1) Preparazione <ol><li> Per iniziare, sede il soggetto in una comoda poltrona davanti allo schermo di un computer. </li><li> Utilizzare un metro a nastro per garantire la distanza tra lo schermo e nasion del soggetto è di 60 cm. </li><li> Infine, una maschera di luce posto di blocco sopra gli occhi del soggetto. </li></ol> 2) Determinazione della soglia fosfene oltre V1/V2 <ol><li> Conoscere soglia fosfene del soggetto sarà importante quando si esplora il comportamento fosfenici più avanti in questo protocollo. Per determinare questa soglia, prima impostare la macchina TMS al 70% della potenza. </li><li> Tenere la serpentina così la parte superiore della figura-8 volti linea mediana del cranio. </li><li> Iniziare singolo impulso stimolazione in un punto circa 3 cm sopra il inion. </li><li> Dopo ogni impulso, chiedere al soggetto di segnalare qualsiasi esperienza fosfenici. </li><li> Iniziare a muoversi la bobina in una piccola griglia di ricerca intorno a questa regione (V1/V2). </li><li> Determinare la posizione in cui stimolazione provoca rapporti fosfene coerente e non ambiguo dal soggetto. E &#39;stato teorizzato che TMS sarà in grado di suscitare fosfeni in circa il 40% della popolazione, quindi c&#39;è una possibilità che il soggetto non riporta nulla. Se questo è il caso, purtroppo, questo esperimento non funziona. Il soggetto dovrebbe essere permesso di lasciare. </li><li> Una volta che il Fosfenico &quot;hotspot&quot; è stato localizzato, regolare la potenza di uscita TMS su e giù fino a quando il soggetto fosfeni rapporti esattamente per 3 di 6 impulsi consecutivi. Questo livello di potenza è del soggetto V1/V2 soglia. Se disponibile, neuronavigazione può essere usato per ottenere una maggiore precisione spaziale in cui localizzare e indirizzare l&#39;hotspot V1/V2. </li></ol> 3) Determinazione della soglia Fosfenico il V5/MT + Complex <ol><li> Utilizzando la stessa procedura di cui sopra e parametri, ci sarà ora determinare la soglia fosfene il V5/MT + complesso. Per individuare questa regione, iniziano in un punto 3 centimetri dorsale e 5 cm lateralmente alla inion del soggetto. </li><li> Dopo ogni impulso, chiedere al soggetto di segnalare qualsiasi esperienza fosfenici. </li><li> Ancora una volta, l&#39;uso di impulsi singoli, iniziare un piccolo modello di griglia di ricerca fino a fosfeni univoca e coerente sono suscitate. </li><li> Infine, regolare la potenza TMS su e giù fino a quando il soggetto fosfeni rapporti esattamente per 3 di 6 impulsi consecutivi. Questo livello di potenza è V5/MT del soggetto + soglia complesso. Ancora una volta, se disponibile, neuronavigazione può essere usato per ottenere una maggiore precisione spaziale per la localizzazione e l&#39;attenzione ai V5/MT + hotspot. </li></ol> 4) Determinazione fosfene comportamento di base <ol><li> Una volta la soglia fosfene è stato determinato per entrambe le aree visive, abbiamo bisogno di misurare fosfenici comportamento di base. Per fare questo, prima rimuovere il soggetto luce di blocco maschera. </li><li> Successivamente, istruire il soggetto a fissare una croce fissazione stabile presentati al centro dello schermo del computer per 60 secondi. </li><li> Sostituire la maschera e, sul punto V1/V2 caldo, generare un unico treno di impulsi per 3 secondi al 120% V1/V2 soglia. </li><li> Attendere cinque secondi e condurre un altro treno. </li><li> Anche in questo caso, attendere cinque secondi e condurre un terzo treno. </li><li> Dopo il terzo treno di impulsi singoli, chiedere al soggetto di descrivere le caratteristiche e la localizzazione di qualsiasi movente suscitato fosfeni. Questa sarà la linea di base. </li><li> Ripetere la stessa procedura il V5/MT + hot spot (ricordatevi di ripristinare il potere TMS al 120% della soglia V5/MT +). </li></ol> 5) Numero Uno Stato <ol><li> Una volta che la linea di base