Diese Arbeit wurde durch ein Grant-Nummer VIDI H64.09.016, der niederländischen Organisation für wissenschaftliche Forschung (NWO) zu CPF finanziert. CPF dankt Dr. Silvina A. Fratantoni für ihre kritischen Anmerkungen und Korrekturen in der letzten Manuskripts. GMRDL / Emmm werden von der niederländischen Technologiestiftung STW (Projekt 12151 und 11350), die Teil der NWO ist, und die teilweise durch das Wirtschaftsministerium gefördert unterstützt. Wir danken der Catherine Kitts und Peter Drent von Nikon Instruments Europe BV für Hilfe und Unterstützung. HX wurde von der Königlichen Niederländischen Akademie der Künste und Wissenschaften (Zuschuss 11CDP10) und WT unterstützt wurde durch Erteilung der Niederländischen Organisation für Wissenschaftliche Forschung (Förder 820.02.006) unterstützt.

<ol>
	<li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J Microsc. 198, 82-87 (2000).</li><li>Yoshihara, Y., De Roo, M., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+formation+and+stabilization.">Dendritic spine formation and stabilization.</a> Curr Opin Neurobiol. 19, 146-153 (2009).</li><li>Dailey, M. E., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+dendritic+structure+in+developing+hippocampal+slices.">The dynamics of dendritic structure in developing hippocampal slices.</a> J Neurosci. 16, 2983-2994 (1996).</li><li>Alvarez, V. A., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+physiological+plasticity+of+dendritic+spines.">Anatomical and physiological plasticity of dendritic spines.</a> Annu Rev Neurosci. 30, 79-97 (2007).</li><li>Jaworski, J., 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=Dynamic+microtubules+regulate+dendritic+spine+morphology+and+synaptic+plasticity.">Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity.</a> Neuron. 61, 85-100 (2009).</li><li>Abbe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beitr&#228;ge+zur+Theorie+des+Mikroskops+und+der+mikroskopischen+Wahrnehmung.">Beitr&#228;ge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung.</a> Archiv f&#252;r mikroskopische Anatomie. 9, 413-418 .</li><li>Gustafsson, M. G., Agard, D. A., Sedat, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I5M:+3D+widefield+light+microscopy+with+better+than+100+nm+axial+resolution.">I5M: 3D widefield light microscopy with better than 100 nm axial resolution.</a> J Microsc. 195, 10-16 (1999).</li><li>Heintzmann, R., Cremer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laterally+modulated+excitation+microscopy:+improvement+of+resolution+by+using+a+diffraction+grating.">Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating.</a> Proc. SPIE 3568 Optical Biopsies and Microscopic Techniques III. , 185-196 (1999).</li><li>Karadagli&#402;&#225;, D., Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+formation+in+structured+illumination+wide-field+fluorescence+microscopy.">Image formation in structured illumination wide-field fluorescence microscopy.</a> Micron. 39, 808-818 (2008).</li><li>Schermelleh, 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=Subdiffraction+multicolor+imaging+of+the+nuclear+periphery+with+3D+structured+illumination+microscopy.">Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.</a> Science. 320, 1332-1336 (2008).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+structured-illumination+microscopy:+wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proc Natl Acad Sci U S A. 102, 13081-13086 (2005).</li><li>Nagerl, U. V., Willig, K. I., Hein, B., Hell, S. W., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+dendritic+spines+by+STED+microscopy.">Live-cell imaging of dendritic spines by STED microscopy.</a> Proc Natl Acad Sci U S A. 105, 18982-18987 (2008).</li><li>Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L., Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+video+microscopy+of+live+cells+by+structured+illumination.">Super-resolution video microscopy of live cells by structured illumination.</a> Nat Methods. 6, 339-342 (2009).</li><li>James, C. D., 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=Aligned+microcontact+printing+of+micrometer-scale+poly-L-lysine+structures+for+controlled+growth+of+cultured+neurons+on+planar+microelectrode+arrays.">Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays.</a> IEEE Trans Biomed Eng. 47, 17-21 (2000).</li><li>Dumitriu, D., Rodriguez, A., Morrison, 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=High-throughput,+detailed,+cell-specific+neuroanatomy+of+dendritic+spines+using+microinjection+and+confocal+microscopy.">High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy.</a> Nat Protoc. 6, 1391-1411 (2011).</li><li>Fitzsimons, C. P., 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=Knockdown+of+the+glucocorticoid+receptor+alters+functional+integration+of+newborn+neurons+in+the+adult+hippocampus+and+impairs+fear-motivated+behavior.">Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.</a> Mol Psychiatry. , (2012).</li><li>van Hooijdonk, L. W., 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=Lentivirus-mediated+transgene+delivery+to+the+hippocampus+reveals+sub-field+specific+differences+in+expression.">Lentivirus-mediated transgene delivery to the hippocampus reveals sub-field specific differences in expression.</a> BMC Neurosci. , (2009).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35, 567-576 (1993).</li><li>Izeddin, I., 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=Super-resolution+dynamic+imaging+of+dendritic+spines+using+a+low-affinity+photoconvertible+actin+probe.">Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.</a> PLoS ONE. 6, (2011).</li></ol>

