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Unregulated private wells are understudied potential sources of community-acquired Legionnaires' disease. Here we conducted a comprehensive survey of 44 homes supplied by private wells in Wake County, North Carolina, quantifying Legionella spp. DNA, Legionella pneumophila DNA, and total bacterial 16S rRNA genes via real-time polymerase chain reaction in hot and cold drinking water samples, along with culturable L. pneumophila via IDEXX Legiolert in cold drinking water samples. Legionella spp. DNA, L. pneumophila DNA and culturable L. pneumophila were detected in 100, 65·5 and 15·9% of the 44 homes, respectively, and culturable levels were comparable to some municipal surveys applying the same methods. Total coliforms and Escherichia coli were monitored as representative faecal indicators and were found in 20·4 and 0·0% of homes. Within certain sample types, Legionella spp. and L. pneumophila gene copy numbers were positively associated with total bacteria (i.e. total 16S rRNA genes) and water softener use, but were not associated with faecal indicator bacteria, inorganic water parameters or other well characteristics. These findings confirm that occurrence of Legionella and L. pneumophila is highly variable in private wells. SIGNIFICANCE AND IMPACT OF THE STUDY: Legionella is the leading identified cause of waterborne disease outbreaks associated with US municipal water systems. While Legionella is known to occur naturally in groundwater, prior efforts to characterize its occurrence in unregulated private wells are limited to sampling at the wellhead and not in the home plumbing where Legionella can thrive. This work documents much higher levels of Legionella in home plumbing versus water directly from private wells and examines factors associated with higher Legionella occurrence.
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Agua Potable/microbiología , Legionella pneumophila/aislamiento & purificación , ADN Bacteriano/genética , Humanos , Legionella pneumophila/clasificación , Legionella pneumophila/genética , Enfermedad de los Legionarios/microbiología , North Carolina , ARN Ribosómico 16S/genética , Ingeniería Sanitaria , Microbiología del Agua , Abastecimiento de AguaRESUMEN
The development of nanoscale electrochemistry since the mid-1980s has been predominately coupled with steady-state voltammetric (i-E) methods. This research has been driven by the desire to understand the mechanisms of very fast electrochemical reactions, by electroanalytical measurements in small volumes and unusual media, including in vivo measurements, and by research on correlating electrocatalytic activity, e.g., O2 reduction reaction, with nanoparticle size and structure. Exploration of the behavior of nanoelectrochemical structures (nanoelectrodes, nanoparticles, nanogap cells, etc.) of a characteristic dimension λ using steady-state i-E methods generally relies on the well-known relationship, λ2 â¼ Dt, which relates diffusional lengths to time, t, through the coefficient, D. Decreasing λ, by performing measurements at a nanometric length scales, results in a decrease in the effective timescale of the measurement, and provides a direct means to probe the kinetics of steps associated with very rapid electrochemical reactions. For instance, steady-state voltammetry using a nanogap twin-electrode cell of characteristic width, λ â¼ 10 nm, allows investigations of events occurring at timescales on the order of â¼100 ns. Among many other advantages, decreasing λ also increases spatial resolution in electrochemical imaging, e.g., in scanning electrochemical microscopy, and allows probing of the electric double layer. This Introductory Lecture traces the evolution and driving forces behind the "λ2 â¼ Dt" steady-state approach to nanoscale electrochemistry, beginning in the late 1950s with the introduction of the rotating ring-disk electrode and twin-electrode thin-layer cells, and evolving to current-day investigations using nanoelectrodes, scanning nanocells for imaging, nanopores, and nanoparticles. The recent focus on so-called "single-entity" electrochemistry, in which individual and very short redox events are probed, is a significant departure from the steady-state approach, but provides new opportunities to probe reaction dynamics. The stochastic nature of very fast single-entity events challenges current electrochemical methods and modern electronics, as illustrated using recent experiments from the authors' laboratory.
