RESUMO
The cellular distribution of the type 3 serotonin receptor (5HT3R) in the rat brain was established immunocytochemically by using a polyclonal antibody raised against a synthetic peptide from the deduced amino-acid sequence of the cloned 5HT3R. The 5HT3R-immunoreactive neurons were found in the forebrain, brainstem, and spinal cord, but within each region, the intensity of the immunoreactivity differed considerably. Within the forebrain, intensely immunoreactive cells were found in layers II-III of the neocortex, anterior olfactory nucleus, hippocampal formation, and amygdala. A few strongly immunoreactive neurons were consistently observed in the caudate putamen, and moderately or weakly labeled neurons were occasionally found in the nucleus accumbens. Within the brainstem, intensely labeled neurons were found in the trigeminal motor (V) and facial (VII) nuclei. Immunostained neurons were detected in the dorsal and the ventral horn of the spinal cord. These results reveal that the 5HT3R-immunoreactive neurons are broadly distributed throughout the rat brain and spinal cord, and suggest that this receptor can subserve significant participation in central nervous system neurotransmission.
Assuntos
Química Encefálica/fisiologia , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Medula Espinal/metabolismo , Animais , Encéfalo/citologia , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Imuno-Histoquímica , Masculino , Mapeamento de Peptídeos , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores 5-HT3 de Serotonina , Medula Espinal/citologiaRESUMO
We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.
Assuntos
Proteínas de Transporte , Hipotálamo Posterior/química , Peptídeos e Proteínas de Sinalização Intracelular , Neurotransmissores/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos , Sequência Consenso , Homeostase , Masculino , Camundongos , Dados de Sequência Molecular , Neurônios/química , Neuropeptídeos/química , Neuropeptídeos/genética , Neurotransmissores/fisiologia , Orexinas , Precursores de Proteínas/química , Precursores de Proteínas/genética , Ratos , Ratos Wistar , Secretina/química , Vesículas Sinápticas/químicaRESUMO
In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca(2+)-signaled exocytosis is effectively blocked by antibodies and peptides to SNAP-25, a key constituent of the vesicle docking complex. In addition, Ca2+ inhibits the ability of synaptotagmin, a synaptic vesicle protein proposed as a calcium sensor and triggering device, to associate with this docking complex. These results support a model in which Ca(2+)-dependent triggering of neurotransmission at central synapses acts after ATP-dependent potentiation of the docking-fusion complex for membrane fusion.
Assuntos
Proteínas de Ligação ao Cálcio , Cálcio/farmacologia , Córtex Cerebral/fisiologia , Exocitose/fisiologia , Ácido Glutâmico/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana , Proteínas do Tecido Nervoso/fisiologia , Peptídeos/farmacologia , Sinaptossomos/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Especificidade de Anticorpos , Western Blotting , Cátions Bivalentes/farmacologia , Exocitose/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Cinética , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/imunologia , Proteína 25 Associada a Sinaptossoma , Sinaptossomos/efeitos dos fármacos , Sinaptotagminas , Tripsina/farmacologiaRESUMO
The present studies were designed to test the learning and memory capacities of transgenic mice with central overexpression of corticotropin-releasing factor in a forced alternation water T-maze task and in the Morris water maze. In T-maze testing, littermate control mice reached a criterion of 70% correct responses after five days of trials, while the performance of transgenic subjects was still random after the same training. In Morris maze testing, control subjects reached the submerged platform significantly faster (F(1.48) = 4.51, P < 0.05) after three days of trials, while the performance of transgenic mice was unimproved over the same period. The deficit in Morris maze performance in transgenic mice was reversed when the platform was visible above the surface of the water. Pre-test administration of the benzodiazepine anxiolytic, chlordiazepoxide (10 mg/kg), before acquisition training also produced a significant (F(4.40) = 16.61, P < 0.001) and persistent improvement in Morris maze performance in transgenic mice when compared to vehicle-treated transgenic litter mates. Finally, there was no evidence of hippocampal cell loss in transgenic brains. The results suggest that corticotropin-releasing factor-overexpressing mice exhibit a profound learning deficit without sensory or motor-related impairments, and that memory plasticity can be restored by anxiolytic pre-treatment. Thus, constitutive overabundance of brain corticotropin-releasing factor may produce hyperemotionality that interferes with learned behaviors. Stress-related disorders characterized by co-morbid deficits in learning/memory may benefit from pharmacological normalization of brain corticotropin-releasing factor systems.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Deficiências da Aprendizagem/genética , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Animais , Masculino , Camundongos , Camundongos Transgênicos , Tempo de Reação/fisiologiaRESUMO
We used in situ hybridization and immunocytochemistry to investigate the presence of GABA in neurons that express the type 3 serotonin receptor (5-HT3R). Quantitative analysis indicated that more than 90% of 5-HT3R expressing cells are GABAergic in the neocortex and hippocampus. The co-existence of 5-HT3R and GABA in cortical and hippocampal neurons indicates that serotonin, via 5-HT3R, can affect GABA release and suggests the participation of 5-HT3R in the inhibitory regulation of forebrain neurons.
