Diese Arbeit wird von NIH NS065183 und Start-up-Fonds von der Rutgers Robert Wood Johnson Medical School unterstützt.

1. Vorbereiten Rat Astrocyte Kultur für konditionierte Medien und Astrocyte-Neuron-Kokulturen. <ol><li> Bereiten Dissektion Puffer (BSS, siehe Tabelle 1 für Rezept) und bei 4 ° C bis zur Verwendung. </li><li> Anesthetize neonatalen Rattenjungen (P0-P2) mit Isofluran in einem 500 ml-Becherglas. </li><li> Bei Jungtieren sind unbeweglich, Spray mit 70% Ethanol und enthaupten. </li><li> Entfernen Sie das Gehirn. <ol><li> Halten Sie den Kopf fest mit einem Paar von Dumont Nr. 5 Pinzette und einer feinen Schere verwenden, um einen Mittelschnitt durch die Haut und Schädel zu machen. </li><li> Expose das Gehirn durch die Reflexion der Schädel an den Seiten, und entfernen Sie das Gehirn in eine Schale mit kaltem BSS. </li></ol></li><li> Trennen Sie die Gehirnhälften aus dem Zwischenhirn und Hirnstamm unter einem Binokular. Entfernen Sie vorsichtig alle Hirnhäute durch die Stabilisierung des Gewebes mit einer Pinzette und sanft zog sich die Hirnhaut mit einer anderen Pinzette. </li><li> Sammeln Sie alle Hemisphären in einem 60 mm-Schale. Mit einer kleinen Schere, zerkleinern Gewebe so fein wie möglich. Übertragen Sie anschließend die zerkleinerten Gewebes auf eine 50 ml konischen Röhrchen. </li><li> 1,5 ml von 1% DNase und 1,5 ml 2,5% Trypsin und 12 ml BSS (Gesamtvolumen 15 ml) zu Gehirnen. Inkubieren in einem Schüttelwasserbad für 15 min bei 37 ° C. Um eine gute Durchmischung zu gewährleisten, ist das Rohr von Hand alle 5 min verwirbelt. </li><li> Überstand durch eine 70 um Zellsieb über ein neues 50 ml konischen Röhrchen. 3 ml FBS zu dieser Überstand. </li><li> Auf die verbleibenden Teile, addieren 13,5 ml BSS und 1,5 ml 2,5% Trypsin und Inkubation im Schüttelwasserbad für weitere 15 min. </li><li> Die restliche Überstand durch die Zelle Sieb und verbinden sich mit dem Überstand aus Schritt 1.8. </li><li> Zentrifugieren bei 1000 UpM für 5 min, um die Zellen zu pelletieren. </li><li> Resuspendieren Pellets in 5 ml Gliazellen Medien. Der Überstand kann wieder zentrifugiert werden, um die restlichen Zellen zu pelletieren. </li><li> Zählen von Zellen mit einer Zählkammer. </li><li> Teller cells in einer Dichte von 1 x 10 7 pro 150 cm 2-Kolben. </li><li> Ändern Sie Medien an die frische Gliazellen Medien am Tag nach der Beschichtung. Danach ernähren Zellen zweimal eine Woche mit Glia-Medien. </li><li> Wenn Zellen&gt; 80% Konfluenz (ca. 10 Tage nach der Beschichtung) erreicht, Gefrierzellen in 90% Pferdeserum und 10% DMSO und halten Aktien in flüssigem Stickstoff. Die Zellen werden mit 2 x 10 6 pro Fläschchen eingefroren. Etwa 5 Fläschchen können aus jedem Kolben eingefroren werden. </li><li> Über 10-14 Tage vor der Einrichtung des Hippocampus-Kultur werden Gliazellen aus den gefrorenen Aktien plattiert. Wir Platte in der Regel fünf 6-Well-Platten, das ist genug, um das Wachstum von 90 Deckgläser von Hippocampus-Neuronen (drei 15 mm runden Deckgläsern pro Well) zu unterstützen. Wir Platte auch eine zusätzliche acht bis zehn 60-mm-Schalen von Gliazellen, die für Anlage N2.1 Medien für die Transfektion verwendet wird. Am Tag vor der neuronalen Kultur, sollten die Zellen mit NB27 Medien zugeführt werden. Die Astrozyten sollte idealerweise&gt; 90% konfluent sein thist der Punkt. Wir finden, dass das Einfrieren stark verringert die Anzahl der Mikrogliazellen in der Astroglia-Kulturen. Da erhöhte Neurotoxizität beobachtet wird, wenn in den Mikroglia Astroglia Kulturen vorhanden ist, verwenden wir immer Astroglia-Kultur aus gefrorenen Beständen, für Co-Kultur mit den Neuronen hergestellt. </li></ol> 2. Neuronale Kultur <ol><li> Saubere Glas-Deckgläser von ersten Spülen in Milli-Q-Wasser 2x. Sie werden dann in konzentrierte Salpetersäure für 24 Stunden mindestens fünfmal insgesamt 2 h eingeweicht und mit Milli-Q-Wasser gespült. Die Deckgläser werden durch Backen in einem Ofen bei 225 ° C für 6 Stunden getrocknet und sterilisiert. </li><li> Überdeckgläser bis 60 mm Schalen nach der Sterilisation. Platzieren vier Punkte sterilem Paraffin nahe der Außenkante jedes Deckglas zu halten Neuronen Gliazellen trennen während Kokultur. </li><li> Deckmantel mit 1 mg / ml Poly-L-Lysin in 0,1 M Borat-Puffer über Nacht bei 37 ° C Inkubator. </li><li> Am nächsten Tag, spülen Deckgläser zweimal in sterilem milliQ Wasser für jeweils mindestens 15 min. Sie werden dann über Nacht in Plattenmedien in 37 ° C-Inkubator inkubiert. </li><li> Euthanize schwangere Ratte (E18) von Isofluran-Narkose, gefolgt von Pneumothorax und einschläfern embryonalen Ratten durch Enthauptung. Gehirn entfernen und zerlegen sich die Hirnhälften wie oben beschrieben. </li><li> Präparieren Sie die hippocampi von der Großhirnhemisphäre. Zeigen alle Hippocampus in eine 15 ml konischen Röhrchen und Verdau mit 0,25% Trypsin (0,5 ml 2,5% Trypsin, 4,5 ml BSS) bei 37 ℃ für 15 min. </li><li> Aufgeschlossene hippocampi in BSS 3x für jeweils 5 Minuten gespült. Sie werden dann mit einer Glaspasteurpipette zerrieben, und die Zelldichte wird unter Verwendung einer Zählkammer bestimmt. Neuronen werden dann in einer Dichte von 2 x 10 5 Zellen pro 60 mm-Schale ausgesät. </li><li> Etwa 2-4 Stunden nach der Beschichtung, Transferdeckgläser mit ange Neuronen zu den Schalen, die Astroglia-Zellen mit Nervenzellen in Richtung der Glia nach unten. Bei DIV3, fügen Cytosinarabinosid dieZellen in einer Endkonzentration von 5 &amp;mgr; M bis Glia-Proliferation zu stoppen. </li></ol> 3. Calcium Transfektion <ol><li> Zustand N2.1 Medien auf Glia der Tag vor der Transfektion. </li><li> Am Tag der Transfektion nehmen Anlage N2.1 Medien aus der Glia und übertragen in eine neue Schüssel. Überdeckgläser mit den Neuronen in der konditionierten Medien N2.1. Lassen Sie ins Gleichgewicht im Inkubator für 10-30 min. </li><li> Einem Satz von sterilen Röhrchen verbinden 1-4 ug DNA, 12,5 &amp;mgr; l 2 M CaCl 2 und sterilem H 2 O für ein Gesamtvolumen von 100 ul. Einem zweiten Satz von Rohren, mit 100 ul 2x HBS. </li><li> Hinzufügen ⅛ Volumen 2 × HBS (12,5 ul) zu einem Zeitpunkt zu dem Röhrchen mit dem CaCl 2 / DNA-Mischung, Vortexen für ein paar Sekunden jedesmal. Ermöglichen, dass die Rohre für 15 min stehen. </li><li> Nach 15 min Inkubation, fügen Sie die Transfektion Mischung tropfenweise zu den Zellen. Inkubieren 1-1,5 Stunden. Eine Schicht aus einem sandartigen Ausfällungen sichtbar sein sollunter dem Mikroskop mit einem 10X-Objektiv. </li><li> Nach Inkubation spülen Deckgläser zweimal mit warmem HBS Waschpuffer. </li><li> Zurück zur ursprünglichen Deckschalen mit Glia, fügen Kynurensäure zu einer endgültigen Konzentration von 0,5 mM. </li><li> Transfizierten Neuronen können live abgebildet oder Immunzytochemie schon am nächsten Tag verarbeitet werden. Neuronen können bis zu drei Wochen nach der Transfektion überleben. </li></ol>

