RESUMO
We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1(b-m3) locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.
Assuntos
Perfilação da Expressão Gênica/métodos , Técnicas de Introdução de Genes/métodos , Vetores Genéticos/genética , Genômica/métodos , Hipoxantina Fosforribosiltransferase/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Citomegalovirus/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Alinhamento de Sequência , Deleção de SequênciaRESUMO
A monoclonal antibody to choline acetyltransferase (ChAT) was utilized for immunocytochemical identification of cholinergic neurons in the basolateral hypothalamus. Light and electron microscopic examination revealed a network of cell bodies, dendrites, and axonal processes dorsolateral to the supraoptic nucleus. Within this region the cells immunoreactive for ChAT receive numerous unlabeled terminals which contact dendrites, cell soma, axons and occasional somatic spines. In a few cases, small ChAT-immunoreactive terminals were observed contacting a cholinergic cell soma or large dendrite. Many ChAT-immunoreactive fibers were directed toward the supraoptic nucleus forming a dense local network but very few of these fibers penetrated deeper than approximately 20 micron into the supraoptic nucleus. A total of 63 ChAT-immunoreactive terminals were mapped within the basal hypothalamus, of which the vast majority contacted unlabeled dendrites immediately dorsolateral to the supraoptic nucleus. Labeled terminals were rare or nonexistent in the medial portions of the hypothalamus or deep within the supraoptic nucleus. This pattern of ChAT terminal densities correlates with the distribution of binding for the muscarinic cholinergic probe, [3H]quinuclidinylbenzilate, but not the binding of the putative nicotinic cholinergic probe, [125I]alpha-bungarotoxin, which is high within the supraoptic nucleus. Thus, the cholinergic neurons of the basal hypothalamus appear to form a network of intrinsic connections which probably represent input to muscarinic cholinergic receptors. No evidence was found to suggest that cholinergic presynaptic terminals were colocalized with the alpha-bungarotoxin binding protein within the supraoptic nucleus.
Assuntos
Fibras Colinérgicas/ultraestrutura , Núcleo Supraóptico/ultraestrutura , Sinapses/ultraestrutura , Animais , Colina O-Acetiltransferase/análise , Fibras Colinérgicas/enzimologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Ratos , Ratos Endogâmicos , Núcleo Supraóptico/enzimologiaRESUMO
This study investigated the influence of ethanol exposure throughout gestation on cholinergic development within the rat striatal region. Pregnant Long-Evans rats were maintained on three diets throughout gestation: A liquid diet in which ethanol accounted for 35-39% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, and a lab chow control diet. At postnatal days 14 and 60 (P14 and P60), the striatal regions of the offspring were analyzed for the number of cholinergic neurons, via choline acetyltransferase (ChAT) immunostaining. The area of the striatum was also measured in these animals. At P14, P21, and P60, ChAT activity was assessed in the same region. These analyses revealed a significant increase in the number of cholinergic striatal neurons at P14 in the animals which had been exposed prenatally to ethanol. This increase was transient, however, with equal numbers of ChAT-positive cells found in all three groups by adulthood (P60). The brain weights of the ethanol-exposed animals were significantly reduced at P14 and P21, but were comparable to controls by P60. There were no significant differences in the striatal area or the overall volume of the region assessed, however, at either P14 or P60. Although there were some increases in ChAT activity across the ages viewed (most notably between P14 and P21), there were no effects of diet on ChAT activity at any age assessed. It is proposed that the increased numbers of cholinergic neurons could be a function of errors in migration, enhanced neurogenesis, diminished cell death, alterations in gene expression, or increased cell survival as a result of alterations in neurotrophic factor production or availability.
