Die Autoren möchten Neurotar AG für technische Hilfe, CIMO Stiftung und FGSN für die finanzielle Unterstützung bedanken.

Verfahren, die tierischen Patienten haben von der Nationalen Tierexperimentierboard, Finnland zugelassen. 1. Vorbereitung der Tiere für Imaging <ol><li> Betäuben eine Maus durch intraperitional (IP) Injektion von Ketamin (0,08 mg pro Körpergewicht) und Xylazin (0,01 mg pro Körpergewicht). Überprüfen Sie die Narkose mit dem hinteren Fuß Prise Reflex. </li><li> Tauchen Augen Tieres in Augentropfen (Viscotears), um die Augen vor dem Austrocknen zu schützen. </li><li> Sie mit der Maus auf einem Heizkissen (Supertech) bei 37 ° C Unterkühlung zu verhindern. </li><li> Reinigen Sie die für die Bildgebung mit 70% Ethanol bezeichnet Fuß-Pad. </li><li> Fügen Sie einen Tropfen Wasser auf der Haut des Fuß-Pad für den Tauch Kopplung zwischen Haut und Deckglas. </li><li> Setzen Kunststoffverpackungsmaterial unter der Hinterpfote, die unter einem Metallring mit Abdeckung Klasse positioniert ist. </li><li> Stellen Sie die Dicke der Kunststoffverpackungsmaterial, um die Haut zu glätten. </li><li> Geben Sie einen Tropfen Wasser auf den Deckglass für das Eintauchen zwischen Deckglas und dem Ziel. </li></ol> 2. Metall Fixateur Ring Vorbereitung <ol><li> Für die Stabilisierung der Haut nutzen die Metall Fixateur. Wir verwenden das Gemeinschaftsgeschmacksmuster geschützt zweiflügelige Fixateur mit einem Metallring (mit freundlicher Genehmigung von Neurotar Ltd, Finnland). Füllen Sie die Spritze mit Sekundenkleber, legte die kleinen Tropfen Sekundenkleber durch die Nadel auf dem Ring und sanft verteilen Sie den Kleber für gleichmäßige Abdeckung der Ringfläche. Es ist wichtig, die minimale Menge von Klebstoff, der nur für eine gleichmäßige Abdeckung benötigt wird gesetzt, da eine zu hohe Menge an Klebstoff kann das Feld der Ansicht zu verkleinern und behindert nachfolgende Bilderzeugung. HINWEIS: Alternativ kann die Kombination aus einem Metallstab (von unten) und Standard-Mikroskopobjektträger aus Glas (als Abdeckung) verwendet werden, um die Haut zu glätten und um eine ordnungsgemäße optische Ankopplung der Baugruppe 14 zu gewährleisten. Zwei Büroklammern verwendet werden, um die Pfote zwischen Glas und Metallstange Oberfläche zu fixieren (Abbildung 4 </sTrong&gt;). </li><li> Bedecken Sie den Ring mit Mikroskop Deckglas (5 mm Durchmesser, Elektronenmikroskopie Science). </li><li> Schrauben Sie den Ring Fixateur an der starren Metallstange, die auf dem motorisierten Mikroskoptisch montaged wird. </li></ol> 3. Bildgebungsverfahren <ol><li> Im Falle der Verwendung THY1-YFP-H-Mäuse, wählen Sie den blauen Teil des Fluoreszenzlampe Spektren Nervenfasern sichtbar zu machen. Finden Sie den Nerv der Interesse an Epi-Fluoreszenz-Modus und konzentrieren sich auf sie. </li><li> Schreiben Sie die Koordinaten eines Punktes für Längs Bildgebung. Verwenden Sie einen Handwurzelunterlage als Bezugspunkt. Während der folgenden Imaging-Sitzungen, erkennen die ersten Handwurzel Pad und dann finden die gleichen stromaufwärts großen Nervenfasern und gehen Sie zurück über die gespeicherten Koordinaten. Es ist wesentlich, um die Fußauflage in der gleichen Richtung senkrecht zu der Metallstange zu positionieren, um dasselbe Bezugssystem zu halten. </li><li> Einschalten des Zwei-Photonen-Modus. Für die Thy1-YFP-H Mäusen ist es geeignet, die 950 nm-Wellenlänge von Laserstrahlung zu verwenden, um die Nervenfasern zu visualisieren. </li><li> Wählen Laserwellenlänge und Emissions Kanäle (450-480 nm für die zweite Generation Erkennung harmonisch, 520-550 nm für die Bildgebung von YFP-markierten Nerven und 580-630 nm zur Detektion von YFP Fluoreszenz und Autofluoreszenz der Haare), mit zu beginnen niedrige Laserleistung (3-5%) auf Lichtschäden und Bleich verhindern. Die typische Spannung an den Photovervielfacherröhren ist 600-700 V für diese Art der Bilderzeugung. </li><li> Stellen Sie die gewünschte Auflösung (512 x 512 oder 640 x 640 für schnelle Messungen Veranstaltungen, 800 x 800 oder 1.024 x 1.024 für die morphologische Bildgebung), axiale Stufe (1-3 Mikrometer), Dicke der Probe und die Zeitintervalle (1-10 min). Die Belichtungszeit ist in der Größenordnung von 1 ms pro Pixel. </li><li> Erste markieren die oberen und unteren Grenzen des Abbildungsvolumens in der Software. </li><li> Erwerben die Referenzbildstapel für die Referenz vor der Läsion. Bei Bedarf ist es möglich, den Ausgangswert während einige Minuten aufzeichnen. </li><li&gt; Mehrmals (mindestens alle 10-15 min) überprüfen Tier Atemfrequenz und Reflexe, bei Bedarf anzuwenden einen zusätzlichen Betrag von Anästhetika. </li><li> Nach der Bild sauber das Pad mit einem Taschentuch und legte die Maus in einem Rückgewinnungskasten bei 36 ° C, bis sie ganz wach. HINWEIS: In der Regel wird die Dauer eines Bildgebungssitzung in Verbindung mit Laser-induzierte Läsion (siehe unten) nicht 1 h überschreiten. Wir empfehlen, zu überprüfen, ob das Tier tief narkotisiert alle 10-15 min; Dies sollte nicht mit der Abbildung selbst stören, sollte aber beachtet werden, wenn experimentelle Verfahren ist geplant werden. Die Dauer der Wirkung einer Einzel Ketamin / xylazyne Dosis ist etwa 30 bis 40 min, so empfehlen wir, gelten zusätzliche ¼ bis ½ Dosis nach 25-30 min. HINWEIS: Es sollte auch berücksichtigt werden, wenn die Experimente plant, dass die Frequenz des Bilderzeugungseinheiten für ein bestimmtes Tier zu 1 Sitzung für 2 Tage nicht überschreiten, um nachteilige Wirkungen zu vermeiden Wiederbewerbs Anästhesie. </li></ol> 4. Laser Läsion <ol><li> Nach dem Erwerb der Referenzbildstapel, verwenden Sie das Bleich Protokoll, um eine Mikro Läsion zu machen. </li><li> Erhöhung der Laserleistung auf 100%. </li><li> Erhöhen Sie die Belichtungszeit auf 100-1000 ms pro Region von Interesse (100-1,000x mehr als bei Standard-Imaging). </li><li> Skizzieren Sie die Fläche für die Läsion. </li><li> Machen die Läsion durch Einschalten der Bleiche. </li><li> Wechseln Sie wieder zu den regulären Bildgebungsmodus und fahren Sie mit Zeitraffer-Aufnahmen. </li></ol> 5. Datenverarbeitung und Analyse <ol><li> Fahren Sie mit Entmischung mit Entmischung in ImageJ Plugin 8 (Joachim Walter, <a href="http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html">http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html</a> ). </li><li> Öffnen Sie das Bild Stapel aufgeteilt und die Kanäle. </li><li> Finden Sie den Bereich frei von den Nerven oder Haare für die Hintergrundmessungen. Setzen Sie ein Region of Interesse in diesem Bereich. </li><li> Skizzieren sorgfältig die Teil der Haare, wo sie sich deutlich von den Nerven ist. </li><li> Skizzieren Sie die Nerven in dem Teil des Bildes, wo es getrennt von der Haar ist. </li><li> Sparen Mischmatrix. </li><li> Gelten gemessenen Mischmatrix für das gesamte Stack. </li><li> Speichern Sie die neuen Bildstapel verarbeitet in TIF-Format *. </li></ol>

