1) Herstellung <ol><li> Um zu beginnen, Sitz der Gegenstand in einem bequemen Stuhl vor einem Computer-Bildschirm. </li><li> Verwenden Sie ein Maßband, um den Abstand zwischen Bildschirm und die Probanden Nasion ist 60 cm zu gewährleisten. </li><li> Schließlich Ort ein Licht-blocking Maske über die Augen des Motivs. </li></ol> 2) Die Bestimmung Phosphene Threshold über V1/V2 <ol><li> Das Wissen des Subjekts Phosphen Schwelle wird wichtig sein, bei der Erkundung phosphenic Verhalten später in diesem Protokoll. Um zu bestimmen, diese Schwelle, zuerst die TMS-Maschine zu 70% Energie. </li><li> Halten Sie die Spule so oben in der Figur-8 Gesichter kranialen Mittellinie. </li><li> Begin single-pulse Stimulation an einem Punkt ca. 3 cm über dem Inion. </li><li> Nach jedem Puls, fragen Sie die vorbehaltlich etwaiger phosphenic Erfahrungen berichten. </li><li> Begin Bewegen der Spule in einem kleinen Suchraster um diese Region (V1/V2). </li><li> Bestimmen Sie den Speicherort, wo Stimulation löst konsequente und eindeutige Phosphen Berichte aus dem Thema. Es wurde vermutet, dass TMS Lage sein wird, Phosphene in rund 40% der Bevölkerung auslösen, so besteht die Chance, das Thema nicht melden wird alles. Wenn dies der Fall ist, leider wird dieses Experiment nicht funktioniert. Das Thema sollte gestattet werden, zu verlassen. </li><li> Sobald die phosphenic &quot;Hotspot&quot; gefunden wurde, stellen Sie die TMS Leistung rauf und runter, bis das Thema berichtet Phosphene für genau 3 von 6 aufeinander folgenden Pulsen. Diese Leistung ist das Thema der V1/V2 Schwelle. Falls verfügbar, können Neuronavigation verwendet werden, um größere räumliche Genauigkeit zu erreichen bei der Ortung und die Ausrichtung der V1/V2 Hotspot werden. </li></ol> 3) Die Bestimmung Phosphenic Threshold über die V5/MT + Complex <ol><li> Mit der gleichen oben genannten Verfahren und Parameter, werden wir nun bestimmen Phosphen Schwelle über die V5/MT +-Komplex. Um diese Region zu finden, an einer Stelle 3 cm dorsal und 5 cm lateral des Subjekts Inion beginnen. </li><li> Nach jedem Puls, fragen Sie die vorbehaltlich etwaiger phosphenic Erfahrungen berichten. </li><li> Auch mit einzelnen Impulsen, beginnen eine kleine Suche Raster bis eindeutige und konsistente Phosphene hervorgerufen werden. </li><li> Schließlich stellen Sie die TMS Leistung rauf und runter, bis das Thema berichtet Phosphene für genau 3 von 6 aufeinander folgenden Pulsen. Diese Leistung ist das Thema der V5/MT + komplexe Schwelle. Auch, wenn verfügbar, Neuronavigation können verwendet werden, um größere räumliche Genauigkeit zu erreichen bei der Ortung und die Ausrichtung der V5/MT + Hotspot werden. </li></ol> 4) Die Bestimmung Baseline Phosphene Behavior <ol><li> Sobald Phosphen Schwelle für beide visuellen Arealen festgestellt worden, müssen wir zu den Ausgangswerten phosphenic Verhalten zu messen. Um dies zu tun, entfernen Sie zunächst das Thema der Licht-blocking-Maske. </li><li> Anschließend weisen Sie den Gegenstand auf eine stabile Fixierung Kreuz in der Mitte des Bildschirms für 60 Sekunden präsentiert starren. </li><li> Tauschen Sie die Maske und über die V1/V2 hot spot, erzeugen einen einzigen Impulsfolge für die 3-Sekunden bei 120% V1/V2 Schwelle. </li><li> Warten Sie fünf Sekunden und führen einen anderen Zug. </li><li> Wieder warten fünf Sekunden und führen ein Drittel zu trainieren. </li><li> Nach der dritten Single Impulsfolge, fragen Sie den Gegenstand zu beschreiben, die Lage und Motiv Eigenschaften einer ausgelöst Phosphene. Dies wird die Grundlinie werden. </li><li> Wiederholen Sie dasselbe Verfahren über die V5/MT + Hot Spot (Sie erinnern sich an die TMS Leistung zu 120% des V5/MT + Schwelle Reset). </li></ol> 5) Condition Number One <ol><li> Sobald die phophenic Grundlinie bestimmt worden ist, entfernen Sie das Thema der lichtundurchlässigen Maske