Assuntos
Fibras Colinérgicas/efeitos dos fármacos , Etanol/farmacologia , Neostriado/citologia , Efeitos Tardios da Exposição Pré-Natal , Ratos Endogâmicos/fisiologia , Fatores Etários , Animais , Contagem de Células , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/enzimologia , Feminino , Imuno-Histoquímica , Masculino , Neostriado/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Gravidez , RatosRESUMO
This study characterized the influence of full-term gestational ethanol exposure on choline acetyltransferase (ChAT)-immunoreactive neurons that project to the hippocampus, within the medial septal (MS) nucleus and the vertical limb of the diagonal band of Broca (DBv). On gestation days 1-22, pregnant dams were fed either a vitamin fortified ethanol-containing liquid diet, pair fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In a previous study, we found that chronic prenatal exposure to ethanol, in this manner, resulted in a significant decline in the ontogenetic upregulation of ChAT activity in the septal area during the second postnatal week, but was followed by recovery to control levels by adulthood. On postnatal days 14 and 60 (P14 and P60) the brains were prepared for ChAT immunocytochemistry. Ethanol exposure had little influence on the number of ChAT-positive neurons in the MS nucleus of animals at either age. Ethanol exposure had no effect on neuronal size or ChAT staining intensity of MS or DBv neurons when compared to chow-fed offspring. Although age-related increases in cholinergic neuronal numbers and decreases in neuronal size were observed between juvenile and adult animals, prenatal ethanol exposure did not appear to influence these postnatal changes in the population as a whole. Overall, these findings suggest that the anatomical maturation of septal cholinergic neurons may be relatively insensitive to prenatal ethanol exposure under conditions of a vitamin-rich dietary supplementation, while biochemical development within this region may be more susceptible to early ethanol influences.
Assuntos
Fibras Colinérgicas/efeitos dos fármacos , Etanol/farmacologia , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Núcleos Septais/citologia , Animais , Contagem de Células , Tamanho Celular/efeitos dos fármacos , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/enzimologia , Feminino , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , Neurônios/enzimologia , Gravidez , Ratos , Ratos Endogâmicos , Núcleos Septais/efeitos dos fármacos , Fatores de TempoRESUMO
During the course of one year, 47 critical care patients with gram-negative bacillary pneumonia at Millard Fillmore Hospital were randomly assigned to aztreonam or tobramycin therapy (two to one). Of these, 40 were fully evaluable for microbiologic and clinical response. All evaluable patients had gram-negative organisms in tracheal aspirate culture specimens and confirmed susceptibility of the organism to both study drugs. There was no difference between the two groups with respect to the percentage of patients who received concurrent antibiotics for gram-positive organisms. More than 60 percent of the patients received mechanical ventilation. Essentially, all had new lung infiltrates as shown by chest radiography, leukocytosis, recent onset of fever, and increased volume of purulent secretions. Half had multilobar pulmonary infiltrates. Their mean age was 73 years, with none under age 50. Most had chronic obstructive pulmonary disease, congestive heart failure, or both. By the prognostic nutritional index criteria, over 70 percent were nutritionally deficient at entry. The majority of infections were caused by Pseudomonas, Enterobacter, Klebsiella, and Escherichia coli. Aztreonam eradicated 92 percent of the causative gram-negative organisms, compared with 57 percent for tobramycin (p less than 0.05). Aztreonam produced a favorable clinical response (cure or improvement) in 93 percent of patients, compared with 50 percent for tobramycin (p less than 0.05). There were no differences in the minor adverse effects observed in the two treatment groups. Overall, aztreonam was superior to tobramycin for treatment of pneumonia due to susceptible gram-negative bacteria in these critical care patients.
Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Infecção Hospitalar/tratamento farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Tobramicina/uso terapêutico , Idoso , Antibacterianos/efeitos adversos , Aztreonam , Ensaios Clínicos como Assunto , Cuidados Críticos/métodos , Método Duplo-Cego , Esquema de Medicação , Quimioterapia Combinada , Feminino , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Pneumonia/microbiologia , Pneumonia/patologia , Distribuição Aleatória , Tobramicina/efeitos adversosRESUMO
BACKGROUND AND METHODS: The incidence and risk factors for erythromycin-induced ototoxicity are unknown. We conducted a prospective, nested case-control study of assessment of auditory function in patients receiving erythromycin versus other antibiotics (control group) for community-acquired pneumonia. Sequential audiograms were performed during antibiotic therapy for both cases and controls by an audiologist unaware of the identity of the therapy administered. Erythromycin serum concentrations were obtained for all patients receiving erythromycin. RESULTS: Symptomatic ototoxicity (tinnitus or hearing loss) confirmed by audiograms was documented in five of 30 patients receiving erythromycin and none of 15 receiving other antibiotics. Ototoxicity was significantly related to high peak concentration and high AUC 0-infinity as a function of decreased total systemic clearance. Ototoxicity occurred only in those patients who received 4 g/day versus 2 g/day or no erythromycin (p = 0.05). Ototoxicity resolved in all patients within 6 to 14 days after discontinuation of therapy. CONCLUSIONS: Erythromycin ototoxicity is dose- and serum concentration-dependent. Patients receiving erythromycin, especially at a total daily dose of 4 g, should be monitored regularly for subjective evidence of sensorineural hearing dysfunction. Ototoxicity is reversible if the diagnosis is made early in the course.