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Técnicas Electroquímicas/instrumentación , Nanotecnología/instrumentación , ADN/química , Difusión , Diseño de Equipo , Cinética , Microelectrodos , Microscopía de Túnel de Rastreo/instrumentación , Modelos Moleculares , Nanopartículas/química , Nanoporos/ultraestructura , Oxidación-Reducción , Procesos EstocásticosRESUMEN
Microiontophoresis is a drug delivery method in which an electric current is used to eject molecular species from a micropipette. It has been primarily utilized for neurochemical investigations, but is limited due to difficulty controlling and determining the ejected quantity. Consequently the concentration of an ejected species and the extent of the affected region are relegated to various methods of approximation. To address this, we investigated the principles underlying ejection rates and examined the concentration distribution in microiontophoresis using a combination of electrochemical, chromatographic, and fluorescence-based approaches. This involved a principal focus on how the iontophoretic barrel solution affects ejection characteristics. The ion ejection rate displayed a direct correspondence to the ionic mole fraction, regardless of the ejection current polarity. In contrast, neutral molecules are ejected by electroosmotic flow (EOF) at a rate proportional to the barrel solution concentration. Furthermore, the presence of EOF was observed from barrels containing high ionic strength solutions. In practice, use of a retaining current draws extracellular ions into the barrel and will alter the barrel solution composition. Even in the absence of a retaining current, diffusional exchange at the barrel tip will occur. Thus behavior of successive ejections may slightly differ. To account for this, electrochemical or fluorescence markers can be incorporated into the barrel solution in order to compare ejection quantities. These may also be used to provide an estimate of the ejected amount and distribution provided accurate use of calibration procedures.
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Sistemas de Liberación de Medicamentos/instrumentación , Iontoforesis , Animales , Electrodos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Despite overall increased production in the last century, it is critical that grazing production systems focus on improving beef and dairy efficiency to meet current and future global food demands. For livestock producers, production efficiency is essential to maintain long-term profitability and sustainability. This continued viability of production systems using pasture- and range-based grazing systems requires more rapid adoption of innovative management practices and selection tools that increase profitability by optimizing grazing management and increasing reproductive performance. Understanding the genetic variation in cow herds will provide the ability to select cows that require less energy for maintenance, which can potentially reduce total energy utilization or energy required for production, consequently improving production efficiency and profitability. In the United States, pasture- and range-based grazing systems vary tremendously across various unique environments that differ in climate, topography, and forage production. This variation in environmental conditions contributes to the challenges of developing or targeting specific genetic components and grazing systems that lead to increased production efficiency. However, across these various environments and grazing management systems, grazable forage remains the least expensive nutrient source to maintain productivity of the cow herd. Beef and dairy cattle can capitalize on their ability to utilize these feed resources that are not usable for other production industries. Therefore, lower-cost alternatives to feeding harvested and stored feedstuffs have the opportunity to provide to livestock producers a sustainable and efficient forage production system. However, increasing production efficiency within a given production environment would vary according to genetic potential (i.e., growth and milk potential), how that genetic potential fits the respective production environment, and how the grazing management fits within those genetic parameters. Therefore, matching cow type or genetic potential to the production environment is and will be more important as cost of production increases.
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Crianza de Animales Domésticos/métodos , Bovinos/fisiología , Crianza de Animales Domésticos/economía , Animales , Cruzamiento , Bovinos/genética , Selección Genética , Estados UnidosRESUMEN
Electrostatic force microscopy (EFM) probes are typically coated in either metal (radius â¼ 30 nm) or highly-doped diamond (radius â¼ 100 nm). Highly-doped silicon probes, which offer a sharpened and stable tip apex (radius â¼ 1-10 nm) and are usually used only in standard atomic force microscopy, have been recently shown to allow enhanced lateral resolution in quantitative EFM and its application for dielectric constant measurement. Here we present the theoretical modelling required to quantitatively interpret the electrostatic force between these sharpened tips and samples. In contrast to a sphere-capped cone geometry used to describe metal/diamond-coated tips, modelling a sharpened silicon tip requires a geometry comprised of a cone with two different angles. Theoretical results are supported by experimental measurements of metallic substrates and â¼10 nm radius dielectric nanoparticles. This work is equally applicable to EFM and other electrical scanned probe techniques, where it allows quantifying electrical properties of nanomaterials and 3D nano-objects with higher resolution.