wieder. </li><li> Weisen Sie die Person in eine stabile Fixierung Kreuz in der Mitte des Bildschirms für 60 Sekunden präsentiert starren. Dieses Mal, anstatt einen leeren Bildschirm, werden wir eine Reihe von bewegenden Punkten an mehreren Standorten rund um das Kreuz einbinden. <img src="/files/ftp_upload/2273/2273fig1.jpg" alt="Abbildung 1" /> Abbildung 1 Die Anpassung Impulse für bedingen:. Einfache Translationsbewegung. Alle Punkte bewegen kohärent entweder nach links oder rechts. Achten Sie darauf, alle Punkte sind in die gleiche Richtung bewegen. Ein Phänomen, in dem Änderungen in der neuronalen Erregbarkeit bei längerer Exposition zu einem Stimuli induziert dient dazu, Verzerrungen der Wahrnehmung der nachfolgend präsentierten Stimuli: Dieser Stimulus sollte erzeugen visuelle Anpassung dienen. </li><li> Nach 60 Sekunden, tauschen Sie die Maske und über die V1/V2 Hotspot, erzeugen eine einzelne Impulsfolge für 3 Sekunden bei 120% V1/V2 Schwelle. </li><li> Warten Sie fünf Sekunden und wiederholen Sie noch zwei weitere Male. </li><li> Nach drei Zügen, fragen Sie den meldepflichtigen die Orte und Motive Eigenschaften einer ausgelöst Phosphene. Falls verfügbar, können die Verwendung einer Augenbewegung Tracking-System in einem dunklen Raum einen Beitrag zur Verwirklichung größerer Präzision und quantitative Überwachung der Augenbewegungen von Probanden während Phosphen elicitations gemacht. </li><li> Wiederholen Sie diesen Vorgang, bis three-train/report phosphene Verhalten auf den Ausgangswert zurück Aktivität. </li><li> Wiederholen Sie diesen Vorgang für die V5/MT + komplexe Hotspot. </li></ol> 6) Condition Number Two <ol><li> In diese zweite Bedingung, wiederholen Sie den Vorgang für bedingen genutzt. Dieses Mal jedoch, anstatt präsentiert das Thema mit einer Reihe von Punkten bewegt in eine einzige Richtung, präsentieren das Thema mit einer Reihe von Punkten jeweils bewegt in seiner relativen Himmelsrichtung weg von einer zentralen Stelle - ähnlich wie bei einem &quot;Star Burst&quot;-Muster . <img src="/files/ftp_upload/2273/2273fig2.jpg" alt="Abbildung 2" /> Abbildung 2 Anpassung Impulse für Bedingung zwei:. Radiale Bewegung. Dots bewegen jeweiligen Himmelsrichtungen entweder zu oder weg von einem zentralen Punkt. </li><li> Nach wie vor nach 60 Sekunden, ersetzen Sie die Maske, führen eine Serie von drei, 120%-Schwelle Züge über V1/V2, für ein Subjekt Bericht bitten, und weiter bis Grundlinie zurück. </li><li> Wiederholen Sie dies für V5/MT +. </li></ol> 7)-Diagramm <ol><li> Als Option nach jedem Zustand (oder nach allen Bedingungen abgeschlossen sind), stellen das Thema auf die Regionen und Motiv Verhalten der einzelnen Reihe von phopsphenes auf eine Grafik zu zeichnen. Dies ist nicht ein wesentlicher Schritt, aber werden Sie mit einem anderen Satz von interpretierbaren Daten zu versorgen. </li></ol> 8) repräsentative Ergebnisse Visuelle Anpassung an die uni-direktionale Motiv Stimuli sollten einen identischen phosphenic Bewegung über V1/V2 entlocken. <img src="/files/ftp_upload/2273/2273fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Beispiele für Phosphene von V1/V2 in der rechten und linken Hemisphäre während der Bedingung ein (basierend auf den Zeichnungen der Probanden) induziert. Die V5/MT + komplexe Phosphene sollte auch betroffen sein, so dass die Phosphen nun als eine Summe der Bewegungsrichtung in der Anpassung Reiz und der Grundlinie Phosphen erscheinen. Allerdings sollten visuelle Anpassung an die Starburst-Muster zu entlocken einem identischen starburst phosphenic Bewegung aus der V5/MT + komplex, aber nicht ändern V1/V2 Grundlinie. <img src="/files/ftp_upload/2273/2273fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Beispiele für Phosphene aus dem V5/MT induzierte +-Komplex in der rechten und linken Hemisphäre während der Zustand zwei (basierend auf den Zeichnungen der Probanden).