Assuntos
Eritromicina/efeitos adversos , Transtornos da Audição/induzido quimicamente , Pneumonia/tratamento farmacológico , Adulto , Idoso , Audiometria , Estudos de Casos e Controles , Eritromicina/farmacocinética , Transtornos da Audição/sangue , Perda Auditiva Bilateral/induzido quimicamente , Perda Auditiva Neurossensorial/induzido quimicamente , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia/sangue , Estudos Prospectivos , Zumbido/induzido quimicamenteRESUMO
Cefmenoxime concentration/effect relationships were retrospectively explored for gram-negative bacteria isolated from 14 critical care patients treated for nosocomial pneumonia. The effects of cefmenoxime concentrations on in vitro growth kinetics of 21 isolated pathogens were studied using the Abbott MS-2 Research System, from which a dynamic response concentration was derived. Serum pharmacokinetic profiles were obtained in each patient. These data were used to calculate the in vivo total area under the curve over dynamic response concentration and the time that cefmenoxime concentrations exceeded the dynamic response concentration for each bacteria. The same determinations were made in 18 patients prospectively treated, except that dosage was optimized on the basis of previous mathematical relations to achieve bacterial eradication in four days. This method of dosage optimization is termed dual individualization. Serial cultures of infected tissues were evaluated to determine the number of days to the eradication of bacteria, and the pharmacokinetic and pharmacodynamic variables were used to describe the bacteriologic response of the original pathogen isolated in pretreatment culture. Bacterial eradication rates could be described from cefmenoxime pharmacokinetics in the patient and from the relation between concentration and bacterial inhibition. Patients who were prospectively treated using these retrospectively derived relationships had a predictable day of bacterial eradication. This, in turn, was associated with a shorter duration of treatment (p less than 0.05). The success of prospective dual individualization is encouraging and suggests that more precise optimization of antibiotic dosage can yield a predictable rate of bacterial eradication from the infection site.
Assuntos
Cefotaxima/análogos & derivados , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Idoso , Cefmenoxima , Cefotaxima/administração & dosagem , Cefotaxima/sangue , Cefotaxima/farmacologia , Computadores , Infecção Hospitalar/sangue , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Feminino , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia/sangue , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Estudos Prospectivos , Estudos RetrospectivosRESUMO
The distribution of glutamate immunoreactivity was mapped within the supraoptic nucleus of the rat hypothalamus utilizing a specific anti-glutamate antibody. Magnocellular neuroendocrine cells of the supraoptic nucleus showed intense immunoreactivity for glutamate which varied with the conditions of fixation. Within the perikarya, reaction product was found associated with the endoplasmic reticulum but not the mitochondria, Golgi, dense bodies or neurosecretory granules. A relatively high density of glutamate-immunoreactive terminals was found in the supraoptic nucleus. These terminals were less affected by fixation condition and were generally found contacting large, glutamate-immunoreactive processes within the ventral dendritic neuropil of the supraoptic nucleus. The pattern and characteristics of glutamate immunoreactivity in the supraoptic nucleus suggested the presence of two distinct glutamate pools. The magnocellular neuroendocrine cells may contain a large, labile metabolic pool of glutamate. These cells, in turn, appear to receive glutamate synaptic input from a more stable pool consistent with suggestions that glutamate may be used as a transmitter within this system.