Assuntos
Córtex Cerebral/citologia , Giro Denteado/citologia , Interneurônios/química , Receptores de Serotonina/genética , Ácido gama-Aminobutírico/fisiologia , Tonsila do Cerebelo/química , Tonsila do Cerebelo/citologia , Animais , Córtex Cerebral/química , Giro Denteado/química , Imuno-Histoquímica , Hibridização In Situ , Interneurônios/fisiologia , Masculino , Bulbo Olfatório/química , Bulbo Olfatório/citologia , Ratos , Ratos Sprague-Dawley , Receptores de Serotonina/análise , Receptores de Serotonina/biossíntese , Núcleos Septais/química , Núcleos Septais/citologiaRESUMO
We developed and characterized 14 polyclonal antibodies against peptides whose sequences were predicted from the type 3 serotonin receptor subunit A (5-HT3R-A) cDNA. One such antiserum, 0165, raised against a peptide corresponding to the large putative intracellular loop, immunoprecipitated in vitro translated 5-HT3R-A protein and recognized both recombinant and neuronal 5-HT3R-A protein by Western blot at a high titer. Furthermore, when antiserum 0165 was used to immunolabel brain sections previously hybridized with a riboprobe specific for 5-HT3R-A transcripts, neuronal co-localization of immunoproduct and transcript was widely found throughout the brain. The study of the distribution of 5-HT3R-A-immunoreactivity in the rat central nervous system with antiserum 0165 revealed intensely immunolabeled neurons in the forebrain (isocortex, olfactory regions, hippocampal formation and amygdala), brainstem (sensory and motor nuclei and nuclei of the reticular formation) and spinal cord (dorsal and ventral horn). At the subcellular level, the 5-HT3R-A was found in endomembranes involved in translation (nuclear envelope and endoplasmic reticulum) and in the dendritic plasma-membrane. The present report is the first description of the 5-HT3R-A immunolocalization in the CNS. The wide distribution of the 5-HT3R-A in the brain and spinal cord based on ligand binding, in situ hybridization and immunolocalization studies support its participation in a large array of central nervous system functions.
Assuntos
Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Receptores de Serotonina/metabolismo , Animais , Imuno-Histoquímica , Hibridização In Situ , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Four structurally related neuron-specific 1B426b mRNAs, designated AMY, BMY, AMZ, and BMZ, have been isolated from rat brain cDNA libraries. The four mRNAs are related to one another by their shared M region and by two pairs of alternative 5' (A, B) or 3' (Y, Z) regions. All four possible combinations were detected. The four transcripts are derived by differential promoter utilization (to generate A or B 5' ends) and alternative splicing (to generate Y or Z 3' ends) of the primary transcripts of the single D2Sutle gene. All four mRNAs were detected in most brain regions, but were enriched within the cortex and hippocampus. In the pituitary only the two A-type and in the adrenal glands only the two B-type mRNAs were detected. In situ hybridization shows a highly heterogeneous distribution across brain regions, paralleling the Northern blot results and additionally identifying the reactive cells as neurons. The cDNAs encode related glycoproteins of 125, 153, 457, and 485 amino acids, which have been detected immunochemically. The AMZ and BMZ proteins show significant sequence similarity with olfactomedin, an extracellular matrix protein of bullfrog olfactory epithelium, suggesting the possibility of a matrix-related function for these rat glycoproteins in neurons and neurosecretory cells.