Die Autoren haben nichts zu offenbaren.

In diesem Artikel wird ein Arbeitsprotokoll, um Bild dendritischen Dornen aus Ratten-Hippocampus-Neuronen primäre Verwendung der SIM in vitro beschrieben. Der primäre Hippocampus Kultur-Methode ist eine Anpassung der von Kaech und Banker 18 beschrieben ursprünglichen Methode. Die Hauptunterschiede sind die Verwendung von Neurobasal/B27 Kulturmedium, das das Erfordernis der Zuführung Astroglia Kulturen eliminiert, und die Zugabe des mitotischen Inhibitor FUDR an Tag 3, der das Überleben von Neuro...

Ein dendritisches Wirbelsäule ist ein kleiner Vorsprung des Neurons Membran. Diese charakteristische Struktur ist darauf spezialisiert, in der Regel erhalten Input von einer einzigen Synapse und stellt die physikalische Kontaktfläche zwischen zwei Nervenzellen. Die meisten funktionell reifen dendritischen Dornen bestehen aus einem kugelförmigen Spitze, genannt Kopf und einem dünnen Hals, der den Kopf mit dem dendritischen Welle verbindet. Allerdings sind nicht statisch, Stacheln und aktiv zu bewegen und ändern ihre Morphologie kontinuierlich auch im erwachsenen Gehirn 2. In einem 2-wöchigen Zeitraum, Ratte primären Hippocampus-Neuronen-Kulturen vom späten embryonalen oder frühen postnatalen Zeit abgeleitet Entwicklung komplexer dendritischen Dornen mit zahlreichen Membran Vorsprünge, die von Anfang an filipodia stachelartigen Strukturen 3 entwickeln. Basierend auf diesem dynamischen Verhalten und andere Merkmale werden dendritischen Dornen haben, um einen anatomischen Substrat für Speicher und synaptische Transmission 4,5 bereitzustellen. Angesichts thE kritischen Rolle, die dendritischen Dorn Größe und Form haben in der synaptischen Funktion, ist es wichtig, ihren Abmessungen exakt zu messen. Stacheln variieren von etwa 200 bis 2000 Nanometer in der Länge und kann leicht mit Hilfe der konfokalen Laserrasterfluoreszenzmikroskopie identifiziert werden. Aber auch andere als die Länge der Wirbelsäule Dimensionen sind in der Regel unter Auflösung der herkömmlichen optischen Systeme &quot;, theoretisch durch Beugung um 200 Nanometer 6 befestigt. Diese Auflösungsvermögen unzureichend zur Abbildung feiner Details, wie die Breite der Wirbelsäule Hals und Kopf. Viel Arbeit wurde gewidmet, um dieses Problem zu lösen und viele relativ neue Super-Resolution-Mikroskopie-Techniken haben erhebliche Fortschritte zur Verfügung gestellt. Insbesondere ist es möglich, die Auflösung über die klassische Grenze ohne Verwerfen jede Emissionslicht durch die Verwendung seitlich Structured Illumination Microscopy (SIM) in einer Weitfeld-, nicht-konfokalen Mikroskop 7-10 zu erreichen. Unter Verwendung dieser Technik in Kombination mit nicht-linear Mikroskopie-Techniken, ist es theoretisch möglich, die laterale Auflösung des Lichtmikroskops von einer unbegrenzten Faktor 11 zu verbessern. Aber in den meisten experimentellen Bedingungen erlaubt SIM zur Auflösungsgrenze um einen Faktor von zwei 1 übertreffen. Andere höchstauflösenden optischen Mikroskopie-Techniken wie Stimulated Emission Depletion (STED) Mikroskopie 12 und Photoaktivierung Lokalisationsmikroskopie (PALM) 12 haben Bildgebung von dendritischen Dornen angewendet. Localization-basierte Verfahren wie PALM erfordern sehr große Zahl von RAW-Bilder zu Super-Auflösung zu erreichen und werden daher in der Geschwindigkeit beschränkt. Andererseits kann STED hohe Abbildungsgeschwindigkeit zu erreichen, wenn auch relativ geringe Photonenzählungen und kleine Sichtfeldern, die nicht für SIM 13 sein kann. In diesem Artikel ist das Ziel, ein Arbeitsprotokoll zum Bild dendritischen Dornen aus Ratten primäre Neuronen im Hippocampus in vitro kultiviert bieten </em&gt; die Verwendung der SIM. Das Protokoll besteht aus zwei unterscheidbaren Phasen: eine erste, bestehend aus einer Einrichtung, Entwicklung, Transfektion und Immunhistochemie von primären Ratten-Hippocampus-Neuronen Kulturen und einer späten Phase gewidmet Bildgebung probieren.