Assuntos
Glutamatos/metabolismo , Núcleo Supraóptico/metabolismo , Animais , Ácido Glutâmico , Imuno-Histoquímica , Masculino , Ratos , Ratos Endogâmicos , Núcleo Supraóptico/ultraestruturaRESUMO
The arborization pattern and postsynaptic targets of corticofugal axons in basal forebrain areas have been studied by the combination of anatomical tract-tracing and pre- and postembedding immunocytochemistry. The anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin was iontophoretically delivered into different neocortical (frontal, parietal, occipital), allocortical (piriform) and mesocortical (insular, prefrontal) areas in rats. To identify the transmitter phenotype in pre- or postsynaptic elements, the tracer staining was combined with immunolabeling for either glutamate or GABA, or with immunolabeling for choline acetyltransferase or parvalbumin. Tracer injections into medial and ventral prefrontal areas gave rise to dense terminal arborizations in extended basal forebrain areas, particularly in the horizontal limb of the diagonal band and the region ventral to it. Terminals were also found to a lesser extent in the ventral part of the substantia innominata and in ventral pallidal areas adjoining ventral striatal territories. Similarly, labeled fibers from the piriform and insular cortices were found to reach lateral and ventral parts of the substantia innominata, where terminal varicosities were evident. In contrast, descending fibers from neocortical areas were smooth, devoid of terminal varicosities, and restricted to the myelinated fascicles of the internal capsule en route to more caudal targets. Ultrastructural studies obtained indicated that corticofugal axon terminals in the basal forebrain areas form synaptic contact primarily with dendritic spines or small dendritic branches (89%); the remaining axon terminals established synapses with dendritic shafts. All tracer labeled axon terminals were immunonegative for GABA, and in the cases investigated, were found to contain glutamate immunoreactivity. In material stained for the anterograde tracer and choline acetyltransferase, a total of 63 Phaseolus vulgaris leucoagglutinin varicosities closely associated with cholinergic profiles were selected for electron microscopic analysis. From this material, 37 varicosities were identified as establishing asymmetric synaptic contacts with neurons that were immunonegative for choline acetyltransferase, including spines and small dendrites (87%) or dendritic shafts (13%). Unequivocal evidence for synaptic interactions between tracer labeled terminals and cholinergic profiles could not be obtained in the remaining cases. From material stained for the anterograde tracer and parvalbumin, 40% of the labeled terminals investigated were found to establish synapses with parvalbumin-positive elements; these contacts were on dendritic shafts and were of the asymmetrical type. The present data suggest that corticofugal axons innervate forebrain neurons that are primarily inhibitory and non-cholinergic; local forebrain axonal arborizations of these cells may represent a mechanism by which prefrontal cortical areas control basal forebrain cholinergic neurons outside the traditional boundaries of pallidal areas.
Assuntos
Axônios/ultraestrutura , Córtex Cerebral/anatomia & histologia , Prosencéfalo/anatomia & histologia , Prosencéfalo/ultraestrutura , Animais , Mapeamento Encefálico , Córtex Cerebral/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Prosencéfalo/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
In the present normal anatomical light and electron microscopic study in the rat, histochemical (Nissl, Timm, Golgi) or immunocytochemical (microtubule-associated protein type 2, glutamate decarboxylase, glutamate receptor subunit 1, synaptophysin) stains were used to analyse neurons embedded within the stria terminalis and their associated neuropil. These cells are closely related to the bed nucleus of the stria terminalis and the centromedial amygdala, and have been termed the "supracapsular part of the bed nucleus of the stria terminalis". The largest part of this neuronal complex is located in the ventrolateral part of the stria, where it appears as a round or oval "lateral pocket" in virtually any type of light microscopic preparation because of its collection of neuronal cell bodies and dense neuropil, in addition to a lacework of unmyelinated axons. A much smaller but still distinct "medial pocket" is located in the medial corner of the stria. The large lateral subdivision of the supracapsular stria terminalis is directly continuous with the lateral bed nucleus of the stria terminalis and extends to the central amygdaloid nucleus, containing a column of neurons that is only broken up into cell clusters at