<ol>
	<li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> J. Neurosci. 30, 6171-6177 (2010).</li><li>Zeitelhofer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+mammalian+neurons+via+nucleofection.">High-efficiency transfection of mammalian neurons via nucleofection.</a> Nat. Protoc. 2, 1692-1704 (2007).</li><li>Janas, J., Skowronski, J., Van Aelst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+delivery+of+RNAi+in+hippocampal+neurons.">Lentiviral delivery of RNAi in hippocampal neurons.</a> Method Enzymol. 406, 593-605 (2006).</li><li>Graham, F. L., vander Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+adenovirus+5+DNA.">A new technique for the assay of infectivity of human adenovirus 5 DNA.</a> Virology. 52, 456-467 (1973).</li><li>Craig, A. M., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+cultured+neurons.">Transfecting cultured neurons.</a> Culturing Nerve Cells. , (1998).</li><li>Alavian, K. N., 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=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13, 1224-1233 (2011).</li><li>Zhang, Y., 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=Modulation+of+synaptic+function+by+VAC14,+a+protein+that+regulates+the+phosphoinositides+PI(3,5)P(2)+and+PI(5)P.">Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P(2) and PI(5)P.</a> EMBO J. 31, 3442-3456 (2012).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Curr. Protoc. Neurosci. Chapter 3, Unit 3 11 (2001).</li><li>Goslin, K. A. H., Banker, G., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Hippocampal+Neurons+in+Low-Density+Culture.">Rat Hippocampal Neurons in Low-Density Culture.</a> Culturing Nerve Cells. , (1998).</li><li>Kohrmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+convenient,+and+effective+method+to+transiently+transfect+primary+hippocampal+neurons.">Fast, convenient, and effective method to transiently transfect primary hippocampal neurons.</a> J. Neurosci. Res. 58, 831-835 (1999).</li><li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca2+-phosphate+transfection+efficiency+in+low-density+neuronal+cultures.">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> Nat. Protoc. 1, 695-700 (2006).</li><li>Goetze, B., Grunewald, B., Baldassa, S., Kiebler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+controlled+formation+of+a+DNA/calcium+phosphate+coprecipitate:+application+for+transfection+of+mature+hippocampal+neurons.">Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons.</a> J. Neurobiol. 60, 517-525 (2004).</li></ol>

