Diese Studien wurden durch Zuschüsse vom MEXT, JSPS KAKENHI Zuschuss Zahlen JP25290009, JP25650120, JP17K07494 und JP17H05984 finanziert.

<ol>
	<li>Feierstein, C. E., Portugues, R., Orger, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+whole+picture:+A+comprehensive+imaging+approach+to+functional+mapping+of+circuits+in+behaving+zebrafish.">Seeing the whole picture: A comprehensive imaging approach to functional mapping of circuits in behaving zebrafish.</a> Neuroscience. 296, 26-38 (2015).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19 (2), 137-141 (2001).</li><li>Muto, 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=Genetic+visualization+with+an+improved+GCaMP+calcium+indicator+reveals+spatiotemporal+activation+of+the+spinal+motor+neurons+in+zebrafish.">Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish.</a> Proc Natl Acad Sci U S A. 108 (13), 5425-5430 (2011).</li><li>Lorent, K., Liu, K. S., Fetcho, J. R., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+space+cadet+gene+controls+axonal+pathfinding+of+neurons+that+modulate+fast+turning+movements.">The zebrafish space cadet gene controls axonal pathfinding of neurons that modulate fast turning movements.</a> Development. 128 (11), 2131-2142 (2001).</li><li>Asakawa, 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=Genetic+dissection+of+neural+circuits+by+Tol2+transposon-mediated+Gal4+gene+and+enhancer+trapping+in+zebrafish.">Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.</a> Proc Natl Acad Sci U S A. 105 (4), 1255-1260 (2008).</li><li>Sternberg, J. R., 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=Optimization+of+a+Neurotoxin+to+Investigate+the+Contribution+of+Excitatory+Interneurons+to+Speed+Modulation+In+Vivo.">Optimization of a Neurotoxin to Investigate the Contribution of Excitatory Interneurons to Speed Modulation In Vivo.</a> Curr Biol. , (2016).</li><li>Arrenberg, A. B., Del Bene, F., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+zebrafish+behavior+with+halorhodopsin.">Optical control of zebrafish behavior with halorhodopsin.</a> Proc Natl Acad Sci U S A. 106 (42), 17968-17973 (2009).</li><li>Orger, M. B., Kampff, A. R., Severi, K. E., Bollmann, J. H., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+visually+guided+behavior+by+distinct+populations+of+spinal+projection+neurons.">Control of visually guided behavior by distinct populations of spinal projection neurons.</a> Nat Neurosci. 11 (3), 327-333 (2008).</li><li>Huang, K. H., Ahrens, M. B., Dunn, T. W., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+projection+neurons+control+turning+behaviors+in+zebrafish.">Spinal projection neurons control turning behaviors in zebrafish.</a> Curr Biol. 23 (16), 1566-1573 (2013).</li><li>Smear, M. C., 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=Vesicular+glutamate+transport+at+a+central+synapse+limits+the+acuity+of+visual+perception+in+zebrafish.">Vesicular glutamate transport at a central synapse limits the acuity of visual perception in zebrafish.</a> Neuron. 53 (1), 65-77 (2007).</li><li>Bianco, I. H., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+transformations+underlying+hunting+behavior+in+zebrafish.">Visuomotor transformations underlying hunting behavior in zebrafish.</a> Curr Biol. 25 (7), 831-846 (2015).</li><li>Trivedi, C. A., Bollmann, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually+driven+chaining+of+elementary+swim+patterns+into+a+goal-directed+motor+sequence:+a+virtual+reality+study+of+zebrafish+prey+capture.">Visually driven chaining of elementary swim patterns into a goal-directed motor sequence: a virtual reality study of zebrafish prey capture.</a> Front Neural Circuits. 7, 86 (2013).</li><li>Jouary, A., Haudrechy, M., Candelier, R., Sumbre, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+2D+virtual+reality+system+for+visual+goal-driven+navigation+in+zebrafish+larvae.">A 2D virtual reality system for visual goal-driven navigation in zebrafish larvae.</a> Sci Rep. 6, 34015 (2016).</li><li>Muto, A., Ohkura, M., Abe, G., Nakai, J., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+visualization+of+neuronal+activity+during+perception.">Real-time visualization of neuronal activity during perception.</a> Curr Biol. 23 (4), 307-311 (2013).</li><li>Del Bene, F., 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=Filtering+of+visual+information+in+the+tectum+by+an+identified+neural+circuit.">Filtering of visual information in the tectum by an identified neural circuit.</a> Science. 330 (6004), 669-673 (2010).</li><li>Semmelhack, J. L., 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=A+dedicated+visual+pathway+for+prey+detection+in+larval+zebrafish.">A dedicated visual pathway for prey detection in larval zebrafish.</a> Elife. 3, 04878 (2014).</li><li>Preuss, S. J., Trivedi, C. A., vom Berg-Maurer, C. M., Ryu, S., Bollmann, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+object+size+in+retinotectal+microcircuits.">Classification of object size in retinotectal microcircuits.</a> Curr Biol. 24 (20), 2376-2385 (2014).</li><li>Romano, S. 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=Spontaneous+Neuronal+Network+Dynamics+Reveal+Circuit's+Functional+Adaptations+for+Behavior.">Spontaneous Neuronal Network Dynamics Reveal Circuit's Functional Adaptations for Behavior.</a> Neuron. 85 (5), 1070-1085 (2015).</li><li>Barker, A. J., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+decision+making+in+the+zebrafish+tectum.">Sensorimotor decision making in the zebrafish tectum.</a> Curr Biol. 25 (21), 2804-2814 (2015).</li><li>Ewert, J. -. 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Fiji Available from: <a target="_blank" href="https://fiji.sc">https://fiji.sc</a> (2017)</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7 (1), 27-41 (1998).</li><li>Mueller, T., Wullimann, M. 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Die Autoren haben keine Interessenkonflikte zu berichten.