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OBJECTIVE: To investigate whether elderly patients, presenting following trauma and requiring immobilisation and imaging of the cervical spine, require increased use of CT and spend longer immobilised when compared to the younger population. METHODS: A retrospective chart review was undertaken of 35 adults aged 18-65â years, and 32 adults over 65â years, requiring cervical spine imaging following trauma. RESULTS: 1 of the 35 younger patients, and 16 of the 32 elderly patients, underwent CT. Elderly patients spent 1:05â h longer immobilised (p<0.005). CONCLUSIONS: Half of the elderly patients underwent CT, and they were immobilised for significantly longer than younger patients. Measures should be adopted to facilitate early diagnosis and mitigate complications of prolonged immobilisation in elderly patients with neck trauma.
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Vértebras Cervicales/diagnóstico por imagen , Vértebras Cervicales/lesiones , Inmovilización/estadística & datos numéricos , Tomografía Computarizada por Rayos X/estadística & datos numéricos , Adolescente , Adulto , Factores de Edad , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
A theoretical analysis of amplitude modulated electrostatic force microscopy (AM-EFM) in liquid media at MHz frequencies, based on a simple tip-sample parallel plate model, is presented. The model qualitatively explains the main features of AM-EFM in liquid media and provides a simple explanation of how the measured electric forces are affected by: the frequency of the applied voltage, the tip-sample distance, the ionic concentration, the relative dielectric constant of the solution, and the relative dielectric constant and thickness of the sample. These results provide a simple framework for the design of AM-EFM measurements for localized dielectric characterization in liquid media.
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We present what is, to our knowledge, the first experimental demonstration of dielectric constant measurement and quantification of supported lipid bilayers in electrolyte solutions with nanoscale spatial resolution. The dielectric constant was quantitatively reconstructed with finite element calculations by combining thickness information and local polarization forces which were measured using an electrostatic force microscope adapted to work in a liquid environment. Measurements of submicrometric dipalmitoylphosphatidylcholine lipid bilayer patches gave dielectric constants of ε(r) ~ 3, which are higher than the values typically reported for the hydrophobic part of lipid membranes (ε(r) ~ 2) and suggest a large contribution of the polar headgroup region to the dielectric response of the lipid bilayer. This work opens apparently new possibilities in the study of biomembrane electrostatics and other bioelectric phenomena.
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Membrana Dobles de Lípidos/química , Microscopía , Nanotecnología , Electricidad Estática , Agua/química , Impedancia Eléctrica , Análisis de Elementos Finitos , SolucionesRESUMEN
BACKGROUND: Since the Food and Drug Administration (FDA) approval of laparoscopic adjustable gastric bands (LAGB) in June 2001, the number LAGB procedures performed in the United States has increased exponentially. This study aimed to benchmark the authors' initial hospital experience to FDA research trials and evidence-based literature. METHODS: Over a 2-year period, 87 consecutive patients with a mean age of 43 years (range, 21-64 years) and a body mass index of 45.6 kg/m2 (range, 35-69 kg/m2) underwent an LAGB procedure at the authors' institution. The authors conducted a retrospective review of the outcomes including conversion, reoperation, mortality, perforation, erosion, prolapse, port dysfunction, excess weight loss, and changes in comorbidities, then compared the data with published benchmarks. RESULTS: Gender, age, and body mass index were comparable with those of other series. Perioperative adverse events included acute stoma obstruction (n = 1) and respiratory complications (n = 2). Delayed complications included gastric prolapse (n = 4) and port reservoir malposition (n = 4). Five bands were explanted. The mean follow-up period was 14 months (n = 79). The mean percentage of excess weight loss was 30% (range, 4.7-69%) at 6 months, 41% (range, 9.6-82%) at 12 months, and 47% (range, 14-92%) at 24 months. Comorbidities resolved included diabetes (74%), hypertension (57%), gastroesophageal reflux disease (55%) and dyslipidemia (38%). CONCLUSIONS: The short-term outcomes for LAGB were comparable with published benchmarks. With adequate weight loss, most patients achieve significant improvement in obesity-related illnesses. With new bariatric accreditation standards and mandates required for financial reimbursement, hospitals will need to demonstrate that their clinical outcomes are consistent with best practices. The authors' early experience shows that LAGB achieves significant weight loss with low mortality and morbidity rates. Despite a more gradual weight loss, most patients achieve excellent weight loss with corresponding improvement of comorbidities within the first 2 years postoperatively.
