Irmgard Weiss uzman teknik destek için teşekkür ederiz. Bu eser DFG kümenin mikroskobu nanometre mesafeden için mükemmel ve moleküler fizyolojisi (CNMPB, B1-7, T.D.) beyin üzerinden tarafından desteklenmiştir.

Hiçbir canlı hayvan çalışmaları yapılmıştır. Hücre kültürleri altında onay yerel hayvan koruma yetkilileri (Tierschutzkommission der Universitätsmedizin Göttingen) tarafından onaylanmış almak için ötenazi hayvanları içeren deneyler T10/30 numara. Deneyler ile onaylanmış protokoller yapılmıştır.
1. birincil hipokampal hücre kültürü
<ol><li>Embriyonik gün 1916,17fare hipokampus Disosiye hücre kültürünü hazırlayın. 12 mm coverslips 50.000-60.000 hücreler/iyi bir yoğunluğu 24-iyi yemekler polyethyleneimine (PEI) ile kaplı hücrelerdeyse plaka. Bir hücre sayım odası ve faz kontrast optik kullanarak yoğunluğu kontrol edin.</li>
	<li>3 gün (gün vitro (DIV) 3) 24-şey plaka %5 CO237 ° C'de kuluçka nöronlar kültür.</li>
	<li>Coverslips iletilen ışık mikroskobu (Örneğin, faz kontrast optik, 10-20 X büyütme) kullanarak hücre sağlık göstergeleri için değerlendirmek. Kontrol için iyi sağlık göstergeleri aşağıdaki: bir açık faz kontrast hale, neurites boncuklu yapıları, olmadan ve hiçbir soma kümeleme veya neurite donatılacak.</li>
</ol>2. transfection
Not: Aşağıdaki iletişim kuralı 3 kuyuları için bir çift-transfection ifade eder. Ancak, zaman 4 kuyular için yeterli miktarda hazırlanan protokol en iyi çalışır.
<ol><li>Transfection arabellek (274 mM NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 15 mM glikoz, 42 mM HEPES) 500 mL bir Erlenmeyer şişeye hazırlayın.<ol>
<li>NaCl 8.0 g, KCl 0,37 gr, 0,095 g Na2HPO4, 1.35 g glikoz ve HEPES 5.0 g 400 ml distile su içinde bir Erlenmeyer flask geçiyoruz.</li>
		<li>PH 6,95 için 1 M NaOH pH metre kullanarak ayarlayın.</li>
		<li>500 ml distile su ile ses seviyesini ve pH metre kullanarak pH kontrol edin.</li>
		<li>20-30 mL aliquots transfection arabelleği aşağıdaki pH değerleri ile pipetting 1 tarafından yapmak transfection arabelleğine M NaOH: 6,96, 6.97, 6.98, 6,99, 7,00, 7,01, 7.04, 7,05, 7,03 7,02 7.06, 7,07, 7,08, 7,09, 7.11. Not: Transfection tampon pH transfection etkinliği için çok önemlidir.</li>
		<li>Transfected hücre en yüksek sayısı transfection arabellek neden test etmek için 6,96 7.11 her pH değeri sınayın. 2.2-2,11 ve doğrulanmış bir plazmid GFP ifade olan transfection yöntemi kullanın. En iyi hangi arabellek çalışır coverslip değerlendirmek her transfection tampon pH değeri başına transfected hücre sayısını belirleyin.</li>
		<li>Aliquot 2 mL microcentrifuge tüpler içine en yüksek transfection verimliliği ile tampon donma ve tüpler-20 ° C'de depolayın</li>
	</ol>
</li>
	<li>İndirimli serum orta, hücre kültürü orta ve 37 ° c su banyosu içinde distile su önceden ısıtmak.</li>
	<li>1.5 mL microcentrifuge tüp transfection karışımı hazırlayın. Steril çalışma koşulları sağlamak için laminar akış başlık altında çalışır.<ol>
<li>Mix 7.5 µL 4 µg her endotoksin içermeyen DNA'ın (Synaptophysin-mOrange ve mGFP/GFP-Rogdi) ile 2 M kalsiyum klorür. Toplam hacmi 1.5 mL microcentrifuge tüp içinde 60 mL ulaşmak için su ekleyin.</li>
		<li>Transfection arabellek 60 mL karışıma ekleyin. En iyi sonuçları elde etmek için transfection tampon dropwise DNA-mix vortex Tarih hafifçe sallayarak süre ekleyin.</li>