Assuntos
Proteínas da Matriz Extracelular/biossíntese , Glicoproteínas/biossíntese , Neurônios/metabolismo , Mucosa Olfatória/metabolismo , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Química Encefálica , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Biblioteca Gênica , Glicoproteínas/química , Glicoproteínas/genética , Hibridização In Situ , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
We have characterized cDNA clones of 1G5, an mRNA highly enriched in the mammalian forebrain that encodes a 504-residue protein found in association with perikaryal membranes and neurites. The protein, which accumulates predominantly postnatally, is associated with vesicles in both axons and dendrites. The sequence of the 1G5 protein highly resembles those of protein kinases with serine/threonine specificity; however, although most residues universally conserved among protein kinases are present, a few signature residues are absent from the 1G5 protein. Furthermore, although recombinant 1G5 protein binds calmodulin in the presence of calcium, it lacks kinase activity with a sample substrate.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Prosencéfalo/metabolismo , Proteínas Quinases/genética , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/fisiologia , Proteínas de Ligação a Calmodulina/genética , Córtex Cerebral/metabolismo , Clonagem Molecular , Sondas Moleculares/genética , Dados de Sequência Molecular , Prosencéfalo/ultraestrutura , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
The distribution of the Ca2+ binding protein parvalbumin was studied in the rat retina with immunocytochemistry using a mouse monoclonal antibody. Specific parvalbumin immunoreactivity was identified within a subpopulation of ganglion cells and a subpopulation of amacrine cells. The topographical data provided by the present study may serve as a basis for a functional characterization of parvalbumin's role in the nervous system.
Assuntos
Parvalbuminas/química , Retina/química , Estilbamidinas , Animais , Corantes Fluorescentes , Gânglios/citologia , Técnicas Imunoenzimáticas , Nervo Óptico/citologia , Ratos , Retina/citologiaRESUMO
The rat 1B1075 mRNA encodes a 533-residue novel chromogranin/secretogranin-like acidic protein that contains an apparent secretion signal, several pairs of tandem basic residues, and internally repeated sequence elements. 1B1075 transcripts are detected, by blotting and in situ hybridization, at the highest levels in the neocortex, hippocampus, cerebellar cortex, selected pontine and diencephalic nuclei, and presumptive pituitary corticotrophs, at lower levels in specific nuclei in most other brain regions, but in none of several other tissues. Utilizing antisera to several nonoverlapping synthetic peptide fragments of the predicted protein sequence, we detect a brain- and pituitary-specific 57-kDa protein in cellular processes and fiber tracts, generally consistent with axonal transport from the cell bodies identified by in situ hybridization. Ultrastructural studies demonstrate that this protein is a component of intraneuronal vesicles in axons and vesicle-like structures in dendrites. Based on these data, we suggest the name Secretogranin III for the 1B1075 gene product. In related collaborative studies, a mouse deleted for the 1B1075-homologous gene has been produced that should allow assessment of its physiological role.
Assuntos
Química Encefálica , Cromograninas/genética , Proteínas do Tecido Nervoso/genética , Hipófise/análise , Proteínas/genética , RNA Mensageiro/genética , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/crescimento & desenvolvimento , Encéfalo/ultraestrutura , Clonagem Molecular , Cricetinae , Diencéfalo/análise , Diencéfalo/ultraestrutura , Técnicas Imunoenzimáticas , Mesencéfalo/análise , Mesencéfalo/ultraestrutura , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Neurônios/análise , Neurônios/ultraestrutura , Hibridização de Ácido Nucleico , Ratos , Ratos Endogâmicos , Rombencéfalo/análise , Rombencéfalo/ultraestrutura , Medula Espinal/análise , Medula Espinal/ultraestrutura , Distribuição TecidualRESUMO
To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.
Assuntos
Regulação da Expressão Gênica , Neurônios/enzimologia , Fosfopiruvato Hidratase/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Encéfalo/metabolismo , Clonagem Molecular , Genes , Óperon Lac , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Testículo/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A rodent cortex-enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex-minus-cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNAs encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine-rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen-like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.
Assuntos
Córtex Cerebral/metabolismo , DNA/metabolismo , Proteínas do Tecido Nervoso/biossíntese , RNA Mensageiro/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Córtex Cerebral/anatomia & histologia , Clonagem Molecular , Biblioteca Gênica , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Neurotoxinas/análise , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , Ratos , Ratos Endogâmicos , Radioisótopos de EnxofreRESUMO
We have identified two nerve growth factor (NGF)-induced polypeptides (Mr 80,000 and 90,000) in PC12 cells that are heat stable and not sulfated. Indirect immunofluorescence localization of these polypeptides in NGF-treated PC12 cells reveals a punctate pattern concentrated in neurites. Immunoperoxidase staining of rat tissue shows that these polypeptides are found throughout the brain in selected subsets of neurons and are absent in the pituitary or adrenal medulla. Several of these characteristics are similar to a recently described NGF-induced secretory protein (VGF8a), which has sequence similarities to secretogranins. The property of NGF inducibility and the distribution of these polypeptides within rat tissues are both novel features for secretogranin proteins.