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Cirugía Bariátrica/estadística & datos numéricos , Hospitalización/estadística & datos numéricos , Laparoscopía/estadística & datos numéricos , Obesidad Mórbida/cirugía , Adulto , Cirugía Bariátrica/métodos , Benchmarking , Boston/epidemiología , Comorbilidad , Complicaciones de la Diabetes/epidemiología , Dislipidemias/epidemiología , Femenino , Estudios de Seguimiento , Reflujo Gastroesofágico/epidemiología , Humanos , Hipertensión/epidemiología , Laparoscopía/métodos , Masculino , Persona de Mediana Edad , Obesidad Mórbida/epidemiología , Complicaciones Posoperatorias/epidemiología , Reoperación/estadística & datos numéricos , Estudios Retrospectivos , Resultado del Tratamiento , Pérdida de PesoRESUMEN
RATIONALE: Nitric oxide synthase (NOS) inhibitors may modulate the discriminative stimulus effects of cocaine because they alter dopamine (DA) release. OBJECTIVES: The effects of the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitro-indazole (7-NI) were examined in experiments designed to better understand the mechanisms that may underlie the interactions between NOS inhibitors and cocaine. METHODS: Rats were trained to discriminate 10 mg/kg cocaine from saline, and then substitution and pretreatment tests with L-NAME and 7-NI were conducted. To determine if the combined effects of NOS inhibitors and cocaine might be related to DA mechanisms and/or to N-methyl-D-aspartate (NMDA) receptor mechanisms, substitution tests with other indirect DA agonists and NMDA antagonists were carried out in the presence and absence of L-NAME. In addition, the roles of the D1 and D2 families of DA receptors in mediating the cocaine-altering effects of L-NAME and 7-NI were examined in antagonism tests using SCH 23390 and haloperidol, respectively. RESULTS: The results demonstrated that neither NOS inhibitor alone substituted for the 10 mg/kg cocaine training dose, but when given as a pretreatment, 100 mg/kg L-NAME as well as 10 mg/kg 7-NI enhanced the discriminative stimulus and rate-decreasing effects of cocaine. L-NAME pretreatment also enhanced the potency of (+)-amphetamine and GBR 12909, but not MK-801, phencyclidine, or NPC 17742, for producing discriminative stimulus and rate-decreasing effects in substitution tests. Further testing showed that the cocaine-enhancing effects of L-NAME and 7-NI were attenuated by doses of haloperidol and SCH 23390 that minimally altered the effects of cocaine alone. CONCLUSIONS: These findings suggest that L-NAME and 7-NI may increase the potency of cocaine and other indirect DA agonists through a central mechanism whereby DA neurotransmission is directly enhanced by NOS inhibition.
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Cocaína/farmacología , Discriminación en Psicología/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Inhibidores Enzimáticos/farmacología , Indazoles/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Benzazepinas/farmacología , Discriminación en Psicología/fisiología , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Haloperidol/farmacología , Masculino , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
The purpose of this study was to determine the extent to which N-methyl-D-aspartic acid (NMDA) antagonists modified the discriminative stimulus effects of cocaine in rats trained to discriminate 5 mg/kg cocaine from vehicle on a fixed-ratio schedule of food presentation as well as the rate-altering effects of cocaine in rats maintained on a fixed-interval schedule of food presentation. NMDA-associated ion channel blockers (dizocilpine, phencyclidine, and magnesium chloride) and competitive NMDA antagonists (NPC 17742 and CGP 37849) displayed similar behavioral effects when administered alone: each drug engendered intermediate levels of cocaine-appropriate responses and rate-dependent effects on food-reinforced operant responding. Selected doses of dizocilpine, magnesium chloride, and phencyclidine given in combination with 1 mg/kg cocaine produced more cocaine-appropriate responses than this dose of cocaine alone. In addition, dizocilpine and magnesium chloride each attenuated the discriminative stimulus effects of higher doses of cocaine. The competitive NMDA antagonists did not appreciably modify the discriminative stimulus effects of any dose of cocaine. Under the fixed-interval schedule, each NMDA antagonist attenuated the effects of 3 mg/kg cocaine, which normally produced maximal increases in response rate. Attenuation of the rate-decreasing effects of the highest dose of cocaine (30 mg/kg) also were observed after pretreatment with dizocilpine and magnesium chloride. These findings demonstrated differences in the way that NMDA-associated ion channel blockers and competitive NMDA antagonists interact with cocaine, and suggest that some NMDA-associated ion channel blockers may either enhance or antagonize the effects of cocaine, depending on the dose and type of behavioral procedure.
