RESUMO
Dendritic spines are the protuberances from the neuronal dendritic shafts that contain excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.
Assuntos
Dendritos/ultraestrutura , Hipocampo/ultraestrutura , Plasticidade Neuronal/efeitos dos fármacos , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/ultraestrutura , Região CA3 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/ultraestrutura , Dendritos/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Giro Denteado/efeitos dos fármacos , Giro Denteado/ultraestrutura , Fluoruracila/farmacologia , Hipocampo/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
The G-protein coupled, protease-activated receptor 1 (PAR1) is a membrane protein expressed in astrocytes. Fine astrocytic processes are in tight contact with neurons and blood vessels and shape excitatory synaptic transmission due to their abundant expression of glutamate transporters. PAR1 is proteolytically-activated by bloodstream serine proteases also involved in the formation of blood clots. PAR1 activation has been suggested to play a key role in pathological states like thrombosis, hemostasis and inflammation. What remains unclear is whether PAR1 activation also regulates glutamate uptake in astrocytes and how this shapes excitatory synaptic transmission among neurons. Here we show that, in the mouse hippocampus, PAR1 activation induces a rapid structural re-organization of the neuropil surrounding glutamatergic synapses, which is associated with faster clearance of synaptically-released glutamate from the extracellular space. This effect can be recapitulated using realistic 3D Monte Carlo reaction-diffusion simulations, based on axial scanning transmission electron microscopy (STEM) tomography reconstructions of excitatory synapses. The faster glutamate clearance induced by PAR1 activation leads to short- and long-term changes in excitatory synaptic transmission. Together, these findings identify PAR1 as an important regulator of glutamatergic signaling in the hippocampus and a possible target molecule to limit brain damage during hemorrhagic stroke.
Assuntos
Astrócitos/metabolismo , Ácido Glutâmico/metabolismo , Receptor PAR-1/agonistas , Algoritmos , Animais , Astrócitos/ultraestrutura , Transporte Biológico , Feminino , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Imageamento Tridimensional , Potenciação de Longa Duração , Masculino , Camundongos , Modelos Biológicos , Método de Monte Carlo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Potenciais Sinápticos , Transmissão SinápticaRESUMO
The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on central nervous system consists of changing expression of estrogen receptors, whereas the result of chronic inflammatory reaction caused by dioxin is occurrence of destructive changes in various organs connected with disturbed metabolism of connective tissue and damage of cells. The aim of the study was to determine the effect of dioxins on function, ultrastructure, and cytological and histological structure of hippocampus, particularly on expression of estrogen receptors in central nervous system as well as to define protective influence of tocopherol (TCP) and acetylsalicylic acid (ASA) on the decrease in activity of proinflammatory effects in central nervous system. It was shown that TCDD contributes to destructive and inflammatory changes along with demyelization of myelin sheaths and atrophy of estrogen receptors in hippocampus. Dioxin contributes to atrophy of estrogen receptors in hippocampus, in which also destructive and inflammatory changes were found along with demyelination of myelin sheaths. Histopathological and ultrastructural image of hippocampus areas in rats, in which both TCP and ASA were used, is characterized by poorly expressed degenerative changes and smaller inflammatory reactivity. Using both TCP and ASA has a protective effect on functions of central nervous system.
Assuntos
Aspirina/administração & dosagem , Sistema Nervoso Central/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Tocoferóis/administração & dosagem , Animais , Aspirina/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Sistema Nervoso Central/ultraestrutura , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Hipocampo/ultraestrutura , Fármacos Neuroprotetores/administração & dosagem , Dibenzodioxinas Policloradas/toxicidade , Ratos , Receptores de Estrogênio/metabolismo , Tocoferóis/metabolismoRESUMO
Neurite outgrowth is a fundamental event in brain development, as well as in regeneration of damaged neurons. Astrocytes play a major role in neuritogenesis, by expressing and releasing factors that facilitate neurite outgrowth, such as extracellular matrix proteins, and factors that can inhibit neuritogenesis, such as the chondroitin sulfate proteoglycan neurocan. In this unit we describe a noncontact co-culture system of hippocampal neurons and cortical (or hippocampal) astrocytes for measurement of neurite outgrowth. Hippocampal pyramidal neurons are plated on glass coverslips, which are inverted onto an astrocyte feeder layer, allowing exposure of neurons to astrocyte-derived factors without direct contact between these two cell types. After co-culture, neurons are stained and photographed, and processes are assessed morphologically using Metamorph software. This method allows exposing astrocytes to various agents before co-culture in order to assess how these exposures may influence the ability of astrocytes to foster neurite outgrowth.