		<li>(RT) Oda sıcaklığında 20 dakika kuluçkaya. Laminar akış hood yanındaki tüp yerleştirerek kuluçka süresi sırasında kuluçka tüp sallayarak kaçının.</li>
	</ol>
</li>
	<li>Laminar akış başlık altında hücre kültür orta ("şartına orta") 1000 mL damlalıklı kullanarak kuyulardan çıkarın ve kuluçka ayrı bir kapta saklayın.</li>
	<li>İndirimli serum orta 500 mL her şey için ekleyin. 20 dakika kuluçka dönemi (adım 2.3.3) bitene 37 ° C ve % 5 CO2 hücreleri kuluçkaya.</li>
	<li>Transfection mix 30 mL birkaç damla pipetting tarafından her şey için ekleyin. Tüpün dibinde tortu atmak.</li>
	<li>Bütün wells transfection karışımı ile temin edildikten sonra orta transfection karışımının dağıtımını sağlamak için 24-şey plaka yavaşça salla.</li>
	<li>60 dakika 37 ° C ve % 5 CO2kuyu kuluçkaya.</li>
	<li>Kaldırmak ve transfection mix atmak ve hücre kültür orta ile üç kez yıkayın. Her şey için hücre kültür orta 1 mL ekleyin ve onları Orta RT. kaldırmak 750 mL 30 saniye için kuluçkaya ve taze orta aynı miktarda ekleyin. Bunu üç kez tekrarlayın. Not: Çamaşır adım önemlidir. Her şey en azından yok orta vardır zaman tutmak (i.e., çıkarma ve iyi iyi değiştirme) ve çamaşır orta yavaşça ekleyin.</li>
	<li>Kaldırmak ve hücre kültür orta atın ve şartına orta iyi-tarafından-da 450 mL ekleyin.</li>
	<li>Nöronlar kuluçka DIV 10 37 ° C ve % 5 CO2 olgun izin.</li>
</ol>3. stimülasyon ve Syt1 alımı
Not: Aşağıdaki protokol için 3 kuyu Anlamazdın uygulanır. İçin depolarizasyon kuyu diğer herhangi bir sayıda tutarları uygun şekilde ayarlayın.
<ol><li>10 x depolarizasyon arabellek (640 mM NaCl, 700 mM KCl, 10 mM MgCl2, 20 mM CaCl2, 300 mM glikoz, 200 mM HEPES, pH 7,4) bir Erlenmeyer şişesi 50 mL hazırlayın. Not: Depolarizasyon arabellek birkaç hafta için 4 ° C'de tutulabilir. Bir sigara depolarize çözüm de kullanılırsa, bir 10 hazırlamak 1290 mM oluşan x Tyrode'nın çözüm NaCl, 50 mM KCl, 10 mM MgCl2, 20 mM CaCl2, 300 mM glikoz, 200 mM HEPES pH 7.4 karşılaştırmak üzere stimülasyon kaynaklı ile kendiliğinden geri dönüşüm geri dönüşüm. 1 x seyreltme sonra Tetradotoksin 1 µM Aksiyon potansiyeli üretimi engellemek için kullanmadan önce ekleyin.<ol>
<li>1.87 gram NaCl, KCl, 0.1 g MgCl2-6 H20, CaCl2-2 H2O 0,15 g ve 3.0 g glikoz-1 H2O ve HEPES 2.38 g 50 ml distile su içinde bir Erlenmeyer flask 2,61 g geçiyoruz. NaOH ve steril-filtre çözümü ile pH ayarlayın. Arabellek 1:10 1 x konsantrasyon ulaşmak için distile suda sulandırmak.</li>
	</ol>
</li>
	<li>%4 paraformaldehyde (PFA) 1 x PBS (pH 7,4) stimülasyon sonra fiksasyon için hazır olun.<ol>
<li>500 mL % 4'lük için 1 x PBS, PFA çözülür paraformaldehyde 450 ml distile H2o 20 g Not: Çözüm Isıtma eriterek kadar hızlandırabilir ancak PFA parçalanır gibi çözüm üzerinde 70 ° C, ısı değil. Uyarı: Toksik, potansiyel olarak kanserojen ve teratojenik PFA. PFA ile çalışırken eldiven, duman başlık altında çalışmak ve sindirim kaçının.</li>
		<li>RT için çözüm soğumaya bırakın ve 50 mL 10 x PBS stok çözeltisi ekleyin. PH 7.4 için NaOH/HCl pH metre kullanarak ayarlayın.</li>
	</ol>
</li>
	<li>1 depolarizasyon arabellek x 600 mL ve hücre kültür ortamına su banyosu 37 ° C'de 10 mL önceden ısıtmak.</li>
	<li>Fare anti-Syt1 antikor (klon 604.2) 1 mL 1 x depolarizasyon arabellek ve girdap 10 saniyeliğine ekleyin.</li>