the most caudal levels of the stria as it drops vertically toward the amygdala. The considerably smaller medial subdivision appears, in turn, to be directly continuous with the medial part of the bed nucleus of the stria terminalis. The medial column tapers off more rapidly than the lateral part, so that as the middle levels are approached, only small interrupted clusters of cells are seen. Solitary neurons can also be found in practically every part of the stria terminalis except among the ventrally located axons of the commissural component. Most of the neurons are small to medium in size, as viewed in transverse sections of the stria, but larger neurons are also encountered. In sections parallel to the stria, many neurons are fusiform in appearance. The dendrites are often aligned in a longitudinal fashion; many of the dendrites related to the cells in the lateral pocket are moderately to densely spined, whereas those in the medial pocket are more sparsely spined. The neuropil in both the lateral and medial pockets is characterized by boutons, bundles of unmyelinated axons, and dendrites. Based on their vesicle content, the boutons are divided into three major types: (A) round or slightly oval, agranular vesicles of uniform size; (B) pleomorphic, agranular vesicles, many of which are flattened; and (C) pleomorphic agranular vesicles, some of which are considerably larger than the ones in type B boutons. Type A boutons establish contacts with both dendritic spines and shafts, whereas types B and C usually contact dendritic shafts and sometimes somata. These synaptic components are similar to those described earlier for the central and medial amygdaloid nuclei. Overall, our results support the contention advanced in 1923 by Johnston [J. comp. Neurol. 35, 337481] that the cells accompanying the stria terminalis are interconnecting columns of a macrostructure encompassing the bed nucleus of the stria terminalis and centromedial amygdala. More recently, it has been appreciated that columns of neurons below the globus pallidus also belong to this macrostructure [Alheid G. F. et al. (1995) In The Rat Nervous System, 2nd edn, pp. 495 578, Academic, San Diego; de Olmos J. S. et al. (1985) In The Rat Nervous System, pp. 223-334, Academic, Sydney], which has been named the "extended amygdala".
Assuntos
Tonsila do Cerebelo/ultraestrutura , Neurônios/ultraestrutura , Núcleos Talâmicos/ultraestrutura , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/metabolismo , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Isoquinolinas , Masculino , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ratos , Sinapses/fisiologia , Sinapses/ultraestrutura , Núcleos Talâmicos/citologia , Núcleos Talâmicos/metabolismoRESUMO
Cultures of septal and hippocampal neurons from fetal rat and dorsal root ganglion neurons from embryonic chick were pretreated with GM1 ganglioside or cycloheximide and then supplemented with toxic concentrations of ethanol. GM1 provided significant protection against ethanol neurotoxicity in each population. The inhibition of protein synthesis by cycloheximide, however, which protects against cell death resulting from withdrawal of neurotrophic factor support, did not ameliorate ethanol-induced neuronal loss.
Assuntos
Encéfalo/metabolismo , Cicloeximida/farmacologia , Etanol/toxicidade , Gangliosídeo G(M1)/metabolismo , Gânglios Espinais/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Neurotoxinas/toxicidade , Animais , Encéfalo/citologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Feto , Gânglios Espinais/citologia , Hipocampo/citologia , Hipocampo/metabolismo , Cinética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Fatores de TempoRESUMO
Dorsal root ganglion (DRG) neurons were cultured with varying concentrations of ethanol and NGF. At low concentrations of NGF (0.1 ng/ml) moderate initial ethanol levels (250 mg/dl) significantly suppressed neurite outgrowth. Higher NGF concentrations (5 ng/ml) protected against this neurotoxicity. At this higher NGF concentration, neuronal survival was not significantly affected by exposure to 0.25-4 g/dl ethanol, although survival was significantly diminished at 5 and 6 g/dl. Neurite outgrowth was a more sensitive indicator of ethanol neurotoxicity in this population, with significant decreases in process extension seen with 1 g/dl ethanol. When cultures were supplemented with 10 ng/ml NGF, however, process elaboration was significantly greater at 1 g/dl ethanol than that measured with 5 ng/ml NGF, and in fact did not differ from NGF controls. These studies indicate that NGF can provide neuroprotective effects against ethanol toxicity under these conditions. The results are discussed in relation to other recent reports of trophic factor neuroprotection.
