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 saklanan bile çözeltinin pH değeri, zaman içinde değişecektir göz önünde bulundurun. Genellikle en fazla bir ay boyunca 4 ° C &#39;de 2 x HBS&#39;de hacimde tutmak ve bir yıla kadar -20 ° C&#39; de. İkinci parametre kültür ortamının pH&#39;ı. Bu tutarlı, N2.1 orta tutmak için rutin bir gecede değil, daha 24 saat Astroglial kültürler üzerinde koşuluna bağlıdır. Buna ek olarak, biz Astroglial kültürleri inci kullanmayı deneyinat konfluansa benzerdir. Glia-işlemi görmüş ortam kullanılması da transfeksiyondan sırasında nöron toksisitesini azaltır. Transfeksiyon tutarlı pH&#39;ın tutmak için önce Koşullu ortam inkübatör içinde dengelenmiş edilir. Kalsiyum fosfat çökeltileri boyutu ve yoğunluğu başarılı transfeksiyon anahtarıdır. Büyük, clumpy çökeltiler genellikle sitotoksisite 10 yol ise küçük çökeltiler, düşük transfeksiyon verimi yol açacaktır. DNA / CaCl 2 ve 2 x 11 HBS karıştırırken tutarlı kalsiyum fosfat çökelti oluşmasını sağlamak için, aralıklı vorteks kullanın. Karıştırma için başka sık kullanılan yöntem, bir Pasteur pipeti 12 kullanarak hava kabarcıkları üfleme geçer. Özellikle transfeksiyon karışımı küçük hacimlerde, daha tutarlı bir çökelti oluşumu vermek üzere aralıklı vorteks bulduk. Dikkate diğer değişkenler DNA&#39;nın kalitesi ve nöro yaşı;ns. Biz transfeksiyon nöronlar div2 ulaştığınızda sürekli çalışmaya başlamak bulmak, ancak verimlilik nöronlar DIV10 ötesinde zaman azalmaya başlar. Son olarak, nöronal kültürler kendilerini sağlık önemli bir parametreyi temsil eder. Biz kokültürü sisteminin kullanımı sağlıklı primer hipokampal kültürlerini oluşturmak için en iyi yol olarak görüyorum. Nöronlar sağlıklı değilse, bunlar uzun transfeksiyonundan sonra hayatta olmaz. Kokültürü sistemi ile, düşük yoğunluklu nöronlar uzun vadeli çalışmalar kolaylaştırır, geçiştirme, sonra 3 hafta boyunca yaşayabilir.

