Diese Arbeit wurde vom SFB 604, DFG MO 1421/2-1 und Krebshilfe 107.089 (HM) unterstützt. AS ist Empfänger eines Young Investigator Award von der Kinder Tumor Foundation (New York, USA).

Die vorliegende Studie wurde nach dem Tierschutzgesetz der Bundesrepublik Deutschland (Tierschutzgesetz der Bundesrepublik Deutschland) durchgeführt und wurde durch das Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz (Thüringer Landesamt für Verbraucherschutz und Lebensmittelsicherheit) zugelassen. 1. Einrichten der Messungen <ol><li> Betäuben die Mäuse durch Isofluran / O 2-Inhalations - zur Einleitung der Anästhesie 3%, 2% für die Wartung Isofluran in 100% Sauerstoff (Abbildung 1). Bestätigen ausreichende Anästhesie durch Testen einfache Reflexe wie Bewegung Reflexe und Prüfung von Sensibilität für Low-grade-Schmerzen. Verwendung der Salbe auf die Augen zu Trockenheit während der Narkose zu vermeiden wird empfohlen, aber nicht unbedingt notwendig, weil das Verfahren dauert in der Regel nur 30 min / Tier insgesamt. Im Falle der Überlebensversuche, verwalten rechtzeitige länger wirkenden Analgetika für die Verwaltung von postoperativen Schmerzen. </li><li> Rasieren Sie das Fell mit einem elektrischen Rasierer Abdeckung der hinteren Gliedmaßen und durchzuführen, Enthaarung mit einem handelsüblichen Haarentfernungscreme während Tiere sind bereits im Analgesie. Um pathogen-freien Status während des gesamten Verfahrens zu erhalten, tragen aseptischen Handschuhe und Instrumente immer sorgfältig mit 70% Ethanol gereinigt. </li><li> Kontrolle der Körpertemperatur Stabilität durch eine Rückkopplung gesteuert Heizplatte und rektale Thermosonde (Abbildung 2). Falls erforderlich, verwenden Sie bitte ein steriles Tuch, um die Wärmeplatte zwischen den Tieren, um eine sterile experimentellen Umgebung halten zu decken. Darüber hinaus ist es empfehlenswert, eine Heizplatte, die elektronisch über einen integrierten Sensor gesteuert wird, die Erwärmungstemperatur, um Gewebeschäden zu vermeiden ≤ 40 ° C zu begrenzen verwenden. <img alt="Figur 1" fo:content-width="4in" fo:src="/files/ftp_upload/51181/51181fig1highres.jpg" src="/files/ftp_upload/51181/51181fig1.jpg" /> Abbildung 1. Experimental Setup, das ein narkotisierten Maus mit rasierten Hinterbeine. </li><li> Nehmen Elektrokardiographie (EKG)-Aufnahmen, die Herzfrequenz als Vitalparameter zu überwachen. Legen Sie drei Elektroden für EKG-Aufzeichnungen, wie folgt: eine Elektrode unter die Haut jedes Vorderbein und eine Elektrode unter der Haut im Nackenbereich. </li><li> Kontakt mit Gel für optimale Leitfähigkeit / Übergangswiderstand Platz Ringelektroden. Die Sensorelektrode (schwarz markiert) an der Position, wo der Gastrocnemius-Muskel seinen maximalen Durchmesser angeordnet. Die Referenzelektrode (rot gekennzeichnet) wird direkt unter der Sensorelektrode platziert. Hinweis: Bitte siehe &#39;Werkstofftabelle &quot;für Ausstattungsdetails. </li><li> Markieren Sie die richtigen Messpositionen mit vorgegebenen, aber konsistente Entfernung zwischen dem proximalen und distalen Ischiasnervenstimulation und die Bleielektrode. Experimentelle Vorschlag: Bei einem Abstand von 4 mm von der Sensorelektrode, wird die distale Stimulation statt.In einem Abstand von 16 mm zur Sensorelektrode wird das proximale