, picrotoxin and 3-mercaptopropionic acid (3-MPA) induced convulsions and on GABA concentrations in cerebellum, whole brain and a synaptosomal fraction of whole brain was investigated. At various intervals after AOAA the rats were either injected with one of the convulsive drugs or sacrificed for analysis of the GABA concentration. AOAA caused a rapid initial (0-30 min) and a later slower increase of GABA in cerebellum and whole brain. In the synaptosomal fraction the GABA accumulation was delayed and less pronounced when compared to the whole brain. The bicuculline induced convulsions were markedly potentiated during the first hour but completely blocked from 2-6 h after AOAA. Picrotoxin showed a somewhat different pattern to bicuculline in the interactions with AOAA. The initial strong potentiation was not observed but the later phase of protection was present. In the interactions with 3-MPA, the effect of AOAA was always protective. The time to onset of convulsions was gradually increased during the first 30 min after AOAA. This protective effect remained practically unchanged up to 6 h after AOAA. However, once started, the convulsions were generally of the same duration and intensity. The results can be interpreted as GABA accumulating after AOAA stimulates GABA receptors to a degree more or less proportional to the whole brain GABA concentration and further that GABA synthetized in neurons is liberated, stimulates inhibitory bicuculline sensitive (predominant) and excitatory bicuculline insensitive receptors and is captured to a large extent by non-neuronal cells.
Assuntos
Acetatos/farmacologia , Ácido Amino-Oxiacético/farmacologia , Encéfalo/metabolismo , Receptores de Superfície Celular/metabolismo , Ácido gama-Aminobutírico/metabolismo , Ácido 3-Mercaptopropiônico/toxicidade , Animais , Bicuculina/toxicidade , Compartimento Celular , Fracionamento Celular , Cerebelo/metabolismo , Interações Medicamentosas , Feminino , Picrotoxina/toxicidade , Ratos , Receptores de GABA-A , Convulsões/induzido quimicamente , Sinaptossomos/metabolismoRESUMO
Asia accounts for more than half of all cases of cervical cancer registered globally and improving prevention is urgently needed. A range of tools and strategies is now available to effectively prevent this disease, including two new prophylactic HPV vaccines approved and recommended for adolescents and young women. However, without communication these tools may have little impact on disease burden. The conferences of the Asia Oceania Research Organisation in Genital Infection and Neoplasia (AOGIN) bring together clinicians and scientists whose work is related to genital infections, particularly HPV, cervical dysplasia and neoplasia, as well as other anogenital cancers, with the aim of improving communication on prevention through human papillomavirus (HPV) vaccination and screening in Asian countries. The scope of this year's AOGIN conference was to extend education to include health workers, family doctors, paediatricians, governmental health agencies, and the general public through patients' testimonials that can reach out to women raising awareness of this silent disease. Community based initiatives and awareness campaigns were also reported, and can empower the people to engage in a dialog with local governments towards prioritization of cancer prevention programs, achieving more for the public than isolated actions. Parents and teachers are encouraged to communicate about these issues within families and schools. Evidence was discussed that males can participate in cervical cancer control as well, and prevention programs involving men should not be neglected as they may reduce genital disease burden in women. Opinion leaders proposed prevention measures to be considered for governmental decisions. While each country develops a locally appropriate policy for cervical cancer control there is a need to revise these programs regularly, as knowledge increases in response to public need, as well as to gather evidence about disease burden and the effectiveness of education and interventions. In conclusion, AOGIN is committed to improve communication with patients, health authorities, professional organizations and opinion leaders towards strengthening cervical cancer prevention in Asia, to achieve a timely steep reduction in this cancer.
Assuntos
Educação em Saúde , Vacinas contra Papillomavirus/uso terapêutico , Neoplasias do Colo do Útero/prevenção & controle , Adolescente , Ásia , Criança , Feminino , Humanos , Vacinação em Massa , Instituições Acadêmicas , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal , Adulto JovemRESUMO
A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.