<table>
	<tbody>
		<tr>
			<td>Fine forceps</td>
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			<td>Big forceps</td>
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			<td>Fine scissor</td>
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			<td>Big scissor</td>
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		</tr>
		<tr>
			<td>Blunt spatula</td>
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		</tr>
		<tr>
			<td>Dissecting microscope with illumination</td>
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		</tr>
		<tr>
			<td>Light microscope</td>
			<td></td>
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		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
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			<td></td>
		</tr>
		<tr>
			<td>Laminar flow cell culture hood</td>
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		</tr>
		<tr>
			<td>High-temperature dry-oven</td>
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		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
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			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator (5% CO2, 37 &#176;C)</td>
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			<td></td>
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			<td>Microcentrifuge</td>
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			<td>Orbital shaker</td>
			<td></td>
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			<td>Nikon structured illumination microscope setup consisting of:</td>
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		</tr>
		<tr>
			<td>Nikon Eclipse Ti research inverted microscope with Perfect Focus System</td>
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		<tr>
			<td>Nikon CFI Apo TIRF 100x oil objective lens (N.A. 1.49)</td>
			<td></td>
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		<tr>
			<td>4 Coherent Sapphire Lasers (458, 488, 514 and 561 nm exitation wavelength)</td>
			<td></td>
			<td></td>
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			<td>SIM Illuminator</td>
			<td></td>
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		</tr>
		<tr>
			<td>Nikon Stage Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>MCL Nano-Drive piezo controller</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Nikon Intensilight C-HGFIE mercury lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SIM Microscope Enclosure temperature control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Andor EM-CCD Camera iXon DU897</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PC with Microsoft Windows 7 Home Edition</td>
			<td></td>
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		</tr>
		<tr>
			<td>Nikon&#8217;s NiS Elements 6.14 SIM software package</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon type A immersion oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy

