Finanzierung: Diese Arbeit wurde durch eine Stiftung Bertarelli Zuschuss (DJL), einem MED-EL Zuschuss (DJL) und ein National Institutes of Health Grants DC01089 (MCB) unterstützt.

HINWEIS: Alle experimentellen Verfahren sind in Übereinstimmung mit dem Animal Care und Verwenden Ausschuss der Massachusetts Eye and Ear Infirmary und der Harvard Medical School, die nationalen Tierpflege Richtlinien befolgen, einschließlich Public Health Service Politik über humane Pflege und Verwendung von Labortieren, die durchgeführt ILAR Guide, und das Tierschutzgesetz. Experimentelle Verfahren nachstehend im Detail Exposition der linken DCN aufgeführt. Verwenden Sie sterile Instrumente während der Durchführung Überleben Chirurgie. 1. Primär Kraniotomie und Rücken Cochlear Nucleus Belichtungs <ol><li> Anästhesie <ol><li> Anesthetize einer Maus im Alter von 8-12 Wochen mit einem Gewicht von 18-24 g, Xylazin 20 mg / kg Ketamin 100 mg / kg über eine intraperitoneale Verabreichung. Überprüfen Sie die Anästhesie durch die Überwachung der Herzfrequenz, Atemfrequenz, sowie toe Prise Rückzug Reflex. Platz Tierarzt Salbe auf die Augen bis zur Trockenheit während der Narkose zu verhindern. Sobald ausreichend unter Narkose, Rasur Haarüberlagert ter Kopfhaut, um ungehinderten Zugang zu der Operationsstelle zu schaffen. </li></ol></li><li> Chirurgische Positionierung <ol><li> Platzieren Sie die Maus sicher in einer Kleintierstereotaktischen Halterung, an Ort und Stelle durch eine Schnauze Klammer gehalten. </li><li> Stellen Sie sicher, dass die Schnauze Klemme locker genug, um für eine ausreichende Atmung aber fest genug, lassen den Kopf der Maus vollständig zu immobilisieren. Wenn die Maus den Kopf locker ist, ist das Tier wahrscheinlich während der Kraniotomie Teil der Verfahren des Kopfhalters fallen. </li><li> Zeigen auditorischen Hirnstamm-Implantat Elektroden in üblicher Weise, die für die Überwachung der Herzfrequenz ermöglicht. Die Atemfrequenz sollte durch Visualisierung überwacht werden. Beachten Sie, dass die normale Herzfrequenz und die Atemfrequenz wird in Abhängigkeit vom Alter des Maus variieren. </li><li> Ein Thermometer ist auf einer homeothermic Heizdecke platziert und über die Platzierung euthermia gewährleistet ist. </li></ol></li><li> Hautschnitt und Exposition der interparietal und occipital Knochen des Schädels <ol><li> Unter einem Microscopa, stellen ein vertikaler Schnitt durch die Haut ausgehend von der Mittellinie, direkt zwischen der Ohrmuschel und sich bis zum Schwanzteil des Hinterhauptbein. Nach der Mittellinie Hautschnitt, verdrängen die Haut seitlich. Visualisieren Sie die Muskeln für den verließ den hinteren Teil des Schädels. </li><li> Entfernen Muskel über dem linken Scheitel, interparietal und occipital Knochen des Schädels durch Exartikulation der Muskelansätze an ihre jeweiligen Knochen mit einem Skalpell oder Iris Schere. Beachten Sie ein kleines Maß an Blutungen entlang der Schnittmuskel Kanten. Minimieren Sie dies durch sanften Druck mit einem Wattestäbchen für 10-15 Sek. </li></ol></li><li> Kraniotomie darüberliegenden Cochlear Nucleus <ol><li> Identifizieren Sie relevanten Nahtlinien, einschließlich sagittalen und Lambdanähte Linien (Abbildung 1, links). </li><li> Mit Rongeure, machen Sie eine Kraniotomie über die Inkabein, links von der Mittellinie, ~ 2 mm kaudal der Lambda-Naht. Diese Region liegt über dem DCN. </li><li> Following Kraniotomie, beachten Sie eine dünne Schicht der Dura, die das Kleinhirn überlagert. Mit einem Skalpell, entfernen Sie die Dura. (Bild 1, Mitte). HINWEIS: Das Entfernen der Dura kann ein kleines Maß an Blutungen. Dieses Verfahren der Verwendung der Skalpellklinge, um die Dura zu entfernen, ist beabsichtigt, um die Menge des Blutverlustes während der Cerebellum Aspiration, die auf der Hirnoberfläche