Assuntos
Astrócitos/ultraestrutura , Neuritos/ultraestrutura , Células Piramidais/ultraestrutura , Animais , Astrócitos/citologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura Livres de Soro , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Células Piramidais/citologia , RatosRESUMO
Most CNS synapses investigated thus far contain a large number of vesicles docked at the active zone, possibly forming individual release sites. At the present time, it is unclear whether these vesicles can be discharged independently of one another. To investigate this problem, we recorded miniature excitatory currents by whole-cell and single-synapse recordings from CA3-CA1 hippocampal neurons and analyzed their stochastic properties. In addition, spontaneous release was investigated by ultrastructural analysis of quickly frozen synapses, revealing vesicle intermediates in docking and spontaneous fusion states. In these experiments, no signs of inhibitory interactions between quanta could be detected up to 1 msec from the previous discharge. This suggests that exocytosis at one site does not per se inhibit vesicular fusion at neighboring sites. At longer intervals, the output of quanta diverged from a random memoryless Poisson process because of the presence of a bursting component. The latter, which could not be accounted for by random coincidences, was independent of Ca2+ elevations in the cytosol, whether from Ca2+ flux through the plasma membrane or release from internal stores. Results of these experiments, together with the observation of spontaneous pairs of omega profiles at the active zone, suggest that multimodal release is produced by an enduring activation of an integrated cluster of release sites.
Assuntos
Hipocampo/metabolismo , Inibição Neural/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Quelantes/farmacologia , Citosol/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Técnica de Fratura por Congelamento , Hipocampo/efeitos dos fármacos , Hipocampo/ultraestrutura , Fusão de Membrana/fisiologia , Método de Monte Carlo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Distribuição de Poisson , Terminações Pré-Sinápticas/metabolismo , Ratos , Tempo de Reação/fisiologia , Processamento de Sinais Assistido por Computador , Processos Estocásticos , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestruturaRESUMO
The release of vesicular glutamate, spatiotemporal changes in glutamate concentration in the synaptic cleft and the subsequent generation of fast excitatory postsynaptic currents at a hippocampal synapse were modeled using the Monte Carlo method. It is assumed that glutamate is released from a spherical vesicle through a cylindrical fusion pore into the synaptic cleft and that S-alpha-amino-3-hydroxy -5-methyl-4-isoxazolepropionic acid (AMPA) receptors are uniformly distributed postsynaptically. The time course of change in vesicular concentration can be described by a single exponential, but a slow tail is also observed though only following the release of most of the glutamate. The time constant of decay increases with vesicular size and a lower diffusion constant, and is independent of the initial concentration, becoming markedly shorter for wider fusion pores. The cleft concentration at the fusion pore mouth is not negligible compared to vesicular concentration, especially for wider fusion pores. Lateral equilibration of glutamate is rapid, and within approximately 50 micros all AMPA receptors on average see the same concentration of glutamate. Nevertheless the single-channel current and the number of channels estimated from mean-variance plots are unreliable and different when estimated from rise- and decay-current segments. Greater saturation of AMPA receptor channels provides higher but not more accurate estimates. Two factors contribute to the variability of postsynaptic currents and render the mean-variance nonstationary analysis unreliable, even when all receptors see on average the same glutamate concentration. Firstly, the variability of the instantaneous cleft concentration of glutamate, unlike the mean concentration, first rapidly decreases before slowly increasing; the variability is greater for fewer molecules in the cleft and is spatially nonuniform. Secondly, the efficacy with which glutamate produces a response changes with time. Understanding the factors that determine the time course of vesicular content release as well as the spatiotemporal changes of glutamate concentration in the cleft is crucial for understanding the mechanism that generates postsynaptic currents.