<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>

Dieses Experiment wird in den Mittelpunkt der staatlichen Abhängigkeit. Neuronen in V1/V2 gedacht werden, um mit einfachen, gerichtete Bewegung entsprechen 7,8 Deshalb Anpassung an die uni-direktionale Motiv Reize die Erregbarkeit der Neuronen registriert diese Bewegung erhöht -. Als solche sollten sie die ersten, die TMS reagieren Puls. Die V5/MT +-Komplex enthält auch Neuronen abgestimmt auf einfache Translationsbewegung, also die Phosphene aus dieser Region induzierte sollte auch durch die Anpassung Sti...

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

Transkranielle Magnetstimulation (TMS) ist eine nicht-invasive neurostimulatory und neuromodulatorischen Technik, die vorübergehend oder dauerhaft modulieren kann kortikalen Erregbarkeit (entweder steigend oder fallend ist) über die Anwendung der lokalisierten Magnetfeldimpulsen. 1,2 Im Bereich der TMS, der Begriff Zustand der Abhängigkeit bezieht sich auf die erste in den Ausgangszustand des jeweiligen neuronalen Region zur Stimulation ausgerichtet. Wie kann gefolgert werden, können die Auswirkungen der TMS (und tun) variieren je nach diesem primären Empfänglichkeit und Ansprechbarkeit der gezielten kortikalen Bereich. 3,4,5 In diesem Experiment werden wir dieses Konzept der staatlichen Abhängigkeit durch die Erhebung und die subjektive Erfahrung des Motivs Phosphene zu untersuchen. Phosphene sind optisch blinkt von kleinen Lichtern durch elektromagnetische Impulse an den visuellen Kortex ausgelöst wahrgenommen. Diese kleinen Lichtern kann davon ausgehen, verschiedene Eigenschaften je nachdem, welche Art von visuellen Kortex wird stimuliert. In diesem speziellen Studie, werden wir gezielt Motiv Phosphene als hervorgerufen durch die Stimulation von V1/V2 und die V5/MT + komplexen visuellen Regionen. 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 diesem Artikel untersuchen wir die Auswirkungen der optisch relevanten staatlichen Abhängigkeit von TMS induzierte Motiv phosphenic Präsentationen.

State-Dependency Auswirkungen auf TMS: Ein Blick auf Motive Phosphene Behavior

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 Aufrufe.  Beth Israel Deaconess Medical Center. In diesem Artikel untersuchen wir die Auswirkungen der optisch relevanten staatlichen Abhängigkeit von TMS induzierte Motiv phosphenic Präsentationen.

Serie: 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.

Die Neurostar TMS Device: Die Durchführung der FDA genehmigten Protokoll für die Behandlung von Depression

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

Forschung

Neurowissenschaften

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.

Kryokonservierung von kortikalen Gewebes Bausteine ​​für die Generierung von hoch angereichertem neuronalen Kulturen

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.

Die Untersuchung der Integration von Adult-geboren 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 ...

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.

Untersuchungen über Veränderungen der hippokampalen Schaltung Funktion Nach 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 ...

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.

Ein Verfahren für die Implantation eines Spinal Kammer für Längs<em&gt; In-vivo-</em&gt; Imaging der Maus Spinal Cord

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

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Live-Imaging von Nikotin induzierte Calcium Signaling und Neurotransmitterausschüttung Zusammen Ventral Hippocampus Axone

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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.

Hochauflösende Quantitative Immungold-Analyse von Membranrezeptoren auf der Netzhaut Ribbon Synapsen

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.

Linear Agarose Kanäle zu studieren<em&gt; Drosophila</em&gt; Larval Krabbeln Verhalten

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.

Kombination von Optogenetik mit künstlichen Mikro-RNAs, die Effekte gen Knockdown präsynaptischen Funktion innerhalb intakte neuronale Schaltkreise zu charakterisieren

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.

State-Dependency Auswirkungen auf TMS: Ein Blick auf Motive Phosphene Behavior

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