und undurchsichtig Identifizierung des DCN Pool wird reduzieren. Gesamtblutvolumen von Maus ist ~ 1,5 ml nicht überschreiten soll&gt; als 15%. Wenn die Blutung diesen Betrag übersteigt, sollte die Maus mit mit Flüssigkeit Ergänzung zur Verfügung gestellt werden. </li><li> Entfernen geronnenem Blut überlagert das Kleinhirn mit dem Wattestäbchen. Optional sanft tropfen Kochsalzlösung mit einem Zahnpunkt auf das Kleinhirn, um Blut zu Blut entfernt. </li></ol></li><li> Kleinhirn-Aspiration <ol><li> Verwendung eines 5 Französisch Saug absaugen seitlichen äußersten Abschnitt der linken Cerebellum über der DCN bis der DCN visBuchs (Abbildung 1, rechts). Entfernen etwa 1/4 bis 1/3 des linken Kleinhirn. Das Wahrzeichen neben dem DCN ist die Ampulle des oberen Bogenganges. HINWEIS: Aspiration des Kleinhirns ist der entscheidende Schritt des Protokolls. Gerichtete Streben des Cerebellum meisten erfolgreich durchgeführt, wenn es in einem einzigen Versuch auftritt und nicht mit Mehrfachdurchgänge des Saug da dies zu Blutungen führen. In Ansaugen zu unterstützen, setzen die Brennebene des Mikroskops an der erwarteten Tiefe des CN, die geringfügig distal der Oberfläche der Kleinhirn wird. Einstellen der Brennebene mit dem CN wird Visualisierung verbessern und ein scharfes Bild. </li><li> Nach Aspiration, weitere Blutungen, Liquor (CSF) Aufbau und Kleinhirn Verschiebung auf der DCN erwartet. Vermitteln 0,5 cc steriler Kochsalzlösung schnell in die Kraniotomie Blutgerinnung verhindern. Eine Kombination aus sanften Abtupfen mit einem Zahnpunkt und Absaugung kann dannverwendet, um abzuräumen einen Weg für die direkte Visualisierung des DCN. Nicht direkt an die Oberfläche des DCN. </li></ol></li></ol> 2. Druckmikroinjektion für Virus-vermittelte Gentransfer und chirurgische Wiederherstellung <ol><li> Nachdem der DCN ist frei von überlagernden Blut und CSF und ist deutlich sichtbar, machen Druck Mikroinjektionen in das DCN mit einem 10 &amp; mgr; l Hamilton-Spritze über einen Zeitraum 2 min. </li><li> Einführung der Nadel mit einem Mikromanipulator, bis die Spitze nicht mehr unter der Oberfläche des DCN sichtbar. </li><li> Für eine optimale Leistung verwenden Sie eine 33 oder 34 Gauge-Nadel (verwenden Sie eine gasdichte Spritze mit der 34-Gauge-Nadel) mit einem flachen Kegel, wie 45 °, um stumpfes Trauma zu minimieren und zu lokalisieren, das Injektionsvolumen im DCN, der ein dünner ist und seichten Hirnstruktur (&lt;300 &amp; mgr; m dick). Hinweis: andere Mikroinjektionsinstrumente genutzt basieren. Idealerweise sollte die Injektions Instrument flexibel sein, um für einige geringfügige ben ermöglichend, wenn nötig, direkt gezielt die DCN. Ferner ist, wie die Maus bewegt sich geringfügig, während des gesamten Verfahrens durch die Atmung, sollte das Gerät robust genug sein, um geringe Mengen an Bewegung zu widerstehen. </li></ol> 3. Chirurgische Wieder <ol><li> Unmittelbar nach der Injektion wieder anzunähern, die Haut und lassen Sie die Maus, um per Standard Recovery-Verfahren zu erholen. Verbleibende Cerebellum und Narbengewebe in den Hohlraum durch Absaugen des Kleinhirns verursacht füllen. Ständig Tiere zu überwachen, bis genügend Bewusstsein wiedererlangt, um Brustlage zu halten. Überwachen Sie Herzfrequenz, Atemfrequenz, sowie die Fähigkeit zu ernähren. HINWEIS: Kein Tier, das einer Operation unterzogen wurde, wird in einen Käfig mit anderen Tieren zurückgegeben, bis vollständig erholt. </li><li> Wenn eine Maus fängt an, im Kreis nach der Operation zu gehen (&lt;5% der Fälle), in der Regel 2-4 Stunden nach der Schließung der Hautschnitt, sofort opfern Sie die Maus, wie es würde wahrscheinlich eine schwierige Zeit Fütterung. Tseine Nebeneffekt ist durch Aspiration des Kleinhirns wahrscheinlich. Das Tier sollte bei Schmerzen nach der Operation überwacht und