<ol>
	<li>Lumpkin, E. A., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sensory+transduction+in+the+skin.">Mechanisms of sensory transduction in the skin.</a> Nature. 445, 858-865 (2007).</li><li>Kennedy, W. R., Wendelschafer-Crabb, G., Johnson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+epidermal+nerves+in+diabetic+neuropathy.">Quantitation of epidermal nerves in diabetic neuropathy.</a> Neurology. 47, 1042-1048 (1996).</li><li>Wu, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morton's+interdigital+neuroma:+a+clinical+review+of+its+etiology,+treatment,+and+results.">Morton's interdigital neuroma: a clinical review of its etiology, treatment, and results.</a> J. Foot Ankle Surg. 35, 112-119 (1996).</li><li>Lauria, G., Lombardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy:+a+new+tool+for+diagnosing+peripheral+neuropathy.">Skin biopsy: a new tool for diagnosing peripheral neuropathy.</a> BMJ. 334, 1159-1162 (2007).</li><li>Cheng, C., Guo, G. F., Martinez, J. A., Singh, V., Zochodne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+plasticity+of+axons+within+a+cutaneous+milieu.">Dynamic plasticity of axons within a cutaneous milieu.</a> J. Neurosci. 30, 14735-14744 (2010).</li><li>Wang, B., Zinselmeyer, B. H., McDole, J. R., Gieselman, P. A., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Imaging+of+Leukocyte+Homing+and+Migration+in+vivo.">Non-invasive Imaging of Leukocyte Homing and Migration in vivo.</a> J Vis Exp. (46), e2062 (2010).</li><li>Sacconi, L., O'Connor, R. P., Jasaitis, A., Masi, A., Buffelli, M., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multiphoton+nanosurgery+of+cortical+neurons.">In vivo multiphoton nanosurgery of cortical neurons.</a> J Biomed Opt. 12, 050502 (2007).</li><li>Robinson, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+injury+to+peripheral+nerves.">Traumatic injury to peripheral nerves.</a> Muscle Nerve. 23, 863-873 (2000).</li><li>Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M., Nerbonne, J. M., Lichtman, J. W., Sanes, J. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+transfer+into+the+dorsal+root+ganglion+of+adult+rats.">Lentiviral gene transfer into the dorsal root ganglion of adult rats.</a> Mol Pain. 7, 63 (2011).</li><li>Yuryev, M., Khiroug, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+longitudinal+investigation+of+individual+nerve+endings+in+the+skin+of+anesthetized+mice+using+in+vivo+two-photon+microscopy.">Dynamic longitudinal investigation of individual nerve endings in the skin of anesthetized mice using in vivo two-photon microscopy.</a> J Biomed Opt. 17, 046007 (2012).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