phophenic è stata determinata, rimuovere il soggetto luce bloccando ancora una volta la maschera. </li><li> Istruire il soggetto a fissare una croce fissazione stabile presentati al centro dello schermo del computer per 60 secondi. Questa volta, invece di uno schermo bianco, ci incorpora una serie di puntini in movimento in diverse località intorno alla croce. <img src="/files/ftp_upload/2273/2273fig1.jpg" alt="Figura 1" /> Figura 1 Adattare stimoli per condizione uno:. Semplice moto traslatorio. Tutti i punti mossa coerentemente sia a sinistra oa destra. Assicurarsi che tutti i punti si stanno muovendo nella stessa direzione. Questo stimolo dovrebbe servire a generare l&#39;adattamento visivo: un fenomeno in cui i cambiamenti nella eccitabilità neurale indotte da esposizione prolungata ad un stimoli serve per polarizzare la percezione degli stimoli presentati successivamente. </li><li> Dopo 60 secondi, sostituire la maschera e, nel corso degli hotspot V1/V2, generare un unico treno di impulsi per 3 secondi al 120% V1/V2 soglia. </li><li> Attendere cinque secondi e ripetere due volte. </li><li> Dopo tre treni, chiedere al soggetto di segnalare i luoghi e le caratteristiche di qualsiasi movente suscitato fosfeni. Se disponibili, l&#39;uso di un sistema di eye tracking movimento in una stanza buia può aiutare a raggiungere una maggiore precisione e il controllo quantitativo del movimenti oculari fatta da soggetti durante elicitations fosfene. </li><li> Continua questa sequenza three-train/report fino maerlcomportamento HeNe ritorna all&#39;attività di base. </li><li> Ripetere la procedura per la V5/MT + hotspot complessi. </li></ol> 6) numero di condizionamento Due <ol><li> In questa seconda condizione, ripetere la stessa procedura utilizzata per condizione uno. Questa volta, però, anziché presentare l&#39;argomento con una serie di puntini in movimento in una direzione singolare, presente il soggetto con una serie di punti ogni movimento nella sua direzione relativa cardinale lontano da un punto centrale - simile ad un &quot;burst stella&quot; modello . <img src="/files/ftp_upload/2273/2273fig2.jpg" alt="Figura 2" /> Figura 2 Adattare stimoli per due condizioni:. Movimento radiale. Punti si muovono in direzioni cardinali rispettivi sia verso o lontano da un punto centrale. </li><li> Come prima, dopo 60 secondi, sostituire la maschera, effettuare una serie di tre, i treni della soglia del 120% rispetto V1/V2, per chiedere un rapporto di soggetto, e continuerà fino al ritorno al basale. </li><li> Ripetere l&#39;operazione per + V5/MT. </li></ol> 7) Schema <ol><li> Come opzione, dopo ogni condizione (o dopo tutte le condizioni sono completate), chiedere al soggetto di disegnare le regioni e le motivazioni del comportamento di ogni serie di phopsphenes su un grafico. Questo non è un passo integrale, ma vi fornirà un altro insieme di dati interpretabili. </li></ol> 8) Risultati Rappresentante L&#39;adattamento visivo al unidirezionale stimoli motivo deve provocare un movimento identico fosfenici oltre V1/V2. <img src="/files/ftp_upload/2273/2273fig3.jpg" alt="Figura 3" /> Figura 3. Esempi di fosfeni indotta dal V1/V2 nell&#39;emisfero destro e sinistro durante la condizione di uno (basato sui disegni di soggetti). Il V5/MT + fosfeni complesso dovrebbe essere colpiti, in modo che il fosfene ora apparirà come una somma di direzione del movimento nello stimolo adattamento e la fosfene basale. Tuttavia, l&#39;adattamento visivo per il modello starburst dovrebbe provocare un identico movimento fosfenici starburst dal V5/MT + complesso, ma non dovrebbe cambiare la linea di base V1/V2. <img src="/files/ftp_upload/2273/2273fig4.jpg" alt="Figura 4" /> Figura 4. Esempi di fosfeni indotta dalla V5/MT + complesso nell&#39;emisfero destro e sinistro durante la condizione di due (basato sui disegni di soggetti).