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Cocaína/farmacología , Aprendizaje Discriminativo/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/farmacología , Aminoácidos/farmacología , Animales , Unión Competitiva , Maleato de Dizocilpina/farmacología , Interacciones Farmacológicas , Cloruro de Magnesio/farmacología , Masculino , Fenciclidina/farmacología , Ratas , Ratas Wistar , Esquema de RefuerzoRESUMEN
Previous studies suggest that magnesium chloride may have discriminative stimulus effects that partially overlap with those of noncompetitive N-methyl-D-aspartate antagonists as well as certain monoamine uptake inhibitors. In our study, rats were trained to discriminate 100 mg/kg magnesium chloride from saline and its discriminative stimulus effects were characterized with respect to N-methyl-D-aspartate receptor and monoamine transporter functions in substitution tests. The discriminative stimulus effects of magnesium chloride were acquired within a moderate number of training sessions and showed dose-related substitution after either subcutaneous (3-300 mg/kg) or intracerebroventricular (0.3-300 microg) administration. The intracerebroventricular administration of magnesium chloride was over 4000 times more potent than its s.c. administration. The monoamine uptake inhibitors cocaine, GBR 12909, talsupram and citalopram fully substituted (> or =90% magnesium-appropriate responses) for magnesium chloride in the majority of subjects tested and the group averages reached a maximum of 72 to 82% responses on the magnesium-appropriate lever. Based on relative potency analysis, the rank order of potency of these four drugs for producing magnesium-appropriate responses was talsupram = cocaine > citalopram = GBR 12909. The N-methyl-D-aspartate receptor antagonists dizocilpine, phencyclidine and NPC 17742 engendered maximum group averages of 49 to 65% responses on the magnesium-appropriate lever. The results suggest that the centrally mediated discriminative stimulus effects of magnesium chloride may be more directly related to interactions with monoamine neurotransmitter functions than to N-methyl-D-aspartate receptor blockade.
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Aprendizaje Discriminativo/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Cloruro de Magnesio/farmacología , Inhibidores de la Captación de Neurotransmisores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Aminoácidos/farmacología , Animales , Maleato de Dizocilpina/farmacología , Masculino , Piperazinas/farmacología , Ratas , Ratas WistarRESUMEN
Cocaine abuse is a major medical and public health concern in the United States, with approximately 2.1 million people dependent on cocaine. Pharmacological approaches to the treatment of cocaine addiction have thus far been disappointing, and new therapies are urgently needed. This paper describes an immunological approach to cocaine addiction. Antibody therapy for neutralization of abused drugs has been described previously, including a recent paper demonstrating the induction of anti-cocaine antibodies. However, both the rapidity of entry of cocaine into the brain and the high doses of cocaine frequently encountered have created challenges for an antibody-based therapy. Here we demonstrate that antibodies are efficacious in an animal model of addiction. Intravenous cocaine self-administration in rats was inhibited by passive transfer of an anti-cocaine monoclonal antibody. To actively induce anti-cocaine antibodies, a cocaine vaccine was developed that generated a high-titer, long-lasting antibody response in mice. Immunized mice displayed a significant change in cocaine pharmacokinetics, with decreased levels of cocaine measured in the brain of immunized mice only 30 seconds after intravenous (i.v.) administration of cocaine. These data establish the feasibility of a therapeutic cocaine vaccine for the treatment of cocaine addiction.