Dendriten sind Vorsprünge, die aus der Dendriten eines Neurons und stellen die primären Ziele von exzitatorischen postsynaptischen Eingänge im Gehirn. Technologische Fortschritte haben diese Strukturen als Schlüsselelemente in Neuron-Konnektivität und synaptische Plastizität identifiziert. Die quantitative Analyse der Wirbelsäule Morphologie mittels Lichtmikroskopie bleibt ein wesentliches Problem aufgrund technischer Einschränkungen mit intrinsischer Brechungsgrenze Lichts verbunden. Dendritischen Dornen können leicht durch konfokale Laser-Scanning-Fluoreszenz-Mikroskopie identifiziert werden. , Mess subtile Veränderungen in der Form und Größe der Stacheln ist jedoch schwierig, weil andere ist als die Länge der Wirbelsäule Dimensionen sind in der Regel kleiner als herkömmliche theoretische Auflösungsgrenze von 200 nm Lichtmikroskopie fixiert optische Auflösung. Mehrere neu entwickelten Super-Resolution-Techniken, um Bild zellulären Strukturen kleiner als die 200 nm, einschließlich dendritischen Dornen verwendet. Tiese Techniken basieren auf klassischen Fernfeld-Operationen auf der Grundlage und ermöglichen die Nutzung der vorhandenen Probenvorbereitung und Bild über die Oberfläche einer Probe. Beschrieben wird hier ein Arbeitsprotokoll zum Super Resolution Structured Illumination Microscopy (SIM), um die Abbildung von dendritischen Dornen in primären hippokampalen Neuronenkulturen gelten. Mögliche Anwendungen der SIM schneiden sich mit denen der konfokalen Mikroskopie. Allerdings präsentieren sich die beiden unterschiedlichen Techniken Anwendbarkeit. SIM bietet eine höhere effektive laterale Auflösung, während der konfokalen Mikroskopie, die auf die Benutzung eines physischen Lochkamera, erreicht Verbesserung der Auflösung auf Kosten der Beseitigung der aus der Fokus Licht. In diesem Protokoll werden die primären Neuronen auf Deckgläschen mit einem Standard-Protokoll, mit DNA-Plasmiden, die fluoreszierende Proteine ​​und abgebildet mit SIM transfiziert, kultiviert. Die hierin beschriebene gesamte Protokoll dauert ca. 2 Wochen, da dendritischen Dornen abgebildet werden nach 16-17 Tagen in vitro, wenndendritischen Entwicklung optimal ist. Nach Beendigung des Protokolls kann dendritischen Dornen in 3D aus der Serie von SIM-Bildstapeln mit spezieller Software rekonstruiert werden.

Beschrieben wird hier eine standardisierte Arbeitsprotokoll zur Abbildung dendritischen Dornen aus Ratten primäre Neuronen im Hippocampus in vitro mit SIM-Karte. Der Protokollablauf sowie die wesentlichen Schritte sind in Abbildung 1 dargestellt. Insgesamt dauert das Protokoll etwa 2 Wochen nach der experimentellen Arbeit in einer ersten Phase der Probenvorbereitung, einschließlich Kultur, Entwicklung und Transfektion von primären Ratten-Hippocampus-Neuronen und Immunhistochemie getrennt, un...

Watch this Scientific Journal Video about Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy at JoVE.com

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dieser Artikel beschreibt ein Arbeitsprotokoll, um Bild dendritischen Dornen von Neuronen im Hippocampus in vitro unter Verwendung Structured Illumination Microscopy (SIM). Super-Resolution-Mikroskopie mit SIM bietet Bildauflösung deutlich über der Lichtbeugungsgrenze in allen drei Raumdimensionen, so dass die Abbildung der einzelnen dendritischen Dornen mit verbesserten Details.

Imaging dendritischen Dornen von Rat Primär Hippocampus Neuronen mit Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

imaging-dendritic-spines-rat-primary-hippocampal-neurons-using

Research

JoVE Journal

Neuroscience

15.7K Aufrufe.  University of Amsterdam.  Dieser Artikel beschreibt ein Arbeitsprotokoll, um Bild dendritischen Dornen von Neuronen im Hippocampus in vitro unter Verwendung Structured Illumination Microscopy (SIM). Super-Resolution-Mikroskopie mit SIM bietet Bildauflösung deutlich über der Lichtbeugungsgrenze in allen drei Raumdimensionen, so dass die Abbildung der einzelnen dendritischen Dornen mit verbesserten Details. 

Serie: Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection und Primärkultur von embryonalen Maus-Hippocampus und kortikalen Neuronen

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Forschung

Neurowissenschaften

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

Fluorescence Recovery After Photobleaching (FRAP) der Fluoreszenz-markierten Proteine ​​in dendritischen Dornen von hippokampalen Neuronen

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminäre Co-Kultur von primären Ratten-kortikalen Neuronen und Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Voltage-sensitive Dye Aufnahme von Axonen, Dendriten und dendritischen Spines einzelner Neuronen in Hirnschnitten

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calciumphosphat-Transfektion von primären Hippocampus-Neuronen

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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 ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Generation von Neurosphären aus gemischten primären Hippocampus und kortikalen Neuronen isoliert aus E14-E16 Sprague Dawley Rat embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

3D-Modellierung von Dendritischen Stacheln mit synaptischer Plastizität

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Bildgebung dendritischer Dornen bei Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

Serielle Block-Face-Rasterelektronenmikroskopie (SBEM) zur Untersuchung dendritischer Stacheln

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...