Assuntos
Encéfalo/metabolismo , Microscopia Eletrônica , Terminações Nervosas/efeitos dos fármacos , Sinaptossomos/análise , Ácido gama-Aminobutírico/análise , Ácido 3-Mercaptopropiônico/farmacologia , Acetilcolinesterase/metabolismo , Animais , Feminino , Glutamato Descarboxilase/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Terminações Nervosas/análise , Terminações Nervosas/enzimologia , Ratos , Ratos Endogâmicos , Succinato Desidrogenase/metabolismo , Sinaptossomos/enzimologia , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura , Ácido Valproico/farmacologiaRESUMO
A cDNA clone of the neural cell adhesion molecule AMOG was isolated from a lambda gt10 library constructed from 8-day-old mouse brain poly(A) + RNA with a 17mer oligonucleotide probe designed from a nonapeptide sequence obtained from tryptic peptides of AMOG. The cDNA clone expressed as a fusion protein that is recognized by polyclonal AMOG antibodies; conversely, polyclonal antibodies prepared against the fusion protein react with AMOG. The clone contains the full sequence derived from the nonapeptide. Of all tissues tested, only brain expresses detectable levels of AMOG by ELISA and Northern blot analyses, indicating a high correlation in expression at the protein and mRNA levels. Both brain and astrocytes express a 3 kb long mRNA, which appears to be encoded by a single gene.
Assuntos
Antígenos de Superfície/genética , Encéfalo/metabolismo , Adesão Celular , Clonagem Molecular , DNA/genética , Adenosina Trifosfatases , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Proteínas de Transporte de Cátions , Moléculas de Adesão Celular , Moléculas de Adesão Celular Neuronais , DNA/isolamento & purificação , Proteínas da Matriz Extracelular , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/análise , Poli A/genética , RNA/genética , RNA MensageiroRESUMO
A novel D3 receptor cDNA containing an insertion of 84 bp has been isolated from a series of alternatively spliced D3 receptor cDNAs by polymerase chain reaction on RNA isolated from rat basal forebrain. Sequence analysis of both subcloned cDNAs and genomic DNA clones, has identified a 84 bp insertion resulting in a 28 amino acid insertion in the first extracellular loop. A comparison of the cDNA sequence of the novel D3 cDNA and the isolated genomic sequence indicates that the novel insertion corresponds to a new exon and arises via an alternative splicing event. The mRNA for the novel insertion could be detected by PCR in several areas of the brain co-localized with the wild type form of the D3 receptor. Moreover, genomic Southern blot analyses suggest that the D3-receptor exon giving rise to this novel D3 mRNA variant might exist in other species.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Éxons , Expressão Gênica , RNA Mensageiro/biossíntese , Ratos Wistar/genética , Receptores de Dopamina D2 , Receptores Dopaminérgicos/biossíntese , Receptores Dopaminérgicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase/métodos , Ratos , Receptores de Dopamina D3RESUMO
Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.
Assuntos
Encéfalo/metabolismo , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/análise , Sequência de Aminoácidos , Animais , Autorradiografia , Sequência de Bases , Fator Neurotrófico Derivado do Encéfalo , Clonagem Molecular , DNA/genética , Biblioteca Gênica , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Radioisótopos de Fósforo , RNA Mensageiro/genéticaRESUMO
Rats were trained for nicotine intravenous infusions in a self-administration paradigm. The effect of nicotine self-administration on regional brain activity was studied by mapping changes of c-fos expression. Specific nicotine effects were determine by comparing the patterning of Fos-like immunoreactivity (Fos-Ll) in nicotine self-administering rats with that in three different control groups. Controls included rats exposed to the same manipulation as nicotine self-administering rats who received intravenous saline instead of nicotine. In addition, two groups of untrained sham-operated rats exposed daily to the same operant boxes were included: one group had the same food restriction used in the operant training, the other was fed ad libitum. Nicotine self-administration, exposure to saline and food restriction increased Fos-Ll in 43, 33 and three brain regions, respectively, when compared with the control group fed ad libitum. Computer-assisted image analysis of Fos-Ll profiles performed on 16 relevant limbic and sensory structures showed that in saline-treated rats a significant (P < 0.01) increase of Fos-Ll profiles was observed in medial prefrontal cortex, lateral septum, core and ventral shell of nucleus accumbens, claustrum, amygdaloid nuclei, paraventricular thalamic nucleus and lateral geniculate nucleus. A significant (P < 0.01) further increase produced by nicotine was found in medial prefrontal cortex and ventral shell of nucleus accumbens. Interestingly, cingulate and piriform cortex, superior colliculus and medial terminal nucleus of the accessory optic tract were specifically activated by nicotine but not saline. These results show that nicotine self-administration activates sensory structures, as well as limbic structures involved in natural rewarding pathways. The results suggest the involvement of restricted terminal regions of the mesocorticolimbic dopaminergic system in the maintenance of nicotine self-administration.