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Cocaína/inmunología , Haptenos/inmunología , Inmunoterapia Activa , Trastornos Relacionados con Opioides/terapia , Vacunas/inmunología , Animales , Cocaína/administración & dosificación , Condicionamiento Operante , Estudios de Evaluación como Asunto , Haptenos/administración & dosificación , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos BALB C , Trastornos Relacionados con Opioides/inmunología , Ratas , Ratas Wistar , Autoadministración , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/inmunologíaRESUMEN
Dose-response functions for selected N-methyl-D-aspartate (NMDA) antagonists and direct and indirect dopamine agonists were compared in rats trained to discriminate either a low (2 mg/kg) or a high (10 mg/kg) dose of cocaine from vehicle. The NMDA-associated ion channel blockers, dizocilpine, phencyclidine and MgCl2, substituted fully for cocaine (> or = 90% cocaine-appropriate responses) in the majority of subjects under the low-dose training condition, but showed little or no substitution for cocaine under the high-dose training condition. The competitive NMDA antagonist [2R,4R,5S-(2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid)] did not substitute for cocaine under either training condition. Cocaine, 1-(2-[bis(4-fluorophenyl)methoxyl)-4-(3-phenylpropyl)piperazine, (+)-amphetamine and the D1 receptor agonist SKF 77434 engendered full substitution for cocaine under both training conditions. Dose-response functions for all four drugs were displaced to the left and average ED50 values were reduced by 3-fold or more under the low-dose compared to the high-dose training condition. The nonselective DA receptor agonist (-)-apomorphine substituted substantially for cocaine only under the low-dose training condition, whereas the D2 receptor agonist (+)-4-propyl-9-hydroxynapthoxazine substituted similarly for cocaine under both training conditions. The results show that change in the training dose of cocaine can affect both the shape and position of the dose-response functions for representative NMDA-associated ion channel blockers and direct and indirect dopamine agonists. The findings further show that under low-dose training conditions, NMDA-associated ion channel blockers can engender cocaine-like stimulus effects comparable to those of direct and indirect dopamine agonists.
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Cocaína/farmacología , Aprendizaje Discriminativo/efectos de los fármacos , Agonistas de Dopamina/farmacología , N-Metilaspartato/antagonistas & inhibidores , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/análogos & derivados , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Animales , Masculino , Oxazinas/farmacología , Piperazinas/farmacología , Ratas , Ratas WistarRESUMEN
The cerebellum is organized into a series of parasagittally aligned bands that may be revealed histologically in the adult mouse by largely complementary immunostaining of Purkinje cells sets with the monoclonal antibodies Zebrin II (ZII; antigen:aldolase C) and P-path (PP; antigen:90-acetyl glycolipids). We compared the normal staining pattern using these markers and an antibody to calbindin with that found in the reeler mutants (rl/rl), in which most Purkinje cell migration is halted beneath the cerebellar white matter. The results revealed that Purkinje cells in reeler mutants, despite their ectopic location in large subcortical masses, show a clear tendency to distribute into alternating zones that either stain for Zebrin II or for P-path, with variable transition zones of mixed labeling. However, the estimated number of zones was fewer than in the normal adult cortex: roughly 7-9 zones are revealed per side in the mutant compared with 14 major divisions in wild type mice. These results raise the possibility that neurons destined to express these markers are segregated during their migration and that the final phase of migration into the cortex might involve further splitting or interdigitation between cell sets expressing the two antigens.
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Glucolípidos/análisis , Proteínas del Tejido Nervioso/análisis , Células de Purkinje/citología , Animales , Anticuerpos Monoclonales , Glucolípidos/inmunología , Inmunohistoquímica , Ratones , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/inmunologíaRESUMEN
The cerebellum is organized into a series of parasagittally aligned bands which are well delineated in the adult mouse by the largely complementary immunostaining of Purkinje cell groups with the monoclonal antibodies Zebrin II (ZII; antigen: aldolase C) and P-path (antigen: 9-O-acetyl gangliosides). We examined the effect of nervous mutation on compartmental organization using these markers and an antibody to calbindin. In nervous mutant, up to 90% of Purkinje cells die in late postnatal development. The size of the cerebellum is about half that of normal, and caudal lobules appear to decrease in size more than anterior ones. Surviving Purkinje cells corresponded to P-path positive ones that were concentrated in two bilateral bands in the vermis and in medial portions of the hemispheres. Only small numbers of ZII positive cells remained, confirming the report by Wassef et al. with Zebrin I antibody. They were primarily located in caudal lobules IX, X and a portion of lobule IV, paraflocculus and flocculus, and their immunoreactivity was weak compared to that of normal. ZII positive cells are dominant in these caudal lobules, while P-path positive cells dominate in rostral lobules in normal mice, and the similar tendency remains in mutant. Thus, the nervous gene action respects not only sagittal compartments delineated by two antibodies, but also rostro-caudal gradient. The cause of the dominant survival of P-path positive cells awaits future study.