In diesem Video zeigen wir die Protokoll-Methode zur nicht-invasiven Längs Zwei-Photonen-Imaging von einzelnen Nervenenden. Die Dynamik der Haut Innervationen in Krankheiten wie Psoriasis und periphere Neuropathie 2, und in traumatischen Verletzungen 9 betroffen. Zwei-Photonen-Bildgebung ermöglicht eine detaillierte Untersuchung der Nervenfasern Strukturen Kollagenmatrix. Die Verwendung von transgenen Mäusen Reporter hilft, die Probleme der Färbung der Nervenfasern ...

A correction was made to <a href="/video/51045">In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin</a>.
Leonard Khiroug was removed as an author.

Erratum: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Kutanen Nervenenden unterziehen dynamischen Veränderungen unter verschiedenen pathophysiologischen Zuständen. Nervenfasern können durch den Prozess der Degeneration und Regeneration oder Umstrukturierung im Zuge von Krankheiten wie periphere Neuropathie 2 oder Morton-Neurom 3 gehen. Nach einer traumatischen Verletzung, ein wichtiger Bestandteil der Nervenenden in der Haut ist Dynamik reinnervation des beschädigten Bereichs. , Ist der gemeinsame Ansatz zur Untersuchung von Nervenenden jedoch ex vivo histologische Schnitte, die Echtzeit-Informationen über die laufenden Prozesse 4 fehlt. Verwendung genetisch kodierte Fluoreszenzmarker, ist es möglich, die Nervenenden in der Haut von lebenden Tieren zu verfolgen, wodurch man reich und deutlich mehr relevante Informationen über die strukturellen Veränderungen. Die Untersuchung der kutanen Nervenenden ist möglich mit herkömmlichen Fluoreszenzmikroskopie jedoch die starke Lichtstreuung des Hautgewebes stark untergräbt die Qualitätder Daten erworben 5. Multiphotonenmikroskopie ermöglicht die Erfassung von Bildern mit hoher Auflösung in den stark streuenden Gewebe durch die nicht-lineare Summierung der Energie der Anregungslichtphotonen was zur Emission von Fluoreszenz nur von dem Brennpunkt des Objektivs. Dieser Effekt führt zu einer robusten Zunahme der Eindringtiefe und die Verbesserung des Signal-zu-Rausch-Verhältnis bei der Messung der Hautgewebe 6. Unter Verwendung des gleichen Lasers, wie für die Bildgebung, ist es möglich, eine selektive Zerlegung der Nervenfasern 7 herzustellen. In dem folgenden Protokoll zeigen wir die Methode der Längs Imaging von Hautnervenenden in vivo in transgenen Mäusen Reporter kombiniert mit selektiven Laser-Läsion mit kommerziell erhältlichen Multiphotonen-Mikroskop-System.