entsprechende Betäubungsmittel sollte der besseren Tier Komfort auf Grund institutioneller Normen werden. Antibiotika kann notwendig sein, wenn Anzeichen einer Infektion. </li></ol> 4. Sekundäre Kraniotomie und Cochlear Nucleus Belichtungs <ol><li> Nach 2-4 Wochen der Heilung und virusvermittelten Gentransfer Inkubation erneut anästhesiert eine vorher injizierten Maus und Schritt 1,1-1,5 optimale Inkubationszeit hängt von der Art des Gens überführt. </li><li> Entfernen Narbengewebe über die Kraniotomiestelle durch eine Kombination von Pinzette, Skalpell und Knochenzangen. </li><li> Nach der Visualisierung der Kraniotomie, identifizieren eine Kombination aus Rest über der DCN Kleinhirn und Narbengewebe. Verwenden Sie einen 5 Französisch Ansaugen darüber liegenden Kleinhirn / Narbengewebe (ähnlich wie das vorherige Kleinhirn Aspiration) anzusaugen. </li><li> Nach Aspiration, erwarten weitere Blutungen, CSF build-up, und die verbleibenden Bits des Kleinhirns. Schnell einzuführen Salzlösung in die Kraniotomie, um die Blutgerinnung zu verhindern. Verwenden Sie eine Kombination aus sanften Zahnpunkt Abtupfen und Absaugung, um den Weg für die direkte Visualisierung des DCN löschen. </li><li> Beachten Sie die Oberfläche des DCN. Führen Optogenetik basierte Physiologie Experimente wie DCN ist jetzt zugänglich. Einführung eines Lichtreizes, zum Beispiel mit einer optischen Faser (2), um eine Optogenetik basierenden Experiment fahren. </li></ol> 5. Histologie <ol><li> Nach Abschluss der Experimente, einschläfern die Maus mit einer Überdosis von Ketamin. Perfundieren der Maus mit normaler Salzlösung, gefolgt von 4% Paraformaldehyd. </li><li> Entpacken Sie die Hirnstamm aus dem Schädel und Post-fix für 2 Stunden. Cryoprotect den Hirnstamm in 30% Saccharose für 24-48 Std. Abschnitt der Hirnstamm mit einem Standard-Kryostaten mit 60 um-Schnitte. </li></ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+the+auditory+pathway.">Optogenetic stimulation of the auditory pathway.</a> J Clin Invest. 124 (3), 1114-1129 (2014).</li><li>Adamantidis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogation+of+dopaminergic+modulation+of+the+multiple+phases+of+reward-seeking+behavior.">Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior.</a> J Neurosci. 30 (31), 10829-10835 (2011).</li><li>Kim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+mimicry+of+the+transient+activation+of+dopamine+neurons+by+natural+reward+is+sufficient+for+operant+reinforcement.">Optogenetic mimicry of the transient activation of dopamine neurons by natural reward is sufficient for operant reinforcement.</a> PloS One. 7 (4), e33612 (2012).</li><li>Britt, J., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogations+of+the+neural+circuits+underlying+addiction.">Optogenetic interrogations of the neural circuits underlying addiction.</a> Curr Opin Neurobiol. 23 (4), 539-545 (2013).</li><li>Abbott, S., Coates, M., Stornetta, R., Guyenet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+c1+and+retrotrapezoid+nucleus+neurons+causes+sleep+state-dependent+cardiorespiratory+stimulation+and+arousal+in+rats.">Optogenetic stimulation of c1 and retrotrapezoid nucleus neurons causes sleep state-dependent cardiorespiratory stimulation and arousal in rats.</a> Hypertension. 61 (4), 835-841 (2013).</li><li>Carter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+arousal+with+optogenetic+modulation+of+locus+coeruleus+neurons.">Tuning arousal with optogenetic modulation of locus coeruleus neurons.</a> Nat Neurosci. 13 (12), 1526-1533 (2010).</li><li>Darrow, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+central+auditory+neurons.">Optogenetic control of central auditory neurons.</a> Assoc. 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Die Autoren haben nichts zu offenbaren.