Konflikte Interesse erklärt.

Es gibt mehrere wichtige Parameter, die sorgfältig für konsequent erfolgreiche Transfektionen 10,11 kontrolliert werden müssen. Der kritischste Parameter zur Calciumphosphat-Transfektion ist der pH-Wert von 2 x HBS, die in unseren Händen variiert üblicherweise zwischen 7,10 bis 7,15. Es wird empfohlen, drei Chargen von Titeln mit pH-Werten in 0,05-Schritten, um die Differenz zwischen den pH-Meter ausmachen. Alternativ stellt die Clontech Säugetier Transfektion Kit 2x HBS, die durchweg guten Wirkungsgrad...

Primären Neuronen eine der schwierigsten Zelltypen zu transfizieren, wie sie sind postmitotischen und sind sehr empfindlich gegenüber Mikroumweltveränderungen. Es gibt vier häufigsten verwendeten Arten von Verfahren zur Expression von exogenen Genen und Kurzhaarnadel-RNAs (shRNAs) in diesen Zellen ein. Jeder hat seine eigenen Vorteile und Nachteile. Zum Beispiel wird in der Regel auf Elektroporation frisch isolierten Neuronen 2 durchgeführt, wie Zellen müssen in Küvetten für die Transfektion übertragen werden. Virus-Infektion kann in der Regel sehr hohen Wirkungsgrad erreichen, 3, ist aber arbeitsintensiv und riskant für die Betreiber. Viele Lipid-vermittelte Transfektion Reagenzien sind kommerziell erhältlich, mit unterschiedlichem Erfolg in Neuronen und verschiedene Ebenen der Zytotoxizität. Calciumphosphat-Transfektion eine bequeme und kostengünstige Methode zur Einführung von Fremdgenen in Neuronen. Die Methode wurde zuerst verwendet, um Adenovirus-DNA in Säuger cel vorstellenls von Graham und van der Eb (1973) 4. Transfektion wurde durch Mischen von Calciumchlorid, die mit rekombinanten DNA in einem Phosphatpuffer durchgeführt. Dies ermöglicht die Bildung von DNA / Calciumphosphat-Präzipitate, die, wenn nach und nach auf eine Monoschicht von Zellen gesunken, sich an die Zelloberfläche werden durch Endozytose aufgenommen und schließlich in den Kern 5. Dieses Verfahren würde die Expression von Fremdgenen eingeführt in der Zielzelle führen. Typische Wirkungsgrade von Calciumphosphat-Transfektion Bereich zwischen 0,5-5% 6-8. Bei sorgfältiger Optimierung und konsistente Ausführung des experimentellen Protokolls ist es jedoch möglich, eine Transfektionseffizienz von nahezu 50% erreichen. Hier beschreiben wir unsere detaillierten Protokoll für die Calciumphosphat-Transfektion von primären Hippocampus-Neuronen, die mit Astroglia-Zellen in einem Sandwich-Format 9 kokultiviert werden.