	<li>Kaldırmak ve hücreleri hücre kültür ortamından atın. 200 mL her şey ve 37 ° C ve % 5 CO2 kuluçka makinesi'de 5 dakika kuluçkaya depolarizasyon-antikor karışımı ekleyin.</li>
	<li>Kaldırmak ve depolarizasyon-antikor mix atmak ve hücre kültür orta ile üç kez yıkayın. Her şey için hücre kültür orta 1 mL ekleyin ve orta RT. kaldırmak 750 mL 30 saniye için kuluçkaya ve taze orta aynı miktarda ekleyin. Üç kere tekrar edin.</li>
	<li>Kaldırın ve hücre kültür orta atmak ve % 4'lük 300 mL ekleyin 1 x PBS PFA. 4 ° C'de 20 dakikadır kuluçkaya</li>
	<li>1 x PBS ile 5 dakika her için üç kez yıkayın. Not: Protokol burada duraklatılmış.</li>
</ol>4. Immunocytochemistry
<ol><li>50 mL engelleme arabelleği hazırlayın. Not: arabellek engelleme-20 ° C'de aylarca tutulabilir.<ol>
<li>2.5 g sükroz ve sığır serum albumin (BSA) 1 g 5 mL 10 x PBS stok çözeltisi geçiyoruz. 1.5 mL % 10 deterjan stok çözeltisi ekleyin. Çözüm kadar tüm bileşenleri düzgün çözünmüş ve distile H2O son hacmi 50 mL erişene dek ekleyin karıştırın. Aliquot çözüm ve depolama için aliquots Don.</li>
	</ol>
</li>
	<li>(Birincil Syt1-antikor türler karşı yönetmen) ikincil, fluorophore birleşince antikor 1: 1000 seyreltme her kuyuda engelleme arabellekte 200 ml seyreltik.</li>
	<li>Kaldırmak ve her şey bir coverslip içeren üzerinden 1 x PBS atın.</li>
	<li>Arabellek-antikor karıştırmak için her şey engelleme 200 mL ekleyin ve RT. az 60 dakika boyunca kuluçkaya Dikkat: ikincil antikorlar ışığa duyarlı olduğundan, tüm adımları ilerlemeye karanlıkta gerçekleştirilmesi gerekir.</li>
	<li>Kuluçka sonra hücreleri üç kez için 1 mL 1 x PBS ile 5 dakika yıkayın.</li>
	<li>Mikroskop slaytlar üzerindeki coverslips ile gömme orta gömün.<ol>
<li>Mikroskop slayt ortamına katıştırma 7 mL damla ekleyin. Coverslip bir şırınga ile kaldırma ve forseps ile kapma 24-şey plaka çıkarın. Dikkat: forseps dikkatli olmalıdır bu yüzden hücreleri coverslip yüzeyi kolayca, zarar görmüş.</li>
		<li>Coverslip PBS kaldırmak ve dikkatli bir şekilde yumuşak bir doku için bir kenar dokunarak kuru için distile su içine daldırma.</li>
		<li>Böylece hücreleri taşıyan yüzey böylece hücreleri gömme orta katıştırma mikroskop slayt yüzleri coverslip gömme orta damlacık üzerine çevirmek.</li>
	</ol>
</li>
	<li>Slaytlar için 1-2 h başlık altında kurumaya bırakın (ışığa maruz önlemek için kapak) ve 4 ° C'de bir mikroskop slayt kutusunda saklamak Not: Protokol burada duraklatılmış.</li>
</ol>5. mikroskobik analizlerin yapıldığı
<ol><li>Coverslips kuru sonra onları objektif ve kamera ile mikroskop altında yerleştirin.</li>
	<li>Kaç piksel gri değerlerden en büyük dağıtım sağlamak için pozlanmış her kanal için çekim hızı ayarlayın. Not: çekim hızı kanallar arasında farklı olabilir iken, coverslips arasında karşılaştırılabilir emin olmak bir kanal için sabit olmalıdır.</li>
	<li>İlgi coverslip (ROIs) 10 bölgeler için çok kanallı görüntüler elde etmek. GFP-floresan, hangi-meli var olmak punctate kontrol ederek yatırım getirisi transfected nöron aksonal işlemlerini içerdiğini denetleyin.</li>
</ol>6. istatistiksel analiz
<ol><li>Görüntüleri .tif dosyaları olarak dışa aktarma. Yanında tıkırtı eğe OpenView18 ilgili resimleri yüklemek | Görüntü dosyasını yükle.</li>