Stimulation aus (Fig. 2) durchgeführt werden. </li></ol><img alt="Figur 2" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig2highres.jpg" src="/files/ftp_upload/51181/51181fig2.jpg" /> 2. Repräsentatives Bild, das die experimentelle Situation unmittelbar vor Beginn der Messungen. Der weiße Pfeil zeigt die Position des Sensor (schwarz) und Referenz (rot) Elektrode am Gastrocnemius-Muskel des linken Hinterbein. Die Stimulation von Nadelelektroden werden an definierten Positionen in Bezug auf den schwarzen Sensorelektrode durchgeführt werden. Der Punkt, der distal Stimulation (schwarze Markierung mit &quot;DS&quot; an der linken Hinterbein) einen Abstand von 4 mm von der Fühlerelektrode; der Ort der proximalen Stimulation (schwarze Markierung mit &quot;ps&quot;) ist 16 mm entfernt. Die rote Linie auf der rechten Hintergliedmaße zeigt die ca.<html> iMate anatomischen Verlauf des Ischiasnervs. Darüber hinaus werden die groben Positionen der relevanten Hinterbeinmuskeln als Landmarken dargestellt. Das Sternchen zeigt die rektale Temperatursonde. 2. Mess <ol><li> Prinzip: Führen Sie eine Reihe von Nervenstimulationen mit Einzel wiederholt erzeugt Rechteckimpulse von 0,1 ms Dauer von Einweg-28 G monopolare Nadelelektroden (Wiederholungsrate 200 ms, siehe Abbildung 3A). Für Offline-Datenanalyse, empfiehlt es sich, gleichzeitig gemeinsam erwerben Stimulationssignale mit der neuromuskulären Funktion Wirkungs-Kurve (3B) durch Nervenstimulation. Durchschnittlich eine Reihe von Maximalreaktionen (&quot;Vertreter der neuromuskulären Funktion response&quot;) für die anschließende Datenanalyse. Um zuverlässige Daten, aufzeichnen und später durchschnittlich mindestens 3 unabhängige, optimale Reaktionskurven pro Stimulationsstelle und Tier zu produzieren. p_upload/51181/51181fig3highres.jpg &quot;src =&quot; / files/ftp_upload/51181/51181fig3.jpg &quot;/&gt; Abbildung 3. Verfahren für die Datenerfassung und Analyse (schematische Darstellung). Wiederholt erzeugten Impulse werden auf den Ischiasnerv über Nadelelektroden (obere Reihe in Abbildung 3A) aufgebracht. Gleichzeitig werden mehrere entsprechende neuromuskuläre Antwort-Kurven aufgezeichnet (untere Reihe in Abbildung 3A). Wenn gemittelt und vergrößert, neuromuskuläre Reaktionskurven durch Stimulationsimpulse (obere Reihe in 3B) zeigen die folgenden charakteristischen Eigenschaften (untere Reihe in Abbildung 3B): Latenz der Signalantwort sowie Dauer und Amplitude des Signals werden angezeigt und können für nachfolgende Berechnungen und Statistiken erhalten werden. Unregelmäßige Signalleitung und / oder suboptimalen Aufnahmen in der Regel in verschiedenen Signal Verformungen führen mit mehr als nur eine positive und eine negative Ablenkung oder Signal Verformung(Bimodale Form) mit reduzierter Amplitude (3C). </li><li> Führen proximalen Stimulation mit einer Nadelelektrode bei bestimmten Position (&quot;PS&quot; in Fig. 2). <ol><li> Um beste Aufnahmebedingungen mit Maximalamplituden zu erreichen, visualisieren die tatsächlichen Wirkungs-Kurven auf die gleichzeitige Stimulation Prozess. Damit ist der Experimentator in der Lage, die Form der Antwortkurven als auch die Größe der Amplitude sofort zu beurteilen. </li><li> Wenn nötig, leicht und genau die Position und / oder den Winkel des