Assuntos
Etanol/farmacologia , Fatores de Crescimento Neural/farmacologia , Sistema Nervoso/efeitos dos fármacos , Animais , Embrião de Galinha , Embrião de Mamíferos/efeitos dos fármacos , Embrião não Mamífero , Gânglios Espinais/embriologia , Concentração OsmolarRESUMO
An ultrastructural analysis of post-embedding glutamate immunocytochemistry within the neural lobe of the pituitary was used to explore the possible role of glutamate within the magnocellular neuroendocrine cells. Relative densities of a colloidal gold marker associated with various cellular and subcellular compartments of the neural lobe were quantified by computer analysis of electron micrographs. Robust glutamate immunoreactivity was observed in both pituicytes (cytoplasm, mitochondria and nucleus) and neurosecretory endings. Within the neurosecretory endings, glutamate staining was specifically localized to the microvesicles with no overlap into the neurosecretory granule population. Stimulation of the vasopressin/oxytocin neurosecretory system by water deprivation increased glutamate content in pituicytes and mitochondria within neurosecretory endings but had little influence on microvesicle glutamate content. The results are consistent with the existence of multiple functional pools of immunoreactive glutamate in both pituicytes and neurosecretory endings. Microvesicles within the neurosecretory endings exhibit many properties of secretory vesicles, appear to be functionally independent of the neurosecretory granules, and have sufficient glutamate immunoreactivity to suggest that this amino acid may be compartmentalized for release in the neural lobe.
Assuntos
Glutamatos/metabolismo , Terminações Nervosas/metabolismo , Sistemas Neurossecretores/metabolismo , Neuro-Hipófise/inervação , Animais , Axônios/imunologia , Axônios/ultraestrutura , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Desidratação/fisiopatologia , Glutamatos/imunologia , Ácido Glutâmico , Imuno-Histoquímica , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Terminações Nervosas/imunologia , Terminações Nervosas/ultraestrutura , Sistemas Neurossecretores/imunologia , Neuro-Hipófise/imunologia , Neuro-Hipófise/ultraestrutura , Ratos , Ratos Endogâmicos , Frações Subcelulares/fisiologia , Frações Subcelulares/ultraestrutura , Fixação de TecidosRESUMO
Although the hypothalamus is generally regarded to have low levels of glutamate receptors, anatomical and physiological studies have provided consistent evidence implicating glutamate as a potential transmitter for the control of neuroendocrine cell activity. To clarify the extent of the contribution of synapses utilizing glutamate for control of vasopressin/oxytocin neuroendocrine cells, we mapped the density and location of glutamate immunoreactive terminals in the supraoptic nucleus and surrounding hypothalamus. Colloidal gold particle densities in presynaptic terminals were measured from electron micrographs of: (1) the magnocellular neuroendocrine cell perikarya (main body of the supraoptic nucleus), (2) the dendritic field of the magnocellular neuroendocrine cells (ventral dendritic neuropil) and (3) the hypothalamic perinuclear zone dorsal to the supraoptic nucleus. In addition, serial sections were stained, alternatively, for glutamate or GABA to determine glutamate staining in GABA cells. Terminals with high glutamate immunoreactivity were clearly distinguished from the glutamate precursor staining found in GABA terminals and were abundant at all rostral-caudal levels within each region. The number of glutamate terminals identified in each region was similar but represented a very high proportion of all terminals in the ventral dendritic neuropil (38%) vs. the main body of the supraoptic nucleus and the perinuclear zone (20-22%). The regional variation in the relative proportion of glutamate terminals was determined largely by differences in the number of non-glutamate terminals within each region. Glutamate and GABA terminals together accounted for over two-thirds of the innervation of vasopressin/oxytocin neuroendocrine cells. No systematic relationship was observed between excitatory and inhibitory inputs on the same cell. These results suggest that glutamate is the predominant excitatory transmitter used for control of vasopressin/oxytocin cells. The relative contribution of glutamate neurotransmission to a particular region will depend, in part, on the number and type of competing non-glutamate terminals.