	<li>Synaptophysin-mOrange görüntüsü olarak Kanal 1, Rogdi-GFP/mGFP resmi gibi 2 kanal ve Kanal 3 olarak Alexa647 floresan görüntü seçin.</li>
	<li>Eşik ROIs.<ol>
<li>Çözümleme ' yi tıklatın | Yer alan puncta üzerinde. Görsel incelenmesi üzerine, diffüz floresan, sadece punctate sinyalleri (temsil eden Synaptophysin-mOrange floresan) Kanal 1 görüntü bırakarak söz konusu değildir eşik ve Delta yoğunluk değerleri seçin. Tüm görüntüler için aynı eşik tutun.</li>
	</ol>
</li>
	<li>Bu ROIs (GFP-Rogdi/mGFP floresan) karşılık gelen kanal 2 ve 3 (Syt1-floresan) her coverslip alanının Şimdi Çalıştır' ı tıklatarak aktarın. Not: ROIs hücre çift transfected ise sadece düşünülmelidir. Bu yöntemde, mOrange ve GFP floresans açıkça görünür olmalıdır.</li>
	<li>Verileri günlük verilerinitıklatarak analiz günlük düzenleyicisine kaydetmek.</li>
	<li>Windows sekmesinin altındaki analiz günlük Düzenleyicisi'ni açın ve her kanal için değerleri kopyalayın. Değerleri ayrı kanallar için bir elektronik tabloya yapıştırabilirsiniz.</li>
	<li>Her iki transfection koşullarında (GFP-Rogdi ve mGFP) ROIs Syt1 kanalda ortalama floresans yoğunluğunu belirlemek.</li>
	<li>Önemli farkları belirlemek için bir öğrenci t-testi gibi uygun istatistiksel testler uygulanır.</li>
</ol>

<ol>
	<li>Koh, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin+I,+a+Ca2++sensor+for+neurotransmitter+release.">Synaptotagmin I, a Ca2+ sensor for neurotransmitter release.</a> Trends in Neurosciences. 26, 413-422 (2003).</li><li>Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Does+Synaptotagmin+Trigger+Neurotransmitter+Release.">How Does Synaptotagmin Trigger Neurotransmitter Release.</a> Annual Review of Biochemistry. 77, 615-641 (2008).</li><li>Perin, M. S., 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=Structural+and+Functional+Conservation+of+Synaptotagmin+(p65)+in+Drosophila+and+Humans.">Structural and Functional Conservation of Synaptotagmin (p65) in Drosophila and Humans.</a> Journal of Biological Chemistry. 266, 615-622 (1991).</li><li>Matteoli, M., Takei, K., Perrin, M. S., S&#252;dhof, T. C., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exo-endocytotic+Recycling+of+Synaptic+Vesicles+in+Developing+Processes+of+Cultured+Hippocampal+Neurons.">Exo-endocytotic Recycling of Synaptic Vesicles in Developing Processes of Cultured Hippocampal Neurons.</a> Journal of Cell Biology. 117, 849-861 (1992).</li><li>Ko, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin-1+performs+neurexin-dependent+and+neurexin-independent+functions+in+synapse+validation.">Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation.</a> The EMBO Journal. 28, 3244-3255 (2009).</li><li>Shinoda, 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=BDNF+enhances+spontaneous+and+activity-dependent+neurotransmitter+release+at+excitatory+terminals+but+not+at+inhibitory+terminals+in+hippocampal+neurons.">BDNF enhances spontaneous and activity-dependent neurotransmitter release at excitatory terminals but not at inhibitory terminals in hippocampal neurons.</a> Frontiers in Synaptic Neuroscience. 6, 27 (2014).</li><li>Ivanova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+controls+localization+and+function+of+CtBP+1+via+binding+to+Bassoon+and+Piccolo.">Synaptic activity controls localization and function of CtBP 1 via binding to Bassoon and Piccolo.