Assuntos
Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Biossíntese de Proteínas , Neoplasias das Glândulas Suprarrenais , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Cromograninas , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Soros Imunes , Técnicas Imunoenzimáticas , Cinética , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Neurônios/citologia , Neurônios/metabolismo , Biossíntese Peptídica , Peptídeos/isolamento & purificação , Feocromocitoma , Proteínas/genética , Proteínas/isolamento & purificação , RatosRESUMO
cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.
Assuntos
Hipocampo/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Sinaptossomos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Expressão Gênica , Biblioteca Gênica , Imuno-Histoquímica , Sistemas de Informação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Sondas de Oligonucleotídeos , Biossíntese de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Proteína 25 Associada a SinaptossomaRESUMO
The mRNA of rat secretory-vesicle protein chromogranin B is abundant in brain, adrenal medulla, and anterior pituitary. The primary translation product predicted from the cDNA sequence of this 2,337-nucleotide transcript corresponds to a hydrophilic 655-residue protein preceded by a signal peptide. Both termini of the mature 75-kD protein show extensive similarity to other chromogranins; the more variable internal region is characterized by glutamic acid clusters and numerous pairs of basic residues. In rodent brain, mRNA accumulation starts around embryonic days 13-14 and peaks by postnatal day 20. In situ hybridization in brain sections shows that the mRNA is enriched in the hippocampal formation, the endocrine hypothalamus, the olfactory system, and in anatomically distinct structures in the pons-medulla.
Assuntos
Cromograninas/genética , Proteínas do Tecido Nervoso/genética , Sistemas Neurossecretores/metabolismo , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Cromogranina B , Clonagem Molecular , Embrião de Mamíferos , Biblioteca Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Especificidade de Órgãos , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Neonatal mice inoculated with lymphocytic choriomeningitis virus (LCMV) have non-lytic persistent neuronal infection and disturbed behavior. We now show that LCMV replicates in neurons containing the neurotransmitter somatostatin without morphologic evidence of injury and that persistent neuronal LCMV infection in mice is attended by a decrease in brain levels of somatostatin mRNA. Brain levels of mRNA for another neurotransmitter peptide, cholecystokinin, are not decreased. These data are the first to localize a virus to a specific neurotransmitter-containing cell during in vivo infection and suggest that persistent viral infections could cause neurologic or psychiatric diseases through selective effects on brain levels of neurotransmitter mRNAs.
Assuntos
Coriomeningite Linfocítica/metabolismo , RNA Mensageiro/metabolismo , Somatostatina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos , Neurônios/metabolismoRESUMO
To study the effects of antithrombotic therapy in experimental stroke, we have characterized a baboon model of acute cerebrovascular thrombosis. In this model an inflatable silastic balloon cuff has been implanted by transorbital approach around the right middle cerebral artery (MCA), proximal to the take-off of the lenticulostriate arteries (LSA). Inflation of the balloon for 3 hours in six animals produced a stereotypic sustained stroke syndrome characterized by contralateral hemiparesis. An infarction volume of 3.2 +/- 1.5 cm3 in the ipsilateral corpus striatum was documented by computerized tomographic (CT) scanning at 10 days following stroke induction and 3.9 +/- 1.9 cm3 (n = 4) at 14 days by morphometric neuropathologic determinations of brain specimens fixed in situ by pressure-perfusion with 10% buffered formalin. Immediate pressure-perfusion fixation following deflation of the balloon was performed in 16 additional animals given Evans blue dye intravenously prior to the 3 hour MCA balloon occlusion. Light microscopy and transmission electron microscopy consistently confirmed the presence of thrombotic material occluding microcirculatory branches of the right LSA in the region of Evans blue stain, but not those of the contralateral corpus striatum. When autologous 111In-platelets were infused intravenously in four animals from the above group prior to the transient 3 hour occlusion of the right MCA, gamma scintillation camera imaging of each perfused-fixed whole brain demonstrated the presence of a single residual focus of 111In-platelet activity involving only the Evans blue-stained right corpus striatum. Focal right hemispheric activity was equivalent to 0.55 +/- 0.49 ml of whole blood, and the occlusion score derived from histologic examination of the microcirculation of the Evans blue-stained corpus striatum averaged 34.8 +/- 2.8. Similar 111In-platelet imaging and histologic scoring experiments carried out in four animals pretreated with the antithrombotic combination heparin and ticlopidine showed marked reduction of both 111In-platelet activity (0.01 +/- 0.03 ml vs. 0.55 +/- 0.49 ml; p less than 0.01) and thrombotic occlusion of the microcirculation (10.8 +/- 7.4 units vs. 34.8 +/- 2.8 units; p less than 0.01) in the right corpus striatum following 3 hours of MCA occlusion. In separate control experiments 111In-labeled autologous platelets were infused after the 3 hour period of right MCA occlusion and subsequent balloon deflation in two animals; no focus of 111In-platelet activity was demonstrated in fixed whole brain.