<table border="0" cellpadding="0" cellspacing="0" width="1231">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
			<td style="width:119px;"></td>
			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td>Could be purchaized from general hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon microscopy of single nerve endings in skin

Nervenenden in der Haut sind in physiologischen Prozessen wie Abfühlen 1 als auch in pathologischen Prozessen, wie neuropathischem Schmerz 2 beteiligt. Ihre enge-Boden-Positionierung erleichtert mikroskopischen Abbildung von Hautnervenenden in lebenden intakten Tier. Mit Multiphotonen-Mikroskopie ist es möglich, feine Bilder Überwindung des Problems der starken Lichtstreuung des Hautgewebes zu erhalten. Reporter transgenen Mäusen, die EYFP unter der Kontrolle der Thy-1-Promotor in Neuronen (einschließlich Peripherie sensorischen Neuronen) exprimieren, sind für die Längsschnittstudien von einzelnen Nervenenden über längere Zeit gut geeignet bis zu mehreren Monaten oder sogar lebenslang. Außerdem wurde unter Verwendung der gleichen Femtosekundenlaser für die Bildverarbeitung, ist es möglich, hochselektive Läsionen der Nervenfasern für die Studien der Nervenfaser Umstrukturierung erzeugen. Hier, in-vivo-Bildgebung stellen wir ein einfaches und zuverlässiges Protokoll für Mehrphotonen und Längslaserbasierte Mikrochirurgie auf der Haut von Mäusen Nervenenden.

Mit dem beschriebenen Verfahren ist es möglich, dieselbe Faser nach der Läsion zu verfolgen und um den Abbau der beschädigten Nervenenden (1) zu untersuchen. Erwerb der Stapel mit der Dicke von 120-150 um ist in der Regel die richtige für die wiederholte Bildgebung während mehreren Tagen um die ganze Faser in das Blickfeld zu halten. Die Läsion kann in der Regel kurz erzeugt werden, wenn das Kunststoffverpackungsmaterial, eingestellt wird, um die Haut zu glätten, so d...

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Das Protokoll für die dynamische Längs Bildgebung und Selective Laser Läsion des Nervenenden in Reporter-transgenen Mäusen wird vorgestellt.

In-vivo Zwei-Photonen-Mikroskopie einzelner Nervenenden in der Haut

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

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Research

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Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

10.7K Aufrufe.  University of Helsinki.  Das Protokoll für die dynamische Längs Bildgebung und Selective Laser Läsion des Nervenenden in Reporter-transgenen Mäusen wird vorgestellt. 

Serie: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In-vivo-Imaging von der Maus Spinal Cord mit Zwei-Photonen-Mikroskopie

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Forschung

Neurowissenschaften

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 Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Die gleichzeitige Zwei-Photonen<em&gt; In Vivo</em&gt; Imaging der synaptischen Eingänge und Postsynaptische Targets in der Maus retrosplenialen Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Die Quantifizierung der zerebrale vaskuläre Architektur unter Verwendung von Zwei-Photonen-Mikroskopie in einem Mausmodell der HIV-induzierten Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

Optische Überwachung der Lebensnervenendigung Beschriften in Haarfollikel Lanceolate Endigungen der<em&gt; Ex Vivo</em&gt; Maus-Ohr-Haut

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Zwei-Photonen-Kalzium Imaging in neuronalen Dendriten in Hirnschnitten

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

In Vivo Zwei-Photon Imaging der kortikalen Neuronen bei Neugeborenen Mäusen

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

In Vivo dreidimensionale zwei-photonische Mikroskopie zur Untersuchung von durchgeführten Gefäßreaktionen durch lokale ATP-Auswurf mit einer Glas-Mikropipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Visualisierung der Proteinkinase Eine Aktivität bei kopffixierten Behaving Mäusen mit In Vivo Zweiphotonen fluoreszenz Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...