</a> The EMBO Journal. 34, 1056-1077 (2015).</li><li>Crawford, D. C., Ramirez, D. M. O., Trauterman, B., Monteggia, L. M., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+molecular+impairment+of+spontaneous+neurotransmission+modulates+synaptic+efficacy.">Selective molecular impairment of spontaneous neurotransmission modulates synaptic efficacy.</a> Nature Communications. 8, 1-14 (2017).</li><li>Riemann, D., Wallrafen, R., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kohlsch&#252;tter-T&#246;nz+syndrome+associated+gene+Rogdi+encodes+a+novel+presynaptic+protein.">The Kohlsch&#252;tter-T&#246;nz syndrome associated gene Rogdi encodes a novel presynaptic protein.</a> Scientific Reports. 7, (2017).</li><li>Kim, 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=Rogdi+Defines+GABAergic+Control+of+a+Wake-promoting+Dopaminergic+Pathway+to+Sustain+Sleep+in+Drosophila.">Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila.</a> Scientific Reports. 7, 1-14 (2017).</li><li>Schossig, A., Wolf, N. I., Kapferer, I., Kohlsch&#252;tter, A., Zschocke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptic+encephalopathy+and+amelogenesis+imperfecta:+Kohlsch&#252;tter-T&#246;nz+syndrome.">Epileptic encephalopathy and amelogenesis imperfecta: Kohlsch&#252;tter-T&#246;nz syndrome.</a> Euopean Journal of Medical Genetics. 55, 319-322 (2012).</li><li>Kraszewski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+Vesicle+Dynamics+in+Living+Cultured+Hippocampal+Neurons+Visualized+with+CY3-Conjugated+Antibodies+Directed+against+the+Lumenal+Domain+of+Synaptotagmin.">Synaptic Vesicle Dynamics in Living Cultured Hippocampal Neurons Visualized with CY3-Conjugated Antibodies Directed against the Lumenal Domain of Synaptotagmin.</a> Journal of Neuroscience. 15, 4328-4342 (1995).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440, 935-939 (2006).</li><li>Petkova, A., Goedecke, N., Korte, M., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligins+mediate+presynaptic+maturation+through+brain-derived+neurotrophic+factor+signaling.">Neuroligins mediate presynaptic maturation through brain-derived neurotrophic factor signaling.</a> bioRxiv. , 262246 (2018).</li><li>Fuchs, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(A)+receptors+can+initiate+the+formation+of+functional+inhibitory+GABAergic+synapses.">GABA(A) receptors can initiate the formation of functional inhibitory GABAergic synapses.</a> European Journal of Neuroscience. 38, 3146-3158 (2013).</li><li>Dresbach, T., 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=Functional+regions+of+the+presynaptic+cytomatrix+protein+Bassoon:+Significance+for+synaptic+targeting+and+cytomatrix+anchoring.">Functional regions of the presynaptic cytomatrix protein Bassoon: Significance for synaptic targeting and cytomatrix anchoring.</a> Molecular and Cellular Neuroscience. 23, 279-291 (2003).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Hippocampal+Neurons+from+Prenatal+Mice.">Isolation and Culture of Hippocampal Neurons from Prenatal Mice.</a> Journal of Visualized Experiments. (65), 4-9 (2012).