Assuntos
Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Método de Monte Carlo , Sinapses/química , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Animais , Simulação por Computador , Condutividade Elétrica , Hipocampo/fisiologia , Hipocampo/ultraestrutura , Cinética , Ratos , Receptores de AMPA/fisiologia , Sinapses/fisiologiaRESUMO
Relatively little information exists regarding the spatial structure of synaptic neuropil in the brain. The present electron microscopic study employs unbiased stereological techniques and Monte Carlo simulations to characterise quantitatively the spatial organisation of synaptic circuitry in the dentate gyrus of the hippocampus, an area of particular importance in mechanisms of learning and the subject of a number of experimental neurobiological models of synaptic plasticity such as long-term potentiation. Firstly, tissue shrinkage/expansion resulting from embedding was assessed by imaging 300-microm thick hippocampal slices in the course of the entire embedding protocol, giving a value of 94.3 +/- 1.1% for distance measures and 84.3 +/- 2.8% for volumetric measures. Secondly, numeric synaptic density, Nv, was estimated using the disector. Thirdly, accumulated area of post-synaptic densities (PSDs) per tissue volume, Sv, and the overall cell membrane area per tissue volume, Sv*, were assessed using unbiased stereological rules coupled with image analysis of single sections. Finally, the mean area of individual PSDs was derived as a ratio Sv/Nv giving: 0.0394 microm2 for axo-spinous PSDs (thus representing approximately 1.3% of total cell membranes) and 0.0769 microm2 for dendritic shaft PSDs (approximately 0.25% of total cell membranes). From these data, the mean nearest neighbour distance between synapses was estimated using Monte Carlo simulations of a random 3D arrangement of synapses constrained by PSD sizes (a truncated Poisson process), giving a value of 0.48-0.51 microm. The physiological importance of the morphometric data obtained is discussed in terms of assessing (i) the role of synaptic environment in modifying synaptic efficacy and (ii) the plausibility of cross talk between synapses in relation to extrasynaptic neurotransmitter diffusion and transient depletion of extracellular Ca2+.
Assuntos
Hipocampo/fisiologia , Hipocampo/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Animais , Membrana Celular/ultraestrutura , Tamanho Celular/fisiologia , Giro Denteado/citologia , Giro Denteado/ultraestrutura , Hipocampo/citologia , Processamento de Imagem Assistida por Computador , Masculino , Microscopia Eletrônica , Modelos Neurológicos , Método de Monte Carlo , Via Perfurante/fisiologia , RatosRESUMO
1. A simulation of fast excitatory synaptic transmission at a hippocampal synapse is presented. Individual neurotransmitter molecules are followed as they diffuse through the synaptic cleft and interact with the postsynaptic receptors. The ability of the model to reproduce published results of patch-clamp experiments on CA3 pyramidal cells is illustrated; parameters of the model that affect the time course and variability of the excitatory postsynaptic current (EPSC) are then investigated. 2. To simulate an EPSC, we release 4,000 neurotransmitter molecules simultaneously from a point source centered 15 nm above a rectangular grid of 14 x 14 postsynaptic receptors. The simulated EPSC at room temperature has a 10-90% rise time of 0.28 ms and a peak open probability of 0.27, and decays with a time constant of 2.33 ms, comparing well with values in the literature. 3. To simulate changes in temperature, we use a 10 degrees temperature coefficient (Q10) for diffusion of 1.3 and apply a Q10 of 3.0 to all the rate constants of the kinetic scheme. At 37 degrees C, the 10-90 rise time is 0.07 ms, the peak open probability is 0.56, and the decay time constant is 0.70 ms. The coefficient of variation (CV) at the peak of the EPSC is 9.4% at room temperature; at 37 degrees C, the CV at the peak drops to 6.6%. 4. We use the diffusion coefficient of glutamine, 7.6 x 10(-6) cm2/s, to model the random movement of glutamate molecules in the synaptic cleft. Slower rates of diffusion increase the peak response and slow the time course of decay of the EPSC. 5. Random variations in release site position have little effect on the time course of the average EPSC or on the CV of the peak response. We simulate a dose-response curve for the effects of releasing between 100 and 7,500 neurotransmitter molecules per vesicle. The half-maximal response occurs for 1,740 molecules. For a simulation with 100 postsynaptic receptors and a diffusion coefficient of 2.0 x 10(-6) cm2/s, 4,000 molecules approaches a saturating dose. 6. Changes to the width of the synaptic cleft, or to the number and spacing of the postsynaptic receptors, have marked effects on the peak height of the simulated EPSC. 7. We extend the model to include a spherical vesicle (50 nm diam) connected to the synaptic cleft by a cylindrical pore 15 nm long. Neurotransmitter molecules are randomly distributed within the vesicle and allowed to diffuse into the synaptic cleft through the pore, which opens to its full diameter in one time step. We find that the pore must open to a diameter of > or = 7 nm within 1 microsecond in order to match the time courses of EPSCs in the literature.