Assuntos
Glutamatos/análise , Hipotálamo/ultraestrutura , Terminações Nervosas/ultraestrutura , Neurônios/ultraestrutura , Núcleo Supraóptico/ultraestrutura , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/análise , Animais , Dendritos/ultraestrutura , Ácido Glutâmico , Masculino , Microscopia Imunoeletrônica , Especificidade de Órgãos , RatosRESUMO
Several studies have reported transient expression of tyrosine hydroxylase in a subpopulation of neurons in the bed nucleus of stria terminalis of preadolescent rats. The tyrosine hydroxylase immunoreactive (TH) neurons, which are of small to medium size and often display a typical bipolar configuration, are confined to the intermediate part of the lateral bed nucleus. By the use of a combination of experimental tracer techniques and immunocytochemical methods, we have demonstrated that these neurons receive a significant number of amygdaloid afferents, which establish mostly symmetric synaptic contacts on the cell bodies and sparsely spined dendritic shafts of the TH neurons. TH neurons also receive a small number of tyrosine hydroxylase-positive terminals of unspecified origin.
Assuntos
Tonsila do Cerebelo/fisiologia , Neurônios/química , Tirosina 3-Mono-Oxigenase/análise , Animais , Tamanho Celular , Imuno-Histoquímica , Masculino , Ratos , Fatores de TempoRESUMO
Pregnant Long-Evans rats were maintained on three diets: a liquid diet in which ethanol accounted for 35-39% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, and a lab chow control diet. At gestation day 18, the fetuses were taken and cultures of septal and hippocampal neurons prepared. Neuronal survival and neurite outgrowth were compared in cultures from the three diet groups, using the following media supplements: ethanol (1.2, 1.8 or 2.4 g/dl), neurotrophic factors (nerve growth factor [NGF] with the septal cultures, basic fibroblast growth factor [bFGF] with the hippocampal cultures), or ethanol plus neurotrophic factors. Both the septal and hippocampal neurons responded to ethanol in a dose-dependent manner. The neurons from both populations from fetuses which had been exposed prenatally to ethanol, however, tolerated considerably higher ethanol concentrations before decreases in survival or outgrowth were seen. These ethanol-exposed neuronal populations were also less responsive to neurotrophic factors: in hippocampal cultures, process outgrowth was significantly enhanced by bFGF in control but not ethanol-derived cultures, and in septal and hippocampal cultures, the neurotrophic factors significantly ameliorated ethanol neurotoxicity in control cultures, but not in those from the ethanol-exposed fetuses. The possible relevance of these observations to the fetal alcohol syndrome is discussed.
Assuntos
Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Etanol/efeitos adversos , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Núcleos Septais/citologia , Animais , Células Cultivadas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Feminino , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Transtornos do Espectro Alcoólico Fetal/veterinária , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fatores de Crescimento Neural/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Gravidez , Ratos , Ratos Endogâmicos , Substância Negra/citologia , Sacarose , Fatores de TempoRESUMO
Extract made from hippocampus of rat pups exposed prenatally to an ethanol-supplemented diet was found to contain more neurotrophic activity at postnatal day 21 than that from animals exposed to control diets, when quantified in a dorsal root ganglion bioassay. This apparent upregulation was specific to hippocampal extract (cerebellar and forebrain/midbrain extracts were also assessed), and to this age (P1, P7, P14 and P60 extracts were also tested). It was suggested that this upregulation may be indicative of, or secondary to, trauma resulting from fetal ethanol exposure. It is speculated that such departures from the normal developmental timetable could contribute to anomalies seen in the fetal alcohol syndrome.