Stimulations-Nadel in Bezug auf den Ischiasnerv zu manipulieren. Diese schonende Optimierung der Stimulationsbedingungen ermöglicht Erreichen konstanter Amplituden mit der größtmöglichen Wert und einem Wirkungs-Kurve von typischen zweiphasigen Form (Abbildung 4). </li></ol></li><li> Führen distalen Stimulation mit einer Nadelelektrode bei bestimmten Position (&quot;ds&quot; in Fig. 2). </li><li> Nach completion der Messung, übertragen Sie die Maus getestet zu einem separaten Käfig, bis er das Bewusstsein wiedererlangt, um ausreichend Brustlage zu halten. Ein Tier nicht unbeaufsichtigt und in der Gesellschaft von anderen Tieren, bis es vollständig von der Narkose erholt. Verwalten Sie rechtzeitig länger wirkenden Analgetika für die Verwaltung von postoperativen Schmerzen. Die systemische Verabreichung von nichtsteroidalen Antirheumatika (NSAR) und Opioide sind für 1-3 Tage empfohlen. </li><li> Alternativ zu opfern, die noch narkotisierten Maus in eine schnelle und humane Art und Weise ohne weitere Schmerzen für das Tier, zB durch Hals-Luxation. </li></ol><img alt="Fig. 4" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig4highres.jpg" src="/files/ftp_upload/51181/51181fig4.jpg" /> Abbildung 4. Illustration, die CMAP Aufnahmen mit Maximalamplituden zu bestimmen. Eine vollständige Anmeldung Serie vorgestellt. (A </strong&gt;) Insertion Punkt mit minimalem CMAP Antwort. (B) Leichte Stimulationsnadel Bewegung führt CMAP Aufnahmen mit maximalen Amplituden. (C) Weitere Änderungen in Nadelplatzierung produzieren CMAP Aufnahmen mit unterschiedlichen Amplituden darunter nahezu maximale Amplitude. (D) Stimulation Nadel-Ersatz mit Serien CMAP Aufnahmen von nahezu maximale Amplitude. Hinweis: Typische Abnahme in CMAP Amplituden während wiederholter Stimulation bei optimalen Stimulationsstelle 12, 13 auf. Sternchen zeigen CMAP Aufnahmen mit für die Mittelung dargestellten Maximalamplituden. 3. Analyse <ol><li> Auszug Nervenleitungsparameter auf der Grundlage der repräsentativen neuromuskuläre Funktion Antwort-Datensatzes unter Verwendung eines geeigneten Software-Paket (zB AtisaPro). Hinweis: Bitte behandeln Sie zeitbezogene Datenermittlung mit besonderer Sorgfalt, da Wendepunkt Bestimmung der Verbindung motorische Aktion Topfrenz (CMAP) Beginn und Beendigung kann schwierig sein. Ein Verfahren, mit prüft Reproduzierbarkeit ist in Fig. 3B, wo die Tangenten an Signalausschläge nach Beginn und vor Beendigung verwendet werden angegeben. </li><li> Berechnen &#39;Signal Latenz &quot;und&quot; CMAP&#39; Werte. <ol><li> Latency &#39;stellt die Zeitverzögerung zwischen Anregung und CMAP Beginn, während der Zeitspanne zwischen CAMP Beginn der erste negative Ausschlag nach Rückkehr zum Ausgangswert wird als initial&#39; CMAP Dauer. Verwenden der Differenz zwischen den distalen und proximalen Latenzen zu Leitungsgeschwindigkeit mit dem Abstand zwischen den distalen und proximalen Stimulationsstellen berechnen. Leitungsgeschwindigkeit = Latenz / Abstand zwischen Stimulationsorte </li><li> CMAP &#39;(Verbindung Motor Aktionspotential) Amplitude stellt die Größe zwischen maximalen positiven und negativen Wendepunkt des CMAP Signal (in mV). CMAP = Wert &quot;positiven Wendepunkt&quot; - negativen Trendwende po-Wert &#39;int &#39; </li></ol></li></ol>