</li><li>Tsuriel, S., 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=Local+sharing+as+a+predominant+determinant+of+synaptic+matrix+molecular+dynamics.">Local sharing as a predominant determinant of synaptic matrix molecular dynamics.</a> PLOS Biology. 4, 1572-1587 (2006).</li><li>Iwabuchi, S., Kakazu, Y., Koh, J., Goodman, K. M., Harata, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Synaptic+Vesicle+Recycling+Using+FM+Dyes+During+Evoked,+Spontaneous,+and+Miniature+Synaptic+Activities.">Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities.</a> Journal of Visualized Experiments. (85), 1-10 (2014).</li><li>Villarreal, S., Lee, S. H., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Synaptic+Vesicle+Endocytosis+in+Cultured+Hippocampal+Neurons.">Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.</a> Journal of Visualized Experiments. (127), 1-8 (2017).</li><li>Kavalali, E. T., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+presynaptic+function.">Visualizing presynaptic function.</a> Nature Neuroscience. 17, 10-16 (2014).</li><li>Opazo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+Intermixing+of+Synaptic+Vesicle+Components+upon+Vesicle+Recycling.">Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling.</a> Traffic. 11, 800-812 (2010).</li><li>Wollebo, H. S., Woldemichaele, B., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+transduction+of+neuronal+cells.">Lentiviral transduction of neuronal cells.</a> Methods in Molecular Biology. 1078, 141-146 (2013).</li><li>Yang, X., Kaeser-Woo, Y. J., Pang, Z. P., Xu, W., S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexin+Clamps+Asynchronous+Release+by+Blocking+a+Secondary+Ca2++Sensor+via+Its+Accessory+&#945;+Helix.">Complexin Clamps Asynchronous Release by Blocking a Secondary Ca2+ Sensor via Its Accessory &#945; Helix.</a> Neuron. 68, 907-920 (2010).</li><li>Wittenmayer, 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=Postsynaptic+Neuroligin1+regulates+presynaptic+maturation.">Postsynaptic Neuroligin1 regulates presynaptic maturation.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13564-13569 (2009).</li><li>Lazarevic, V., Schone, C., Heine, M., Gundelfinger, E. D., Fejtova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Remodeling+of+the+Presynaptic+Cytomatrix+upon+Homeostatic+Adaptation+to+Network+Activity+Silencing.">Extensive Remodeling of the Presynaptic Cytomatrix upon Homeostatic Adaptation to Network Activity Silencing.</a> Journal of Neuroscience. 31, 10189-10200 (2011).</li><li>Kraszewski, K., Daniell, L., Mundigl, O., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+synaptic+vesicles+in+nerve+endings+monitored+by+recovery+from+photobleaching+of+synaptic+vesicle-associated+fluorescence.">Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence.</a> Journal of Neuroscience. 16, 5905-5913 (1996).</li><li>Hua, Y., Sinha, R., Martineau, M., Kahms, M., Klingauf, 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+common+origin+of+synaptic+vesicles+undergoing+evoked+and+spontaneous+fusion.">A common origin of synaptic vesicles undergoing evoked and spontaneous fusion.</a> Nature Neuroscience. 13, 1451-1453 (2010).</li><li>Han, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Glycosylation+Is+Essential+for+Vesicular+Targeting+of+Synaptotagmin+1.">N-Glycosylation Is Essential for Vesicular Targeting of Synaptotagmin 1.