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), yaklaşık 50% olduğu gösterir. Astroglial kokültürü sistemi ile, nöronlar sağlıklı kalır ve transfeksiyonundan sonra normal olarak gelişir. 2 10 gün PSD-95-GFP yapı ile transfeksiyondan sonra, DIV15 bir hipokampal nöron göstermektedir. Nöron nöronlar sağlıklı gösterir, çok sayıda olgun mantar şeklinde dendritik dikenler geliştirmiştir. Yüksek transfeksiyon verimliliği ve bu protokolün minimal toksisite birincil suaygırı gen fonksiyonlarını incelemek için değerli bir araç yaparCampal nöronlar. Son olarak, bu protokol, potansiyel olarak, beyindeki sinir hücrelerinin diğer türlerine kültürleri, hem de diğer zor transfekte hücre tipleri transfekte edilmesi için adapte edilebilir. <img alt="Şekil 1" fo:content-width="6in" fo:src="/files/ftp_upload/50808/50808fig1highres.jpg" src="/files/ftp_upload/50808/50808fig1.jpg" width="600px" /> Şekil 1.. Hipokampus nöronlar alanında 28 nöronların yüksek dışarı transfeksiyon verimlilik. 16. olumlu göstererek, Div4 GFP ile transfekte. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50808/50808fig1highres.jpg" target="_blank">resmi büyütmek için buraya tıklayın</a> . <img alt="Şekil 2," fo:content-width="6in" fo:src="/files/ftp_upload/50808/50808fig2highres.jpg" src="/files/ftp_upload/50808/50808fig2.jpg" width="600px" /> Şekil 2. PSD ile transfekte hipokampal nöron-95-GFP çok sayıda olgun mantar şeklinde dendritik dikenler gösteren DIV15 de,. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50808/50808fig2highres.jpg" target="_blank">resmi büyütmek için buraya tıklayın</a> . Tablo 1. Tampon / Medya Tarifler. <table border="1"><tbody><tr><td> Reaktif </td><td> Bileşenleri </td></tr><tr><td> Glial Medya </td><td> 425 ml MEM 5 ml GlutaMAX 5 ml sodyum piruvat, 100 mM 15 ml% 20 glukoz 25 ml FBS (son% 5) 25 ml buzağı serumu (son% 5) 5 ml Penisilin / Streptomisin </td></tr><tr><td> 0.1 M Borat Tampon </td><td> 2.48 g borik asit 3.9 g sodyum tetraborat (boraks) 800 ml H2O milliQ 8.5 NaOH ile pH sterilize </td></tr><tr><td> Kaplama Medya </td><td> 425 ml MEM 5 ml GlutaMAX 5 ml sodyum piruvat, 100 mM 15 ml% 20 glucose 50 ml FBS </td></tr><tr><td> NB27 Medya </td><td> 485 ml Neurobasal Media 5 ml GlutaMAX 2 ml B27 takviyesi (50x) </td></tr><tr><td> Diseksiyon Tampon (BSS) </td><td> 50 ml HBSS 10x 5 ml 1 M HEPES, pH 7.3 5 ml Penisilin / Streptomisin 440 ml H2O </td></tr><tr><td> N2.1 Medya </td><td> 425 ml MEM 5 ml GlutaMAX 5 ml sodyum piruvat, 100 mM 15 ml% 20 glukoz MEM içinde 50 ml ovalbumin,% 1 5 ml N2 takviyesi </td></tr><tr><td> 2 x HEPES tamponlu tuz (HBS) steril </td><td> 274 mM NaCl 9.5 mM KCI 15 mM glukoz 42 mM HEPES 1.4 mM Na 2 HPO 4 3 farklı pH (7.05, 7.10, 7.15) toplu halde hazırlayın </td></tr><tr><td> Yıkama Tamponu (HEPES Tamponlu Tuzu) </td><td> 50 ml HBSS 10x 5 ml 1 M HEPES, pH 7.3 445 ml H2O</td></tr><tr><td> 50 mM Kynurenic asit </td><td> 1x PBS Kynurenic asit 0.473 g çözülür. Çözeltisi içine almak için NaOH damla ekleyin. Daha sonra pH 7.0 &#39;a geri ayarlamak için HCI kullanımı </td></tr></tbody></table> Tablo 2. Kültür hazırlanması için zaman çizgisi. <table border="1"><tbody><tr><td> Gün </td><td> Eylem </td></tr><tr><td> Başlamadan önce </td><td> Astrocyte kültürünü hazırlayın ve hücreleri aşağı dondurma. Astrosit kültürünün bir grup genellikle hipokampal kültürlerin birkaç ay destekleyebilir. </td></tr><tr><td> 1. Hafta Pazartesi </td><td> Astrositleri çözülme. </td></tr><tr><td> Hafta 1 Salı </td><td> Astrositleri besleyin. </td></tr><tr><td> Hafta 1 Cuma </td><td> Astrositleri besleyin. </td></tr><tr><td> Hafta 2 Pazartesi </td><td> Astrositleri besleyin. Daha sonra 24 saat boyunca nitrik asit emmek H, 2 O lamelleri durulayın. </td></tr><tr><td> Hafta 2 Salı </td><td> Rinse 5x lamelleri. Kuru lamelleri sterilize. </td></tr><tr><td> Hafta 2 Çarşamba </td><td> Lamelleri parafin noktalar yerleştirin. Coat poli-L-lizin ile lamelleri. </td></tr><tr><td> Hafta 2 Perşembe </td><td> Steril H2O kaplı lamelleri durulayın, sonra 37 ° C de bir gece ortam kaplama lamelleri inkübe NB27 için astrositlerden üzerine medya değiştirin. </td></tr><tr><td> Hafta 2 Cuma </td><td> Hipokampal diseksiyon ve kaplama. </td></tr></tbody></table>

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

Nanyang Technological University

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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Prenatal Fareler gelen Hipokampal Nöronlar izolasyonu ve Kültürü

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

Hastalık Patogenez Eğitim için Bir Araç Olarak Nöronlar içine Antikor Transfeksiyon

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Eşzamanlı Elektrofizyolojik Kayıt ve Suprakiazmatik Nucleus Nöronlar Kalsiyum Görüntüleme

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

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 ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Ratiometric kalsiyum görüntüleme Caenorhabditis Elegans davranışlar içinde bireysel nöronların

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...