Assuntos
Envelhecimento/metabolismo , Etanol/farmacologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Bioensaio , Feminino , Fatores de Crescimento Neural/metabolismo , Gravidez , Ratos , Ratos EndogâmicosRESUMO
Chick embryos were chronically exposed to either ethanol (approximately 30 mg/d) or saline, from E4-E13. Homogenate extract was prepared from forebrain tissue from E16 experimental and control embryos and was applied to cultured dorsal root ganglia (DRG). Neurotrophic activity in the forebrain extract (FBX) was significantly reduced in the ethanol-treated embryos compared to saline controls, both in terms of influences on neuronal survival and process elaboration. In addition, E8-9 DRGs from embryos exposed to ethanol from E4 were less viable in the presence of NGF than were those from controls. DRG survival in the presence of E16 FBX (from untreated embryos) was not different following ethanol treatment, but neurite production was significantly reduced. These results suggest that neurotrophic factor content and responsiveness may be appreciably altered following chronic prenatal ethanol exposure. Such alteration could underlie certain CNS anomalies seen in the fetal alcohol syndrome.
Assuntos
Etanol/farmacologia , Gânglios Espinais/embriologia , Fatores de Crescimento Neural/fisiologia , Prosencéfalo/embriologia , Animais , Embrião de Galinha , Técnicas de Cultura , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/ultraestrutura , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/fisiologiaRESUMO
Chick embryos were given daily injections of ethanol (approximately 30 mg/day) either chronically from embryonic days 4 to 15 (E4-E15) or E18, or acutely from E15 to E18. Untreated and saline-injected embryos served as controls. Although morphological indicators of developmental age were not different among groups, chronic ethanol reduced weights of several brain regions. Similar to rodent models of prenatal ethanol exposure, chronic ethanol treatment reduced cerebellar Purkinje cell numbers compared to controls. Chronic but not acute ethanol exposure resulted in a significant reduction in choline acetyltransferase activity in the optic tectum (OT) and forebrain (FB) compared with controls. This study demonstrates that the chick embryo is a viable model to investigate the effects of ethanol exposure on CNS development. Unlike the mammalian fetus, the avian embryo is isolated from maternal interactions and may prove more useful in investigating the mechanisms by which ethanol directly influences brain development.
Assuntos
Encéfalo/embriologia , Cerebelo/embriologia , Colina O-Acetiltransferase/metabolismo , Etanol/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Contagem de Células , Cerebelo/efeitos dos fármacos , Embrião de Galinha , Etanol/administração & dosagem , Etanol/sangue , Tamanho do Órgão , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/embriologia , Prosencéfalo/enzimologia , Células de Purkinje/citologia , Células de Purkinje/efeitos dos fármacos , Colículos Superiores/efeitos dos fármacos , Colículos Superiores/embriologia , Colículos Superiores/enzimologia , Fatores de TempoRESUMO
Total blood cholesterol (TBC) levels and contributory risk factors in an adolescent population were investigated. Existing TBC screening records were reviewed on 452 10th grade students in two schools. The sample consisted of 52% males and 48% females whose mean age was 15.47 years. Blood samples were analyzed by the Reflotron. Risk factors investigated included age, gender, ethnicity, individual and family history of high cholesterol, history of high blood pressure, smoking tobacco products, and oral contraceptive use. The sample mean for TBC was 150.61. The only significant factors identified by ANOVA were gender and use of oral contraceptives. Females had higher TBC levels than males, and females who used oral contraceptives had higher TBC levels than nonusers.
PIP: Total blood cholesterol levels were screened by a fingerstick-desktop method in 452 10th grade students from 2 schools as part of a cholesterol task force that included health education on risk factors. The children averaged 15.47 years. The Reflotron analyzer (Boehringer Mannheim Diagnostics) used non-fasting blood. The overall mean TBC level was 150.61 mg/dl. By risk groups, 76% were low risk, with TBC levels ranging from 100-169; 11% were moderate risk, ranging 1270-185; and 12% were high risk, with levels ranging from 170-400. Girls' levels averaged 160.15, significantly higher than boys' 141.95, consistent with prior published reports. The 10 girls using oral contraceptives had a mean TBC of 188.20, compared to 158.85 for nonusers. There were no significant differences for other potential risk groups: ethnicity, history of high blood pressure, current smoking, individual or family history of high cholesterol. School A had a higher socioeconomic background than School B. The mean cholesterol level for School A was 149.02, compared to 152.08 for School B. Schools need more health education on risk factors and lifestyle, and preventive health intervention.