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Die Autoren haben nichts zu offenbaren.

Die beschriebenen Protokoll bietet eine einfache und zuverlässige Methode, um Ischiasnerv Leitungseigenschaften an narkotisierten Mäusen ohne die Notwendigkeit, den Nerv der Zinsen aussetzen zu bestimmen. Dennoch bewirkt diese experimentelle Verfahren Gewebeverletzungen durch Nadelpunktion. Es ist daher eine sinnvolle Option, um die Tiere nach Abschluss der Aufnahmen zu opfern. , Im Vergleich zu anderen invasiven Verfahren, die die Belichtung des Nerven vor Aufnahmen erfordern, ist jedoch Gewebeschäden vergleichsweis...

Elektrophysiologische Messungen sind ein unverzichtbares Werkzeug für die Untersuchung der funktionellen Integrität der peripheren Nerven in beiden klinischen und Laborumgebungen. Beim Menschen wird eine große Anzahl von neuromuskulären Störungen und Neuropathien diagnostisch beruhen auf elektrophysiologischen Messungen. Durch Messen Nerven Eigenschaften wie Leitungsgeschwindigkeit oder mögliche Amplituden des Signals ist es möglich, den ungefähren Ausgangs von peripheren Nervenerkrankungen zu charakterisieren. Die Nervenleitungsgeschwindigkeit ist stark abhängig von der schnellen Signalausbreitung durch Myelinisierung aktiviert. Daher demyelinisierende Prozesse zeigen in der Regel verringert Leitungsgeschwindigkeiten 4. Die Verbindung Motor Aktionspotential (CMAP) - Korrelation mit der Anzahl der Funktions Axone - ist ein Indikator für axonalen Schaden, wenn deutlich reduziert 5. Somit wird mittels der elektrophysiologischen Methoden die Ätiologie von peripheren Nervenschädenunterschieden werden kann, wie zum erblichen Neuropathien 6,7, diabetische Neuropathie 8,9, chronisch inflammatorische demyelinisierende Polyneuropathie (CIDP) 10, 11 oder metabolische Neuropathien. Normalerweise in der Anwendung am Menschen nicht-invasive Aufnahmen auf der Nervus ulnaris oder bevorzugt werden. Bei Mäusen ist es einfach zu Nerven Eigenschaften der Ischiasnerv, der größte Nerv des peripheren Nervensystem (PNS), die sowohl große analysieren - und Kleinkaliber Axone der motorischen und sensorischen Systems. Die wie hier gezeigt Verfahren ist eine schnelle, einfache und zuverlässige Methode, um alle für die Elektrophysiologie an peripheren Nerven in der intakten Maus relevanten Standardwerte zu messen. Durch die Aufnahmen aus einer geschützten Organismus werden physiologischen Bedingungen des Nerven-Umgebung gewährleistet.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Concentric Needle Electrodes (Stimulation)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td style="width:119px;">9013S0901</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital Ring Electrodes (Recording)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td>9013S0302</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ToM - Tower of Measurement (A/D converter)</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AtisaPro, Data acquisition &#38; analysis software</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HSE-Stimulator T</td>
			<td colspan="2">Hugo Sachs Elektronik, Hugstetten, Germany</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurements on mouse sciatic nerves