Dieses Papier beschreibt die Technik der direkten Visualisierung des DCN im Mausmodell für die Manipulation des zentralen auditorischen Systems. Das skizzierte Ansatz der direkten Visualisierung bietet erhebliche Vorteile gegenüber dem Haupt Alternative, die stereotaktische Ansätze sind. In erster Linie die direkte Visualisierung des DCN ermöglicht sofortige Bestätigung der Website des Hirnstamms, während stereo Ansätze nicht direkte Visualisierung leisten. In Experimenten, die längere Inkubationszeiten erforder...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

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			<td height="21" style="height:21px;width:197px;">Stereotaxic holder</td>
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			<td height="21" style="height:21px;width:197px;">Scalpel blade #11</td>
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			<td>10011-00</td>
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			<td height="21" style="height:21px;width:197px;">5 French suction</td>
			<td style="width:227px;">Symmetry Surgical</td>
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			<td height="21" style="height:21px;width:197px;">Dental Points</td>
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			<td>100-8170</td>
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			<td height="51" rowspan="2" style="height:52px;width:197px;">10 &#181;l Hamilton syringe</td>
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			<td height="42" rowspan="2" style="height:42px;width:197px;">34 gauge, needle</td>
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			<td height="21" style="height:21px;">Rongeurs</td>
			<td>Fine Surgical Tools</td>
			<td>16021-14</td>
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</table>

direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

Untersuchung über die Verwendung von virusvermittelten Gentransfer zu verhaften oder rückwärts Hörverlust hat sich weitgehend auf die Umfangs Hörsystem verbannt worden. Nur wenige Studien haben den Gentransfer mit dem zentralen auditorischen Systems untersucht. Dorsalen cochlearen Nukleus (DCN) des Hirnstamms, die Neuronen zweiter Ordnung der Hörbahn enthält, ist eine potentielle Stelle für einen Gentransfer. In diesem Protokoll wird ein Verfahren zur direkten und maximale Exposition des murinen DCN über einen posterioren Fossa Ansatz demonstriert. Dieser Ansatz ermöglicht entweder akut oder das Überleben der Operation. Nach direkten Visualisierung des DCN, eine Vielzahl von Experimenten sind möglich, einschließlich Injektion in die Opsinen Cochleariskern und nachfolgende Stimulation durch eine optische Faser zu einer Blaulicht-Laser gekoppelt ist. Andere Neuro Experimenten wie elektrische Stimulation und neuronalen Injektor Kurven sind denkbar. Die Höhe der Visualisierungstion und die Dauer der Stimulation erreichbar machen diesen Ansatz für eine Vielzahl von Experimenten.

Teilweise Cerebellar Aspiration den Zugang zu der Cochlear Nucleus Nachdem die Haut und Muskeln, die über dem Schädel sind, Schädeloberfläche Sehenswürdigkeiten wie den koronaren und lamda Nahtlinien entfernt, zeigen die ungefähre Lokalisierung der Kraniotomie. Nach Kraniotomie mit Rongeure wird das Kleinhirn visualisiert. Sorgfältige Absaugung der kleinen Teil des Kleinhirns zeigt Visualisierung des CN, die dann injiziert werden kann (Abbildung 1). <p cl...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Direkte Visualisierung der Murine Rücken Cochlear Nucleus für optogenetische Stimulation der Hörbahn

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

Research

JoVE Journal

Neuroscience

9.1K Aufrufe.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

Serie: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Mit dem Theta-Burst-Protokoll zu Mechasnism der Plastizität in Individuen sich mit Fragile X-Syndrom und Autismus

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Forschung

Neurowissenschaften

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 ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

Electrode Positionierung und Montage in Transkranielle Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualisierung von Propriozeptoren in<em&gt; Drosophila</em&gt; Larven und Puppen

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

LWL-Implantation für chronische optogenetische Stimulation des Hirngewebes

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetische Stimulation von Escape Verhalten in<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetische Stimulation des Nager Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Optogenetische Stimulation des Hörnervs

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Die Analyse der Genexpression Veränderungen im Hippocampus Ratte nach Tiefenhirnstimulation des Anterior Thalamuskern

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In - vitro - Modellierung von Cancerous Neural Invasion: The Dorsalwurzelganglion Modell

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...