1) Preparação <ol><li> Para começar, o banco do sujeito em uma cadeira confortável na frente de uma tela de computador. </li><li> Use uma fita métrica para garantir a distância entre a tela e násio do sujeito é de 60 cm. </li><li> Finalmente, coloque uma máscara de bloqueio de luz sobre os olhos do sujeito. </li></ol> 2) Determinar Threshold fosfeno sobre V1/V2 <ol><li> Sabendo limiar fosfeno do sujeito será importante quando se explora o comportamento phosphenic mais tarde neste protocolo. Para determinar esse limite, primeiro defina a máquina TMS a 70% de energia. </li><li> Segure a bobina de modo que o topo da linha média cifra-8 faces cranial. </li><li> Comece-estimulação único pulso em um ponto cerca de 3 cm acima do ínion. </li><li> Após cada pulso, pergunte o assunto para relatar qualquer experiência phosphenic. </li><li> Começam a se mover a bobina em uma grade pequena pesquisa em torno desta região (V1/V2). </li><li> Determinar o local onde a estimulação provoca consistente e inequívoca relatórios fosfeno do assunto. Foi teorizado que a TMS não será capaz de provocar fosfenos em cerca de 40% da população, por isso há uma chance de o assunto não irá relatar nada. Se este for o caso, infelizmente, esta experiência não vai funcionar. O assunto deve ser autorizado a sair. </li><li> Uma vez que o phosphenic &quot;hotspot&quot; foi localizado, ajustar a potência TMS cima e para baixo até que o assunto fosfenos relatórios para exatamente 3 de 6 pulsos consecutivos. Esta potência é o limiar do sujeito V1/V2. Se estiver disponível, neuronavegação pode ser utilizado para alcançar maior precisão espacial quando localizar e orientar a hotspot V1/V2. </li></ol> 3) Determinar Threshold Phosphenic sobre o V5/MT + Complex <ol><li> Usando o mesmo procedimento acima mencionado e parâmetros, vamos agora determinar o limiar fosfeno sobre o V5/MT + complexo. Para localizar esta região, começa em um ponto três centímetros dorsal e 5 cm lateral para ínion do sujeito. </li><li> Após cada pulso, pergunte o assunto para relatar qualquer experiência phosphenic. </li><li> Novamente, utilizando pulsos simples, comece um padrão de grade pequena pesquisa até fosfenos inequívoca e consistente são eliciadas. </li><li> Finalmente, ajustar a potência TMS cima e para baixo até que o assunto fosfenos relatórios para exatamente 3 de 6 pulsos consecutivos. Esta potência é V5/MT do sujeito + limiar complexo. Novamente, se neuronavegação, disponível podem ser usados ​​para alcançar uma maior precisão espacial quando localizar e orientar a V5/MT + hotspot. </li></ol> 4) determinar o comportamento da linha de base fosfeno <ol><li> Uma vez limiar fosfeno foi determinada para as duas áreas visual, precisamos medir o comportamento phosphenic linha de base. Para fazer isso, primeiro remova a máscara do sujeito-luz de bloqueio. </li><li> Em seguida, instrua o sujeito a olhar para uma cruz fixação estável apresentado no centro da tela do computador por 60 segundos. </li><li> Substituir a máscara e, sobre o ponto V1/V2 quente, gerar um trem de pulso único para 3 segundos a 120% V1/V2 limiar. </li><li> Espera de cinco segundos e realizar outro trem. </li><li> Mais uma vez, espera de cinco segundos e realizar um terceiro trem. </li><li> Depois que o trem de pulsos terceiro single, pedir ao sujeito para descrever as características de localização e motivo de qualquer provocou fosfenos. Esta será a linha de base. </li><li> Repita este mesmo procedimento ao longo dos V5/MT + hot spot (lembre-se de repor o poder TMS a 120% do limiar + V5/MT). </li></ol> 5) Um número de condição <ol><li> Uma vez que a linha de base phophenic foi determinada, remova a máscara do sujeito-luz de bloqueio, mais uma vez. </li><li> Instruir o sujeito a olhar para uma cruz fixação estável apresentado no centro da tela do computador por 60 segundos. Desta vez, ao invés de uma tela em branco, vamos incorporar uma série de pontos em movimento em vários locais ao redor da cruz. <img src="/files/ftp_upload/2273/2273fig1.jpg" alt="Figura 1" /> Figura 1 Adaptação estímulos para uma condição:. Movimento de translação simples. Mover todos os pontos de forma coerente tanto para a esquerda ou direita. Certifique-se de todos os pontos estão se movendo na mesma direção. Este estímulo deve servir para gerar adaptação visual: um fenômeno em que as mudanças na excitabilidade neural como induzidas pela exposição prolongada a um estímulo serve para polarizar a percepção dos estímulos apresentados posteriormente. </li><li> Após 60 segundos, troque a máscara e, ao longo dos hotspot V1/V2, gerar um trem de pulso único durante 3 segundos em 120% V1/V2 limiar. </li><li> Espera de cinco segundos e repita mais duas vezes. </li><li> Depois de três trens, pedir o assunto para relatar os locais e as características de qualquer motivo provocou fosfenos. Se estiver disponível, o uso de um sistema de rastreamento de movimento dos olhos em um quarto escuro pode ajudar a atingir uma maior precisão e monitorização quantitativa dos movimentos dos olhos feitos por indivíduos durante elicitations fosfeno. </li><li> Continue essa seqüência até three-train/report phospcomportamento HeNe retorna à atividade inicial. </li><li> Repita este procedimento para o V5/MT + hotspot complexo. </li></ol> 6) Número Dois Condição <ol><li> Nesta segunda condição, repita o mesmo procedimento utilizado para uma condição. Desta vez, porém, ao invés de apresentar o assunto com uma série de pontos em movimento em uma direção singular, apresentar o assunto com uma série de pontos a cada movimento em sua direção em relação cardinal de distância de um ponto central - semelhante a uma &quot;explosão da estrela&quot; padrão . <img src="/files/ftp_upload/2273/2273fig2.jpg" alt="Figura 2" /> Figura 2 Adaptação estímulos para a condição dois:. Movimento radial. Pontos se movem em direções cardeais respectivos ou em direção ou para longe de um ponto central. </li><li> Como antes, após 60 segundos, troque a máscara, realizar uma série de três, trens limite de 120% sobre V1/V2, para pedir um relatório em questão, e continuar até a volta de linha de base. </li><li> Repita o procedimento para + V5/MT. </li></ol> Diagrama 7) <ol><li> Como uma opção, depois de cada condição (ou depois de todas as condições forem concluídas), pedir ao indivíduo para desenhar as regiões e comportamento motivação de cada série de phopsphenes em um gráfico. Isto não é um passo essencial, mas irá lhe fornecer um outro conjunto de dados interpretáveis. </li></ol> 8) Resultados Representante Adaptação visual aos estímulos uni-direcional motivo deve provocar um movimento idêntico phosphenic mais V1/V2. <img src="/files/ftp_upload/2273/2273fig3.jpg" alt="Figura 3" /> Figura 3. Exemplos de fosfenos induzidos a partir de V1/V2 no hemisfério direito e esquerdo durante uma condição (baseado nos desenhos de indivíduos). O V5/MT + fosfenos complexos também devem ser afetados, de modo que o fosfeno agora aparecerá como uma soma de direção de movimento no estímulo de adaptação e os fosfeno linha de base. No entanto, a adaptação visual para o padrão starburst deve causar um movimento idêntico phosphenic starburst do V5/MT + complexo, mas não deve mudar a linha de base V1/V2. <img src="/files/ftp_upload/2273/2273fig4.jpg" alt="Figura 4" /> Figura 4. Exemplos de fosfenos induzidos a partir da V5/MT + complexo no hemisfério direito e esquerdo durante a condição de dois (baseado nos desenhos de indivíduos).

