Bu çalışma NIH hibe NS065183 ve Rutgers Robert Wood Johnson Tıp Okulu&#39;nda start-up fonları tarafından desteklenmektedir.

1.. Koúullanmıú Medya ve Astrosit-nöron cocultures Rat Astrosit Kültür hazırlanıyor. <ol><li> (BSS tarifi için bakınız Tablo 1) diseksiyonu tampon hazırlayın ve 4 ° C&#39;de mağaza kullanıma hazır olana kadar. </li><li> 500 ml beher izofluran ile yenidoğan sıçan yavrular (P0-P2) anestezisi. </li><li> Yavrular hareketsiz olduğunda,% 70 etanol ile sprey ve başını kesmek. </li><li> Beyin kaldırmak. <ol><li> Dumont # 5 forseps bir çift ile sıkıca kafa tutun, ve deri ve kafatası yoluyla bir orta hat kesi yapmak için ince makas kullanın. </li><li> Yanlara kafatası yansıtarak beyin Açığa ve soğuk BSS içeren bir çanak içine beyin kaldırmak. </li></ol></li><li> Diseksiyon kapsamında diensefalon ve beyin sapından hemisferdeki ayırın. Dikkatle forseps bir çift doku stabilize ve yavaşça forseps başka bir çift ile meninks uzağa çekerek tüm meninksler kaldırmak. </li><li> Tüm hemisferlerdir toplayın in bir 60 mm çanak. Küçük bir makas, kıyma doku gibi ince mümkün olduğunca kullanma. Daha sonra, 50 ml konik tüp kıyılmış doku aktarın. </li><li> 1,5 1% DNase I&#39;in ml ve 1.5 ml% 2.5 tripsin ve beyin BSS (15 ml toplam hacim) 12 ml ilave edilir. 37 ° C&#39;de 15 dakika boyunca çalkalanan bir su banyosu içinde inkübe Iyi karışmasını sağlamak için, tüp elle her 5 dakikada girdaplanır. </li><li> Yeni bir 50 ml konik tüp üzerinde 70 mikron hücre süzgecinden süpernatant aktarın. Bu yüzer 3 ml FBS ekleyin. </li><li> Kalan parçalar için, BSS 13.5 ml ve 1.5 ml% 2.5 tripsin ekleyin ve 15 dakika boyunca çalkalama su banyosu içinde inkübe edilir. </li><li> Hücre süzgecinden kalan süpernatan aktarın ve Aşama 1.8 &#39;den süpernatan ile birleştirir. </li><li> Pelet hücreleri 5 dakika boyunca 1000 rpm&#39;de santrifüj. </li><li> Glial ortam 5 ml içinde süspanse peletler. Üst faz, geri kalan hücreler pellet haline getirmek için tekrar santrifüj edilebilir. </li><li> Hemasitometre kullanarak hücreleri saymak. </li><li> Plaka cel1 x 10 7 cm başına 150 2 şişesi yoğunluğunda ls. </li><li> Gün sonra kaplama taze glial medya medya değiştirin. Daha sonra, glial ortamı ile iki kez bir hafta hücreleri besleme. </li><li> Hücreler&gt;% 80 ortak akışkanlığa (yaklaşık 10 gün sonra kaplama) ulaştığı zaman,% 90 at serumu ve% 10 DMSO içinde hücrelerini dondurma ve sıvı azot içinde stok tutmak. Hücreler 2 flakon başına x 10 6 dondurulur. Yaklaşık olarak 5 şişeler her şişeden dondurulabilir. </li><li> Yaklaşık 10-14 gün hipokampal kültürünü kurmadan önce, glial hücreler dondurulmuş stoklardan kaplanmıştır. Biz genellikle hipokampalinden 90 lamelleri (kuyu başına üç 15 mm yuvarlak lamelleri) büyümesini destekleyecek kadar beş 6-iyi plakaları, plaka. Ayrıca, transfeksiyon için klima N2.1 ortamı için kullanılır glial hücreler ek bir sekiz ila on 60 mm yemekler, plaka. Bir gün önce kültür nöronal hücreler, NB27 ortam ile beslenmelidir. Astrocytes ideal th de&gt;% 90 birleşen olmalıdırnoktasıdır. Bu büyük ölçüde dondurma astroglial kültürlerde mikroglia sayısını azaltır bulabilirsiniz. Mikroglia Astroglial kültürlerde mevcut olduğunda artan nörotoksisite görülmektedir yana, biz her