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Embolia e Trombose Intracraniana/etiologia , Animais , Plaquetas , Encéfalo/patologia , Artérias Cerebrais/patologia , Circulação Cerebrovascular , Transtornos Cerebrovasculares/etiologia , Índio , Embolia e Trombose Intracraniana/patologia , Masculino , Papio , Próteses e Implantes , RadioisótoposRESUMO
The rat brain-specific polypeptide 1B236 was previously characterized by molecular cloning and nucleotide sequence determination of its mRNA. It has been shown to exist in rat brain in discrete neuronal circuits, primarily as a 100,000 Da glycoprotein. We now have determined the time course of expression of 1B236 mRNA and protein in rat brain during fetal and postnatal development, detecting 1B236 mRNA by RNA blotting and assaying 1B236 protein by electroblotting and radioimmunoassay with antibodies against synthetic peptides. By both indices, expression of the 1B236 gene products is found to be a relatively late event in neuronal development. 1B236 mRNA is first detectable in extracts of whole rat brain at Postnatal Day 5 (PD 5) and increases to a maximum concentration at PD 25. In extracts of dissected brain regions, 1B236 mRNA is first detectable at PD 5 in hindbrain and cerebellum, at PD 9 in midbrain/diencephalon, but not until PD 13 in telencephalon. The appearance of 1B236 protein follows a very similar time course to that of its mRNA in both whole brain and dissected brain regions, suggesting that the expression of the protein during development is regulated largely by transcription of its mRNA. The pattern of 1B236 expression was confirmed by immunocytochemical localization of 1B236 protein: Immunoreactive material can be detected first in spinal cord at PD 3-PD 5 and then appears in progressively more rostral brain regions in increasingly older animals. Several brain regions, however, that do not contain 1B236 immunoreactivity in the adult, such as optic nerve and somatic efferent cranial nerve nuclei, show transient expression of 1B236 during postnatal development.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/crescimento & desenvolvimento , Regulação da Expressão Gênica , Genes , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Histocitoquímica , Peso Molecular , Gravidez , RNA Mensageiro/análise , Radioimunoensaio , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
The 318-amino acid, carboxy-terminal sequence of the putative brain-specific polypeptide 1B236 was deduced from the nucleotide sequence of its cloned brain-specific mRNA. Antisera raised against selected synthetic peptide fragments of this protein were used to map the cellular location of the presumptive gene product in the brains of normal or colchicine-pretreated adult rats. Antisera directed against any of three C-terminally located, but nonoverlapping, nonhomologous, synthetic peptide segments (P5, P6, or P7) produced virtually identical maps of intensely immunoreactive neuropil staining. The immunoreactivity was distributed heterogeneously and was most pronounced within olfactory, somatosensory, and limbic systems, and was more modest in certain motor and auditory structures. In colchicine-pretreated rats, large, multipolar perikarya were observed within the amygdala, caudate-putamen, cingulate, parietal, and piriform cortices, as well as in particular diencephalic and pontine nuclei. Smaller immunoreactive neurons with more limited dendritic extensions were observed in the olfactory bulb, the cerebellar cortex, and the dorsal horn and intermediolateral cell columns of the spinal cord. No immunoreactivity was observed in visceral structures innervated by the autonomic nervous system or in non-neural tissues. In addition to the virtually superimposable maps produced by antisera to all three synthetic fragments selected from the C-terminus of 1B236, some uniquely reactive sites were seen. Antisera to the most N-terminal of the three synthetic immunogens (P5) were reactive with neurons of the medial trapezoid nucleus and in nerve terminals surrounding the deep cerebellar nuclei. Antisera against the most C-terminal synthetic immunogen (P7) were reactive with neurons of the paraventricular and supraoptic hypothalamic nuclei. These data demonstrate that the 1B236 protein is located within selected neuronal elements within functionally related cellular circuits established more formally by other methods. Our data show that protein 1B236-immunoreactive cells share at least the expression of this protein and suggest that these cells may also be related epigenetically or evolutionarily. These data, together with other subcellular, ultrastructural, and electrophysiological properties of 1B236, suggest that this protein could be considered as a prohormone capable of yielding several final candidate transmitter products.