Assuntos
Hipocampo/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Simulação por Computador , Difusão , Hipocampo/ultraestrutura , Método de Monte Carlo , Neurotransmissores/farmacocinética , Técnicas de Patch-Clamp , Células Piramidais , Temperatura , Fatores de TempoRESUMO
Using confocal microscopy in conjunction with microdrop application of Dil, we have imaged and measured individual dendritic spines of living hippocampal CA1 pyramidal neurons in acute brain slices, before and approximately 3 hr after induction of long-term potentiation by chemical means. Statistical analysis of changes in the length of individual spines, and comparison with results of Monte Carlo simulations, suggests that two forms of structural change occur in chemically induced long-term potentiation: growth of a subpopulation of small spines, and angular displacement of spines. These changes could provide a structural basis for the expression of long-term potentiation.
Assuntos
Dendritos/fisiologia , Dendritos/ultraestrutura , Hipocampo/fisiologia , Hipocampo/ultraestrutura , Potenciação de Longa Duração , Algoritmos , Animais , Técnicas In Vitro , Masculino , Microscopia Confocal , Modelos Neurológicos , Método de Monte Carlo , Ratos , Ratos Sprague-Dawley , Estimulação QuímicaRESUMO
We quantified the synaptic density in the entorhinal cortex (Brodmann area 28) in autopsy material from 10 individuals with Alzheimer's disease and compared them to 11 age-matched, postmortem-matched control subjects without dementia, using standard electron microscopy. The statistical data showed no change in synaptic density between control and Alzheimer subjects, in either lamina III or V of the cortex. There were no correlations between synaptic density and synaptic apposition length or density of senile plaques. The entorhinal cortex stands in marked contrast to other cortical areas that show a significant decline in synaptic numbers with Alzheimer's disease. This preservation of synaptic numbers may be related to a plasticity response that is greater in the entorhinal area than in other areas of the cortex.
Assuntos
Doença de Alzheimer/patologia , Hipocampo/patologia , Sinapses/ultraestrutura , Idoso , Autopsia , Hipocampo/ultraestrutura , Humanos , Microscopia Eletrônica , Emaranhados Neurofibrilares/patologia , Emaranhados Neurofibrilares/ultraestrutura , Tamanho do Órgão , Valores de ReferênciaRESUMO
The distribution and ultrastructure of Alzheimer's neurofibrillary tangles (ANT) in the brain stem, hypothalamus, and Ammon's horn were studied in four patients with postencephalitic parkinsonism of Economo type (PEPE). The distribution of ANT was as previously reported; the pattern of distribution resembled to that of amine-containing nerve cells. Ultrastructurally, ANT revealed twisted tubules (TT), but straight tubules (ST) of 150 A width were also found in the locus ceruleus of three cases; sometimes, TT and ST were mixed in a single neuron. Whether the coexistence of TT and ST in the locus ceruleus is a characteristic ultrastructural feature of ANT in PEPE or a regional peculiarity could not be determined. Ultrastructurally, ANT in PEPE were identical to those found in the brains of patients with Alzheimer's disease or senile dementia.