zaman nöronlar ile kokültür için dondurulmuş stokları hazırlanan Astroglial kültür kullanın. </li></ol> 2. Nöron Kültürü <ol><li> MilliQ su 2x ilk durulayarak temizleyin cam lamelleri. Daha sonra, 2 saatlik bir toplam değerin en az beş kez 24 saat için konsantre nitrik asit ile ıslatılmış ve milliQ su ile durulanır. Lameller, 6 saat boyunca 225 ° C&#39;de bir fırın içinde pişirme ile kurutuldu ve sterilize edilir. </li><li> Transferi sterilizasyonu sonrasında 60 mm yemekleri lamelleri. Nöronlar kokültür sırasında glial hücrelerden ayrı tutmak için her lamel dış kenarına yakın steril parafin dört nokta yerleştirin. </li><li> 37 ° C inkübatör içinde bir gece boyunca 0.1 M borat tamponu / ml poli-L-lisin, 1 mg ile kaplayın lamelleri. </li><li> Ertesi gün, iki kez steril m lamelleri durulamaen az 15 dakika her biri için illiQ su. Daha sonra 37 ° C kuluçka makinesi içinde kaplama ortam içinde gece boyunca inkübe edilir. </li><li> Pnömotoraks ardından izofluran anestezi ile gebe sıçan (E18) Euthanize ve başı kesilerek embriyonik sıçan euthanize. Beyin çıkarın ve yukarıda tarif edildiği gibi serebral hemisfer teşrih. </li><li> Serebral yarımkürede hippocampi parçalara ayır. 15 ml konik bir tüp içine tüm hipokampları yerleştirin ve 15 dakika için 37 ℃ da% 0.25 tripsin (0.5 ml% 2.5 tripsin, 4.5 ml BSS) ile sindirmek. </li><li> Sindirilen hipokampi 5 dakika her biri için BSS 3x durulanır. Daha sonra, bir cam Pasteur pipeti ile tritüre edilir ve hücre yoğunluğu, bir hemositometre kullanılarak belirlenir. Nöronlar, daha sonra 60 mm tabak başına 2 x 10 5 hücre yoğunluğunda tohumlanır. </li><li> Yaklaşık 2-4 saat sonra kaplama, transfer nöronlar glia doğru aşağı bakacak şekilde, Astroglial hücreleri içeren yemekleri bağlı nöronlar ile lamelleri. DIV3 anda, için sitozin arabinosid ekle5 uM bir nihai konsantrasyonda hücre glia çoğalmasını durdurmak. </li></ol> 3. Kalsiyum Transfection <ol><li> Gliadaki transfeksiyondan önce günde Durumu N2.1 medya. </li><li> Transfeksiyon gününde, glia kapalı durumunu N2.1 ortamı almak ve yeni bir çanak içine aktarın. Transferi klimalı N2.1 medya içine nöronlar ile lamelleri. 10-30 dakika boyunca kuluçka makinesi içinde dengeye olsun. </li><li> Steril tüplere bir set için, 1-4 ug DNA, 100 ul toplam hacim için 2 M CaCI2 12.5 ul, ve steril H2O birleştirir. Tüpler bir ikinci dizi için, 2 x 100 ul HBS ekleyin. </li><li> Bir kaç saniye için her girdap, CaCl2 / DNA karışımını ihtiva eden bir tüp, bir seferde 2x HBS&#39;de ⅛ hacmi (12.5 ul) ilave edin. Tüpler 15 dakika boyunca bekletin. </li><li> 15 dakika inkübasyondan sonra, hücrelere transfeksiyon karışımı damla damla ilave edildi. 1-1.5 saat boyunca inkübe edin. Kum gibi çökeltilerin tabakası görünür olmalıdır10X objektif kullanılarak mikroskop altında. </li><li> İnkübasyondan sonra, sıcak HBS yıkama tamponu ile iki defa lamelleri yıkayın. </li><li> , Glia orijinal yemeklere lamelleri Dönüş 0.5 mM&#39;lik nihai bir konsantrasyona kadar Kynurenic asit ekleyin. </li><li> Transfekte nöronların canlı görüntüsü ya da erken ertesi gün olarak İmmünositokimya için işlenebilir. Nöronlar transfeksiyonundan sonra üç hafta kadar yaşayabilir. </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>