<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>

Nessun conflitto di interessi dichiarati.

Questo esperimento viene al cuore dello stato di dipendenza. Neuroni in V1/V2 si pensa a corrispondere con semplici, movimento direzionale 7,8 Pertanto, l&#39;adattamento alle uni-direzionale stimoli motivo aumentato l&#39;eccitabilità dei neuroni registrare questo movimento -. Quanto tali, dovrebbero essere i primi a reagire al TMS impulsi. Il V5/MT + complesso contiene anche neuroni sintonizzati semplice moto traslatorio, quindi il fosfeni indotto da questa regione dovrebbe essere interessata dallo stimolo...

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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

Stimolazione magnetica transcranica (TMS) è un non-invasiva e tecnica neurostimulatory neuromodulatori che possono transitoriamente o durevolmente modulare l&#39;eccitabilità corticale (sia aumentando o diminuendo esso) attraverso l&#39;applicazione localizzata di impulsi di campo magnetico. 1,2 Nell&#39;ambito della TMS, il termine dipendenza dello stato si riferisce alla prima le condizioni originarie della regione di particolare mirati per la stimolazione neurale. Come si può dedurre, gli effetti della TMS può (e fare) variano in base a questa suscettibilità primaria e la reattività del territorio mirati corticale. 3,4,5 In questo esperimento, esamineremo questo concetto di stato di dipendenza attraverso l&#39;elicitazione e l&#39;esperienza soggettiva dei fosfeni movente. Fosfeni sono visivamente percepito lampi di piccole luci attivati ​​da impulsi elettromagnetici alla corteccia visiva. Queste piccole luci possono assumere caratteristiche diverse a seconda di quale tipo di corteccia visiva viene stimolato. In questo studio particolare, saremo targeting fosfeni come motivo ha suscitato attraverso la stimolazione dei V1/V2 e le regioni visive V5/MT + complessa 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

In questo articolo esaminiamo gli effetti di dipendenza stato visivamente rilevanti TMS indotta presentazioni fosfenici movente.

Stato-dipendenza Effetti sulla TMS: uno sguardo al comportamento Motive fosfene

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 Views.  Beth Israel Deaconess Medical Center. In questo articolo esaminiamo gli effetti di dipendenza stato visivamente rilevanti TMS indotta presentazioni fosfenici movente.

Video: 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.

Il dispositivo TMS NeuroStar: Condurre la FDA ha approvato protocollo per il trattamento della depressione

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

Ricerca

Neuroscienze

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.

Crioconservazione di tessuto corticale blocchi per la generazione di altamente arricchito Culture neuronale

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.

Studiare l&#39;integrazione degli adulti nati Neuroni

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.

Indagini su Alterazioni della funzione del circuito ippocampale seguito lieve trauma cranico

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.

Una procedura di impianto del Camera spinale per longitudinale<em&gt; In Vivo</em&gt; Imaging del cavo del mouse spinale

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.

Risoluzione alta analisi quantitativa Immunogold di Recettori di membrana a retina nastro Sinapsi

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.

Utilizzando canali lineari Agarose per studiare<em&gt; Drosophila</em&gt; Comportamento larvale 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.

Combinando optogenetica con microRNA artificiali per caratterizzare gli effetti del Gene atterramento su funzione presinaptica all'interno di circuiti neuronali intatto

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 di FM in bicicletta alla sinapsi: confronto tra alto potassio depolarizzazione, elettrica e stimolazione Channelrhodopsin

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.

Effetti del Neurotrauma indotta da Blast il roditore pressurizzato Middle arterie cerebrali

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