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Gangliósidos/biosíntesis , Mutación/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Células de Purkinje/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Biomarcadores , Muerte Celular/fisiología , Cerebelo/anatomía & histología , Cerebelo/citología , Gangliósidos/genética , Gangliósidos/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos C3H , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Células de Purkinje/efectos de los fármacosRESUMEN
During normal development of the mammalian forebrain, the paired cerebral hemispheres are initially separated midsagittally by the connective tissue-filled longitudinal fissure. During subsequent stages, the hemispheres fuse as basal lamina is remodeled and fibroblasts are eliminated from the fissure to create new central nervous system (CNS) territory in the midline. Two axon pathways, the corpus callosum and dorsal callosal stria, eventually use this region as part of their pathway. In order to assess the possible role of glial cells in the fusion process and in the guidance of axons in this and several other areas of the forebrain, we have analyzed the developing brain in timed cat and mouse embryos with immunohistochemical and morphological techniques. With the use of astroglial-specific antibodies and electron microscopy, we have visualized two distinct, primitive astroglial structures associated with the cerebral midline, and seven more associated with other specific brain regions. The way in which one of these structures moves as a column along the hemispheric midline in synchrony with seam formation suggests the possibility that during morphogenesis of the telencephalon, astrocytes may aid in the fusion process. In addition, the compact assemblage, early appearance and location of this and the other glial structures in relation to well defined neuroanatomical landmarks or axon pathways suggest that they may transiently compartmentalize relatively large regions of the CNS and organize certain developing fiber systems by acting as guides or barriers at critical stages of ontogeny.
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Astrocitos/ultraestructura , Axones/ultraestructura , Encéfalo/embriología , Vías Nerviosas/embriología , Animales , Biomarcadores , Encéfalo/citología , Gatos/embriología , Fusión Celular , Cuerpo Calloso/embriología , Edad Gestacional , Ratones , Ratones Endogámicos C3H/embriología , Ratones Endogámicos C57BL/embriología , Morfogénesis , Vías Nerviosas/ultraestructura , Prosencéfalo/embriologíaRESUMEN
In order to characterize differences in growth patterns of axons as they elongate toward their targets and during the initial stages of terminal arbor formation within the targets, we examined the primary visual system of fetal and newborn hamsters using three morphological methods: the Cajal-deCastro reduced silver method, the rapid Golgi technique, and anterograde transport of HRP. Axons emerge from the retina between the 10th and 11th embryonic days (E10-E11). The front of retinal axons crosses the chiasm, extends over the primitive dorsal nucleus of the lateral geniculate body (LGBd) by E13, and advances to the back of the superior colliculus (SC) by E13.5-E14. The rate of axon growth during this advance is nearly 2 mm/day. Collateral sprouts appear on axons around E15.5. In the LGBd and SC, these sprouts arise from multiple sites along the parent axons. Only one or a few of the sprouts continue to grow and branch, while others are eliminated. The net rate of axon collateral advance in this second phase is an order of magnitude slower than during the stage of axon elongation. Thus, formation of CNS projections may involve two qualitatively distinct modes of axon growth. The arborization mode contrasts with the elongation mode by the presence of branching, a lack of fasciculation and a slower average rate of extension. The stereotypic direct advance of axons during elongation also differs from the remodelling which occurs during arborization. The delay between axon arrival at targets and onset of arborization could be a reflection of axons "waiting" for a maturational change to occur in the retina or in targets. Arborization in the LGBd and SC is initiated around the same time, implicating the former possibility. However, a slower differentiation of retinal arbors in the SC, in addition to morphological differences of arbors in the two structures, suggests that alterations in substrate factors also play a critical role in triggering the early stages of arbor formation.