<table border="0" cellpadding="0" cellspacing="0" width="879">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">Dumont #5 forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Fine scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td>straight / sharp 8.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Vannas-Tubinger spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15008-08</td>
			<td>angled / sharp / 8.5 cm / 5 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Standard pattern forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11000-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Student surgical scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">91401-14</td>
			<td>blunt / 14.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15006-09</td>
			<td>angled to side / 9 cm / 10 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Isoflurane</td>
			<td style="width:207px;">Webster Veterinary</td>
			<td style="width:119px;">14043-225-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Poly-L-lysine</td>
			<td>Sigma</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Sigma</td>
			<td>M2279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">100 mM Sodium pyruvate</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Glucose</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x HBSS</td>
			<td>Sigma</td>
			<td>H4385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">1 M HEPES, pH 7.3</td>
			<td>Gibco/Invitrogen</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX</td>
			<td>Gibco/Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal Media</td>
			<td>Gibco/Invitrogen</td>
			<td>21130-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement (50x)</td>
			<td>Gibco/Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Gibco/Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovalbumin</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">2.5% Trypsin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15090-046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">DNase I</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Cytosine arabinoside</td>
			<td style="width:207px;">Calbiochem/EMD Millipore</td>
			<td align="right" style="width:119px;">251010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Kynurenic acid</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium phosphate transfection of primary hippocampal neurons

Calciumphosphat-Präzipitation ist eine bequeme und kostengünstige Methode zur Transfektion von kultivierten Zellen. Mit Optimierung ist es möglich, dieses Verfahren auf Fest transfizierbaren Zellen wie primäre Neuronen verwendet werden. Hier beschreiben wir unsere detaillierten Protokoll für die Calciumphosphat-Transfektion von Neuronen im Hippocampus mit Astroglia-Zellen kokultiviert.

Wenn die verschiedenen Parameter der Transfektion optimiert und sorgfältig von Experiment zu Experiment gesteuert, ist es möglich, Transfektionseffizienzen von bis zu 50% zu erhalten. Fig. 1 zeigt ein Feld von Neuronen, die mit GFP auf DIV4 transfiziert sind. Das Feld enthält insgesamt 28 Neuronen, von denen 16 wurden transfiziert. Dies entspricht einem Wirkungsgrad von über 50%. Eine Probenahme von anderen Bereichen auf dem gleichen Deck zeigt den Gesamtwirkungsgrad liegt bei 50% (Daten nicht gezei...

Watch this Scientific Journal Video about Calcium Phosphate Transfection of Primary Hippocampal Neurons at JoVE.com

Calcium Phosphate Transfection of Primary Hippocampal Neurons

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 ...

calcium-phosphate-transfection-of-primary-hippocampal-neurons

Research

JoVE Journal

Neuroscience

25.1K Aufrufe.  Rutgers University.  Calciumphosphat-Präzipitation ist eine bequeme und kostengünstige Methode zur Transfektion von kultivierten Zellen. Mit Optimierung ist es möglich, dieses Verfahren auf Fest transfizierbaren Zellen wie primäre Neuronen verwendet werden. Hier beschreiben wir unsere detaillierten Protokoll für die Calciumphosphat-Transfektion von Neuronen im Hippocampus mit Astroglia-Zellen kokultiviert. 

Serie: Calcium Phosphate Transfection of Primary Hippocampal Neurons

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

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using 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. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

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. ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Primärkultur von Maus dopaminergen Neuronen

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluoreszenz- und Biolumineszenz-Bildgebung subzellulärer Ca<sup&gt; 2+</sup&gt; Im Alter von Neuronen im Hippocampus

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Low-Density Primäre Hippocampus Kultur

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Messung der synaptischen Vesikel Endozytose in kultivierten Hippocampal Neuronen

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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 ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Verwendung von primär kultivierten Hippocampus-Neuronen zur Untersuchung der Anordnung von Axon-Anfangssegmenten

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

Einfache und reproduzierbare Low-Density-Primärkultur mit gefrorenem Bestand embryonaler Hippocampus-Neuronen

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Im lebenden Organismus Calcium-Bildgebung neuronaler Ensembles in Netzwerken primärer sensorischer Neurone in intakten dorsalen Wurzelganglien

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...