Obwohl der zwei-Photonen-Laser eine hervorragende räumliche Auflösung hat, speziell Abtragen einzelne Neuronen, sollte große Vorsicht genommen werden, um unerwünschte Beschädigungen auf das Hirngewebe durch Hitze zu vermeiden. Der wichtigste Schritt bei der Ablation Experiment soll die optimale Höhe der Laserbestrahlung bestimmen. Unzureichende Bestrahlung schlägt fehl, die Neuronen zu töten. Zu viel Bestrahlung Hitzeschäden wird das umliegende Gewebe, was zu unerwünschten Effekten führt. Optimale Bereich der ...

Um zu verstehen wie Verhalten von neuronaler Aktivität im Gehirn entsteht, ist es notwendig, die neuronalen Schaltkreise zu identifizieren, die bei der Erzeugung von diesem Verhalten beteiligt sind. Das Larvenstadium der Zebrabärbling bietet ideale Tiermodell für die Funktion des Gehirns zu studieren mit dem Verhalten verbunden, weil ihre kleine, durchsichtige Gehirne neuronalen Aktivität mit einer zellulären Auflösung in einem breiten Bereich der untersuchen ermöglichen die Gehirn beobachtend Verhalten1. Bildgebung neuronaler Aktivität in bestimmten Nervenzellen ist möglich durch die Erfindung der genetisch codierten Kalzium (Ca) Indikatoren (GECIs) wie GCaMP2geworden. GCaMP transgenen Zebrafisch haben sich als nützlich für Verhalten durch die Durchführung von Ca Bildgebung im Verhalten der Tiere3neuronale funktionskreis zuordnen.
Während Ca Bildgebung Korrelationen zwischen neuronaler Aktivität und Verhalten nachweisen kann, um zu zeigen, Kausalität, Unterdrückung der neuronalen Aktivität und testen ihre Consequence(s) auf das Verhalten sind wichtige Schritte. Es gibt verschiedene Möglichkeiten, dies zu erreichen: Nutzen der genetischen Mutation, die bestimmte neuronale Schaltkreise4, Ausdruck von Neurotoxinen in spezifischen Neuronen5,6, Verwendung von optogenetische Tools wie Halorhodopsin7, verändert und Laser-Ablation von gezielten Neuronen8,9. Laser-Ablation ist besonders geeignet für die Beseitigung der Aktivität in eine relativ kleine Anzahl von spezifischen Neuronen. Irreversiblen Beseitigung von neuronaler Aktivität durch das Töten von Neuronen erleichtert die Beurteilung Verhaltensstörungen folgen.
Ein interessantes Verhalten, das auf das Larvenstadium im Zebrafisch beobachtet werden können ist Beutefang (Abb. 1A). Dieses optisch geführte, zielgerichtetes Verhalten bietet eine günstige Experimentiersystem für die Untersuchung der Sehschärfe10, antwortauslösung Transformation11,12,13, visuelle Wahrnehmung und Anerkennung von Objekte14,15,16,17,18, und Entscheidungsfindung19. Wie wird Beute erkannt durch Raubtiere und wie Beute Erkennung führt um zu Beute fangen Verhalten wurde eine zentrale Frage in Neuroethologie20. In diesem Artikel konzentrieren wir uns auf die Rolle der Pretecto Hypothalamus Schaltung von Projektionen aus einem Kern in der pretetto gebildet (Nucleus Pretectalis Superficialis pars Magnocellularis, im folgenden einfach als die pretetto gemerkt), die ILH. Laser-Ablation von der pretetto zeigte sich Beute erfassen Aktivität reduzieren und neuronalen Aktivität in der ILH, die mit der Beute visuelle Wahrnehmung21ist, abzuschaffen. Hier, laser Protokolle für die Durchführung von-Ablation und testen seine Wirkung mit Ca2 + Bildgebung und Verhaltensstörungen Aufnahme im Zebrafisch, die Larven beschrieben werden.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">NuSieve GTG Agarose</td>
			<td style="width:223px;">Lonza</td>
			<td style="width:361px;">Cat.#50080</td>
			<td style="width:383px;">low-melting temperature agarose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6 cm petri dish</td>
			<td>FALCON</td>
			<td>Product#:351007</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dissecting needle</td>
			<td>AS ONE Corporation</td>
			<td>Cat. No. 2-013-01</td>