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Axones/fisiología , Retina/embriología , Retina/crecimiento & desarrollo , Vías Visuales/embriología , Vías Visuales/crecimiento & desarrollo , Envejecimiento , Animales , Animales Recién Nacidos , Transporte Axonal , Axones/ultraestructura , Cricetinae , Embrión de Mamíferos , Cuerpos Geniculados/embriología , Cuerpos Geniculados/crecimiento & desarrollo , Edad Gestacional , Peroxidasa de Rábano Silvestre , Mesocricetus , Neuronas/fisiología , Quiasma Óptico/embriología , Quiasma Óptico/crecimiento & desarrollo , Coloración y Etiquetado , Colículos Superiores/embriología , Colículos Superiores/crecimiento & desarrolloRESUMEN
A monoclonal antibody, RC1, has been generated which provides a selective and sensitive immunohistochemical marker of radial glial cells and related cell forms during development of the mouse CNS. Beginning on embryonic day E10, immunocytochemistry performed on cryostat sections stains throughout the CNS a subpopulation of cells in the ventricular zone with radial processes that terminate with endfeet at the pial surface. These processes become fasciculated and attain maximal densities by E12-14 in the spinal cord and lower brainstem and by E14-16 in the midbrain, cerebellum and forebrain. Fasciculation is especially prominent for a subclass of these cells at the midline of the brainstem and spinal cord. As nuclear and cortical structures develop, the trajectories of the radial fiber fascicles undergo systematic and region-specific distortions in their initially simple linear configuration, in the process maintaining a consistent spatial registration of germinal ventricular zones with distal sites of assembly of post-migratory neurons. In the late fetal period, radial glial progressively disappear and scattered immature astrocytes bearing multiple fine processes appear in most regions of the CNS. In the spinal cord, a transitional unipolar radial form is identified in the emerging ventral and lateral funiculi between E13 and E17. In the cerebellum, precursors to the unipolar Bergmann glial cell are identified by E15, and in the retina, precursors of the bipolar Müller cell are identified by E16. Postnatally, RC1-stained radial glia become sparse, and after one week, immunoreactive cells include only ependymal cells, hypothalamic tanycytes, Bergmann glia, Müller cells, a unipolar radial form in the dentate gyrus, and a subpopulation of white matter astrocytes. These results suggest that radial cells of astroglial lineage comprise a diverse set of cell classes which subserve multiple functions in the developing and adult brain.
Asunto(s)
Anticuerpos Monoclonales , Sistema Nervioso Central/crecimiento & desarrollo , Neuroglía/fisiología , Animales , Tronco Encefálico/citología , Tronco Encefálico/crecimiento & desarrollo , Sistema Nervioso Central/citología , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Diencéfalo/citología , Diencéfalo/crecimiento & desarrollo , Femenino , Hipotálamo/citología , Hipotálamo/crecimiento & desarrollo , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Nervio Óptico/citología , Nervio Óptico/crecimiento & desarrollo , Embarazo , Ramos Subendocárdicos/fisiología , Ratas , Ratas Endogámicas , Retina/citología , Retina/crecimiento & desarrollo , Médula Espinal/citología , Médula Espinal/crecimiento & desarrollo , Telencéfalo/citología , Telencéfalo/crecimiento & desarrollo , Tálamo/citología , Tálamo/crecimiento & desarrolloRESUMEN
A monoclonal antibody, C2, raised against chick embryo spinal cord, is shown by a solid phase immunoabsorbent assay to recognize a molecular species associated with neurofilaments extracted from adult mouse and rat brain. As immunoreactivity is lost following pre-treatment with alkaline phosphatase, the antibody probably recognizes a phosphorylated protein. Immunocytochemical staining in fetal mouse indicates that this antigen is expressed selectively in axons from the earliest stages of their development. Neuronal somata tend to show only weak immunoreactivity. The C2 antibody allowed visualization of the spatiotemporal pattern of axonal growth in the retina, neocortex and cerebellum with greater resolution than in previous light microscopic descriptions. The concept that the leading process of some classes of migratory neurons becomes transformed into an axon is supported by the expression of C2 immunoreactivity in radially ascending processes from principle neuron classes in the fetal retina and cerebellum.