Elektrophysiologische Untersuchungen ermöglichen eine rationelle Einteilung der verschiedenen neuromuskulären Erkrankungen und der Hilfe, zusammen mit neuropathologischen Techniken, im Verständnis der Pathophysiologie ein. Hier beschreiben wir eine Methode, um elektrophysiologische Untersuchungen an Maus Ischiasnerven in vivo durchzuführen. Die Tiere werden mit Isofluran, um Analgesie für die getesteten Mäuse gewährleisten betäubt und ungestört Arbeitsumgebung während der Messungen, die ca. 30 min / Tier zu nehmen. Eine konstante Körpertemperatur von 37 ° C wird durch eine Heizplatte gehalten und kontinuierlich durch eine rektale Thermosonde 2 gemessen. Zusätzlich wird ein Elektrokardiogramm (ECG) routinemßig während der Messungen, um aufgezeichnet, um den physiologischen Zustand der untersuchten Tiere zu überwachen. Elektrophysiologische Aufzeichnungen werden auf den Ischiasnerv, der größten Nerv-ten durchgeführtE peripheren Nervensystem (PNS), die Versorgung der Hintergliedmaße Maus sowohl mit motorischen und sensorischen Nervenbahnen. In unserem Protokoll, bleiben Ischiasnerven in situ und müssen daher nicht extrahiert oder ausgesetzt werden, so dass Messungen ohne negative Nervenreizungen zusammen mit aktuellen Aufnahmen. Mit entsprechenden Nadelelektroden 3 führen wir sowohl die proximalen und distalen Nervenstimulationen, die Registrierung der Potentiale übertragen mit Messelektroden an Wadenmuskulatur. Nach der Datenverarbeitung, zuverlässig und sehr konsistent Werte für die Nervenleitgeschwindigkeit (NLG) und der Verbindung Motor Aktionspotential (CMAP), die Schlüsselparameter für die Quantifizierung des Brutto peripheren Nervenfunktionsfähigkeit, erreicht werden kann.

<html> Wir haben eine Reihe von in-vivo-elektrophysiologische Messungen an Ischiasnerven von 12 Mäusen in dieser Studie insgesamt: 6 Tieren jedes Geschlechts. Die Messungen wurden mit der dargestellten Protokoll durchgeführt und lieferte die folgenden Ergebnisse: Sowohl männliche als auch weibliche Mäuse zeigen eine mittlere Ischiasnervenleitungsgeschwindigkeit von ca. 20 m / s (Abbildung 5). Dies steht im Einklang mit anderen Messungen in der Literatur. Darüber...

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves at JoVE.com

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Messungen der Nervenleitungseigenschaften in vivo beispielhaft ein leistungsfähiges Werkzeug, um verschiedene Tiermodellen für neuromuskuläre Erkrankungen zu charakterisieren. Hier präsentieren wir eine einfache und zuverlässige Protokoll, mit dem elektrophysiologische Analysen über Ischiasnerven von betäubten Mäusen durchgeführt werden.

In-vivo-Elektrophysiologische Messungen an Maus Ischiasnerven

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

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In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

33.0K Aufrufe.  Fritz Lipmann Institute.  Messungen der Nervenleitungseigenschaften in vivo beispielhaft ein leistungsfähiges Werkzeug, um verschiedene Tiermodellen für neuromuskuläre Erkrankungen zu charakterisieren. Hier präsentieren wir eine einfache und zuverlässige Protokoll, mit dem elektrophysiologische Analysen über Ischiasnerven von betäubten Mäusen durchgeführt werden. 

Serie: In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Elektrophysiologische Messungen von einer Moth Riechsystem

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Forschung

Neurowissenschaften

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Die Transfektion von Maus retinalen Ganglienzellen durch In vivo Elektroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Elektrophysiologische Messungen und Analyse von Nozizeption in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Funktionelle Bildgebung mit Ultraschall-Blut-Hirnschranke Störung und Mangan-MRT

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo Zwei-Photonen-Imaging Of Experience-abhängigen molekularen Veränderungen in kortikalen Neuronen

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In vivo - Gentransfer auf Schwann - Zellen in der Nagerischiasnerv durch Elektroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Analyse von Immunzellen in einzelnen Ischias Nerven und Dorsal Root Ganglion aus einem einzigen Mausklick mit Durchflusszytometrie

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

In Vivo Elektrophysiologische Messungen der zusammengesetzten Muskel Aktionspotential aus die Vorderbeine in Maus-Modellen der Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

In Vivo Calcium Imaging der Seitenlinie Haarzellen im Larvenstadium Zebrafisch

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...