			<td>https://keystone-lab.com/en/item/detail/404142</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LSM7MP</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>two-photon laser scanning microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">W Plan-Apochromat 63x/1.0</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>63X objective lens</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Imager.Z1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>an epi-fluorescence microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ZEN</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Image acquisition software for confocal microscopes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Secure-Seal Hybridization Chamber Gasket, 8 chambers, 9 mm diameter x 0.8 mm depth</td>
			<td>Molecular Probes</td>
			<td>Catalogue # S-24732</td>
			<td>Used as a recording chamber in Ca imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Imageing Chambers</td>
			<td>Grace Bio-Labs</td>
			<td>CoverWell Imaging Chambers PCI-A-2.5</td>
			<td>Used as a behavioral recording chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">surgical knife</td>
			<td>MANI</td>
			<td>Ophthalmic knife MST15</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ORCA-Flash4.0</td>
			<td>Hamamatsu Photonics</td>
			<td>model:C11440-22CU</td>
			<td>a scientific CMOS camera</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;">HCImage</td>
			<td>Hamamatsu Photonics</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">image acuisition software</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Hard Disk Recording module</td>
			<td>Hamamatsu Photonics</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">An software module that enables saving the movie files onto a hard disc drive in a short time</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SZX7</td>
			<td>Olympus</td>
			<td></td>
			<td>stereoscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DF PL 0.5X</td>
			<td>Olympus</td>
			<td></td>
			<td>objective lens for SZX7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Point Grey Grasshopper3 4.1 MP Mono USB3 Visio</td>
			<td>FLIR Systems, Inc.</td>
			<td>Product No. GS3-U3-41C6NIR-C</td>
			<td>CMOS camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XIMEA xiQ camera</td>
			<td>XIMEA</td>
			<td>Product No. MQ042RG-CM</td>
			<td>CMOS camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">a ring LED light</td>
			<td>CCS</td>
			<td>Model: LDR2-100SW2-LA</td>
			<td>White LED</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;"></td>
			<td style="width:223px;"></td>
			<td style="width:361px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nylon mesh 32&#181;m</td>
			<td>Tokyo Screen</td>
			<td>N-No.380T</td>
			<td>http://www.tokyo-screen.com/cms/sta20347/</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Nylon mesh 13&#181;m</td>
			<td>Tokyo Screen</td>
			<td>N-No. 508T-K</td>
			<td style="width:383px;">http://www.tokyo-screen.com/cms/sta20347/</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:316px;">Metal seive 150 micron aperture</td>
			<td>Tokyo Screen</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">http://www.tokyo-screen.com/cms/sta20341/#ami</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">Metal seive 75 micron aperture</td>
			<td>Tokyo Screen</td>
			<td></td>
			<td>http://www.tokyo-screen.com/cms/sta20341/#ami</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">EBIOS</td>
			<td>Asahi Food &#38; Healthcare, Co. Ltd.</td>
			<td></td>
			<td>dry beer yeast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">LabVIEW</td>
			<td>National Instruments</td>
			<td></td>
			<td>an integrated development environment for programming</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;">Mai-Tai HP</td>
			<td>Spectra Physics&#160;</td>
			<td></td>
			<td>two-photon laser&#160;</td>
		</tr>
	</tbody>
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ablation of a neuronal population using a two-photon laser and its assessment using calcium imaging and behavioral recording in zebrafish larvae