Ilgi çatışması ilan etti.

Dikkatle sürekli başarılı transfections 10,11 için kontrol edilmesi gereken birkaç önemli parametre vardır. Kalsiyum fosfat transfeksiyonu için en önemli parametre bizim elimizde genellikle 7,10-7,15 arasında değişmektedir 2x HBS, pH değeridir. Biz pH metre arasındaki farkı açıklamak için 0.05 artışlarla pH değerleri ile hisse senetleri üç toplu yapmanızı öneririz. Seçenek olarak ise, Clontech memeli transfeksiyon kiti sürekli iyi bir verim elde edilir 2x HBS sağlar. Dondurucuda ...

Birincil nöronlar postmitotic ve mikro çevre değişikliklerine karşı çok hassas olarak transfekte etmek zor hücre türlerinden biridir. Bu hücrelerde 1 ekzojen genlerin ve kısa saç tokası RNA (shRNAs) ekspresyonu için yöntemler yaygın olarak kullanılan dört tipi vardır. Her biri kendine özgü avantajları ve dezavantajları vardır. Transfeksiyon için hücreler, küvetler içine transfer edilmelidir Örneğin, elektroporasyon, genellikle taze olarak izole edilen nöronlar 2 üzerinde gerçekleştirilir. Virüs enfeksiyonu genellikle çok yüksek verim 3 ulaşmak, ancak daha fazla emek-yoğun operatörler için ve riskli olabilir. Birçok lipid-aracılı transfeksiyon reaktifleri nöronlar ve sitotoksisite farklı düzeylerde başarı değişen derecelerde, ticari olarak temin edilebilir. Kalsiyum fosfat transfeksiyonu, nöronlar içinde yabancı genlerin sokulması için uygun ve ekonomik bir yöntemi temsil eder. Yöntem, ilk memeli hücre içine adenovirüs DNA&#39;nın katılması için kullanılmıştırGraham ve Van Der Eb (1973) tarafından ls 4. Transfeksiyon, bir fosfat tampon maddesi içinde rekombinant DNA ile kalsiyum klorür karıştırılarak gerçekleştirilmiştir. Bu, yavaş yavaş hücrelerin bir mono-tabakası üzerinde ayrıştırılarak DNA / kalsiyum fosfat oluşumu, hücre yüzeyine bağlı, endositoz ile içeri alınır ve son olarak da çekirdek 5 girmektedir, ki çökelir sağlar. Bu işlem, hedef hücre içine yabancı genlerin ekspresyonuna sebep olur. 0.5-5% 6-8 arasında, kalsiyum fosfat transfeksiyonu aralık tipik verimleri. Bununla birlikte, dikkatli bir optimizasyon ve deneysel protokol sürekli uygulama ile, hemen hemen% 50 bir transfeksiyon verimine ulaşmak mümkündür. Burada bir sandviç biçiminde 9 astroglial hücreleri ile kültürlenmiş primer hipokampal nöronlar, kalsiyum fosfat transfeksiyonu için, detaylı bir protokol açıklar.

<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

Kalsiyum fosfat çökeltme kültürlenmiş hücrelerin transfeksiyonu için uygun ve ekonomik bir yöntemdir. Optimizasyonu ile, primer nöronlar gibi sert-transfect hücreleri üzerinde bu yöntemi kullanmak mümkündür. Burada astroglial hücreleri ile kültürlenmiş hipokampal nöronlar kalsiyum fosfat transfeksiyonu için, detaylı bir protokol açıklar.

Transfeksiyon, farklı parametreler optimize edilmiş ve dikkatli bir deney için deney kumanda edilir, bu% 50&#39;ye kadar bir transfeksiyon verimi elde etmek mümkündür. 1 Div4 üzerine GFP ile transfekte edilir nöronların bir alanı göstermektedir. Bu alan 16 transfekte edilmiştir, bunlar arasında 28 nöron, bir toplam içerir. Bu% 50&#39;in üzerinde bir verimlilik gösterir. Aynı lamel başka alanların bir örnekleme genel verimlilik (veriler gösterilmemiştir), yaklaşı...

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

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Primer hipokampal nöronlar kalsiyum fosfat transfeksiyonu

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 Görüntüleme.  Rutgers University.  Kalsiyum fosfat çökeltme kültürlenmiş hücrelerin transfeksiyonu için uygun ve ekonomik bir yöntemdir. Optimizasyonu ile, primer nöronlar gibi sert-transfect hücreleri üzerinde bu yöntemi kullanmak mümkündür. Burada astroglial hücreleri ile kültürlenmiş hipokampal nöronlar kalsiyum fosfat transfeksiyonu için, detaylı bir protokol açıklar. 

Video: 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.

Embriyonik Fare Hipokampal ve Kortikal nöronlar primer Nucleofection ve Kültür

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

Nörobilim

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.

Yapısal Aydınlatma Mikroskobu kullanılarak Rat İlköğretim hipokampalinden Dentritik Spine görüntüleme

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.

Fare dopaminerjik nöronlar İlköğretim Kültür

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.

Subselüler Ca Floresans ve Biyoparlaklık Görüntüleme<sup&gt; 2+</sup&gt; Yaşlı Hipokampal Nöronlar

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.

Düşük Yoğunluk İlköğretim Hipokampal Nöron Kültürü

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.

Sinaptik vezikül endositoz kültürlü Hipokampal nöronlar içinde ölçme

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.

E14-E16 Sprague Dawley Sıçan Embriyosu İzole Karışık Primer Hipokampal ve Kortikal Nöronlardan Nörosfer Lerin Üretimi

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.

Axon İlk Segmentlerinin Montajını İncelemek için Birincil Kültürlü Hipokampal Nöronların Kullanımı

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.

Embriyonik Hipokampal Nöronların Dondurulmuş Stoğunu Kullanarak Kolay ve Tekrarlanabilir Düşük Yoğunluklu Birincil Kültür

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.

İn Vivo Bozulmamış Dorsal Kök Ganglialarında Primer Duyusal Nöronların Ağlarında Nöronal Toplulukların Kalsiyum Görüntülenmesi

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