<ol>
	<li>Pascual-Leone, A., Davey, M., Wassermann, E. M., Rothwell, J., Puri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Transcranial Magnetic Stimulation. , (2002).</li><li>Walsh, V., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transcranial Magnetic Stimulation: A Neurochronometrics of Mind. , (2005).</li><li>Silvanto, J., Muggleton, N. G., Cowey, A., Walsh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+adaptation+reveals+state-dependent+effects+of+transcranial+magnetic+stimulation.">Neural adaptation reveals state-dependent effects of transcranial magnetic stimulation.</a> European Journal of Neuroscience. 25, 1874-1881 (2007).</li><li>Silvanto, J., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-Dependency+of+Transcranial+Magnetic+Stimulation.">State-Dependency of Transcranial Magnetic Stimulation.</a> Brain Topography. 21, 1-10 (2008).</li><li>Silvanto, J., Cattaneo, Z., Battelli, L., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+cortical+excitablility+determines+whether+TMS+disrupts+or+facilitates+behavior.">Baseline cortical excitablility determines whether TMS disrupts or facilitates behavior.</a> Journal of Neurophysiology. 99, 2725-2730 (2008).</li><li>Silvanto, J., Muggleton, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+validity+of+the+TMS+state-dependency+approach:+targeting+functionally+distinct+motion-selective+neural+populations.">Testing the validity of the TMS state-dependency approach: targeting functionally distinct motion-selective neural populations.</a> Neuroimage. 40, 1841-1848 (2008).</li><li>Tootell, R. B., Rappas, J. B., Kwong, K. K., Malach, R., Born, R. T., Brady, T. J., Rosen, B. R., Belliveau, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+human+MT+and+related+visual+cortical+areas+using+magnetic+resonance+imaging.">Functional analysis of human MT and related visual cortical areas using magnetic resonance imaging.</a> Journal of Neuroscience. 15, 3215-3230 (1995).</li><li>Singh, K. D., Smith, A. T., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+frequency+and+direction+sensitivities+of+human+visual+areas+measured+using+fMRI.">Spatiotemporal frequency and direction sensitivities of human visual areas measured using fMRI.</a> Neuroimage. 12, 550-564 (2000).</li><li>Rutchmann, R. M., Schrauf, M., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+activation+during+dichoptic+presentation+of+optic+flow+stimuli.">Brain activation during dichoptic presentation of optic flow stimuli.</a> Exp Brain Res. 134, 533-537 (2000).</li><li>Morrone, M. C., Tosetti, M., Montanaro, D., Fiorentini, A., Cioni, G., Burr, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cortical+area+that+responds+specifically+to+optic+flow+revealed+by+fMRI.">A cortical area that responds specifically to optic flow revealed by fMRI.</a> Nat. Neuroscience. 3, 1322-1328 (2000).</li></ol>