Um die Rolle einer Subpopulation von Neuronen im Verhalten zu erkennen, ist es wichtig, die Folgen der Blockierung der Aktivität in lebenden Tieren zu testen. Laser-Ablation von Neuronen ist eine wirksame Methode für diesen Zweck, wenn Neuronen mit fluoreszierenden Sonden selektiv beschriftet sind. In der vorliegenden Studie werden Protokolle für Laser Abtrag einer Subpopulation von Neuronen mit ein zwei-Photonen-Mikroskop und Testen ihrer funktionalen und Verhaltensstörungen Folgen beschrieben. In dieser Studie wird Beute Erfassung Verhalten im zebrafischlarven als ein Studienmodell verwendet. Die Pretecto-Hypothalamus Schaltung ist bekannt, dieses optisch getriebene Beute fangen Verhalten zugrunde liegen. Zebrafisch pretetto wurden Laser abgetragen und neuronaler Aktivität in der minderwertigen Lappen des Hypothalamus (ILH; das Ziel der pretectal Projektion) wurde untersucht. Beute Erfassung Verhalten nach pretectal Ablation wurde ebenfalls getestet.

Spezifische Neuronen wurden genetisch beschriftet mit EGFP oder GCaMP6s, deren Ausdruck in Gal4 Linien Gefahren wurden. Eine Gal4 Linie gSAIzGFFM119B wurde verwendet, um einen Kern im pretectal Bereich (magnozelluläre oberflächlichen pretectal Zellkern) und einer Subpopulation der Riechkolben Neuronen zu beschriften. Eine weitere Gal4-Linie, hspGFFDMC76A, wurde verwendet, um die ILH beschriften. Wir Laser-abgetragen pretectal Neuronen bilateral (Abbildung 2...

Watch this Scientific Journal Video about Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae at JoVE.com

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

Hier präsentieren wir ein Protokoll, um eine genetisch beschrifteten Subpopulation von Neuronen durch einen zwei-Photonen-Laser von Zebrafisch-Larven abtragen.

Ablation von neuronalen Einwohner mit einem zwei-Photonen-Laser und ihre Bewertung mit Kalzium Imaging und Verhaltensstörungen Aufnahme in Zebrafischlarven

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

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JoVE Journal

Neuroscience

8.7K Aufrufe.  SOKENDAI (The Graduate University for Advanced Studies). Hier präsentieren wir ein Protokoll, um eine genetisch beschrifteten Subpopulation von Neuronen durch einen zwei-Photonen-Laser von Zebrafisch-Larven abtragen.

Serie: Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

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

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

Automatisierte Hochdurchsatz-Analysen in Behavioral Zebrafischlarven

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

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 ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Zwei-Photonen-<em&gt; In vivo</em&gt; Imaging von dendritischen Dornen in der Maus Cortex Mit einem ausgedünnten Schädel Vorbereitung

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Längerer Inkubation von Akute neuronales Gewebe für Elektrophysiologie und Calcium-Bildgebung

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

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+ ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Studium der Funktion des Gehirns bei Kindern mit Magnetoenzephalographie

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupille Zwei-Photonen-In-vivo-Bildgebung der Netzhaut der Maus

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Maus Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

Ablation von neuronalen Einwohner mit einem zwei-Photonen-Laser und ihre Bewertung mit Kalzium Imaging und Verhaltensstörungen Aufnahme in Zebrafischlarven

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