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Esta experiência começa no coração de dependência do Estado. Neurônios em V1/V2 são pensados ​​para corresponder com o movimento, simples direcional 7,8 Portanto, a adaptação aos estímulos uni-direcional motivo aumento da excitabilidade dos neurônios registrar este movimento -. Como tal, devem ser os primeiros a reagir ao TMS pulso. O V5/MT + complexo também contém neurônios ajustado ao movimento de translação simples, portanto, o fosfenos induzidos a partir desta região também deve ser ...

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<td>Light Blocking Eye Mask</td>
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<td>Any Single Pulse Capable TMS Device</td>
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state-dependency effects on tms:  a look at motive phosphene behavior

A estimulação magnética transcraniana (TMS) é um neurostimulatory não-invasivo e técnica neuromoduladores que pode transitoriamente ou duradoura modular a excitabilidade cortical (aumentando ou diminuindo-a), através da aplicação de pulsos localizados campo magnético. 1,2 No campo da TMS, o termo dependência de estado refere-se à condição inicial, base da região neural alvo de estimulação. Como pode ser inferido, os efeitos da TMS podem (e fazem) variam de acordo com esta susceptibilidade primária e capacidade de resposta da área alvo cortical. 3,4,5 Nesta experiência, vamos examinar este conceito de dependência de estado através do levantamento e da experiência subjetiva de fosfenos motivo. Fosfenos são visualmente percebidos flashes de luzes pequenas desencadeada por pulsos eletromagnéticos para o córtex visual. Estas pequenas luzes pode assumir características variadas, dependendo de qual tipo de córtex visual está sendo estimulada. Neste estudo particular, que será alvo fosfenos motivo como suscitou através da estimulação de V1/V2 e as regiões V5/MT + complexo visual. 6

Watch this Scientific Journal Video about State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior at JoVE.com

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Neste artigo, vamos examinar os efeitos da dependência do Estado visualmente relevantes sobre TMS induzida apresentações phosphenic motivo.

Estado de dependência-Efeitos sobre a TMS: Um olhar sobre Comportamento fosfeno Motive

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

state-dependency-effects-on-tms-a-look-at-motive-phosphene-behavior

Research

JoVE Journal

Neuroscience

10.5K visualizações.  Beth Israel Deaconess Medical Center. Neste artigo, vamos examinar os efeitos da dependência do Estado visualmente relevantes sobre TMS induzida apresentações phosphenic motivo.

Vídeo: State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

O dispositivo NeuroStar TMS: Conduzindo a FDA Aprovado protocolo para o tratamento da depressão

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Neurociência

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Criopreservação de tecido cortical Blocos de Geração de culturas altamente enriquecido Neuronal

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

Estudar a Integração da Adult-nascido Neurônios

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Investigações sobre alterações da função Circuito Hippocampal Após lesão cerebral traumática leve

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Um Processo de Implantação de uma Câmara Spinal para Longitudinal<em&gt; In Vivo</em&gt; Criação de Imagens do Cordão do rato Spinal

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

High-Resolution Análise Quantitativa immunogold de Receptores de Membrana na retina fita Sinapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Usando Canais Linear de agarose para o Estudo<em&gt; Drosophila</em&gt; Comportamento larval Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinação de Optogenetics com microRNAs Artificial para caracterizar os efeitos do Gene Knockdown na função pré-sináptica dentro circuitos neuronais intacta

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

Tintura de FM ciclismo a sinapse: comparando a despolarização de potássio alto, elétrica e Channelrhodopsin estimulação

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Efeitos de Neurotrauma induzida por explosão em roedores pressurizado artérias cerebrais de médio

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...