</a> Neuron. 41, 85-99 (2004).</li><li>Kwon, S. E., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+is+dispensable+for+sorting+of+synaptotagmin+1+but+is+critical+for+targeting+of+SV2+and+synaptophysin+to+recycling+synaptic+vesicles.">Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV2 and synaptophysin to recycling synaptic vesicles.</a> Journal of Biological Chemistry. 287, 35658-35668 (2012).</li><li>Afuwape, O. A. T., Wasser, C. R., Schikorski, T., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pool-specific+modification+of+neurotransmitter+release+by+intravesicular+free+radical+generation.">Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.</a> The Journal of Physiology. 595, 1223-1238 (2017).</li><li>Sara, Y., Virmani, T., De&#225;k, F., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+pool+of+vesicles+recycles+at+rest+and+drives+spontaneous+neurotransmission.">An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission.</a> Neuron. 45, 563-573 (2005).</li><li>Wilhelm, B. G., Groemer, T. W., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same+synaptic+vesicles+drive+active+and+spontaneous+release.">The same synaptic vesicles drive active and spontaneous release.</a> Nature Neuroscience. 13, 1454-1456 (2010).</li><li>Bacci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+blockade+of+glutamate+receptors+enhances+presynaptic+release+and+downregulates+the+interaction+between+synaptophysin-synaptobrevin-vesicle-associated+membrane+protein+2.">Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.</a> Journal of Neuroscience. 21, 6588-6596 (2001).</li><li>Piccoli, G., 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=LRRK2+Controls+Synaptic+Vesicle+Storage+and+Mobilization+within+the+Recycling+Pool.">LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool.</a> Journal of Neuroscience. 31, 2225-2237 (2011).</li><li>Tracy, T. E., Yan, J. J., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+knockdown+of+AMPA+receptors+reveals+a+trans-synaptic+signal+for+presynaptic+maturation.">Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation.</a> The EMBO Journal. 30, 1577-1592 (2011).</li><li>Truckenbrdot, S., Viplav, A., Jaehne, S., Vogts, A., Denker, A., Wildhagen, H., Fornasiero, E. F., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+synaptic+vesicles+are+inactivated+by+contamination+with+SNAP25.">Ageing synaptic vesicles are inactivated by contamination with SNAP25.</a> bioRxiv. , 172239 (2017).</li></ol>

Yazarlar ifşa gerek yok.

Orada üç deneyleri rutin olarak sinaptik vezikül (SV) geri dönüşüm çalışırdım. İlk iki kullanımını içerecek bir) Floresan styryl Boyayıcılar FM1-(hangi organelleri endositoz sırasında alınır ve ekzositozu sonra yayımlanan membranlar dahil) 43 gibi; ve b) fluorescently rekombinant SV proteinler (ki, overexpression proteinaceous geri dönüşüm makineleri dahil) öğesini. Ekli fluorophores onların floresans pH bağlı olarak değiştirirseniz, bir SV asidik iç ve ekstraselüler ortamın pH aras?...

Presynaptic özelliklerini değiştirmek, sinaptik plastisite sırasında veya Yanıt pertürbasyon sinaptik fonksiyon olarak nasıl sinaptik vezikül geri dönüşüm eğitim belirlemede önemlidir. Synaptotagmin-1 (Syt1) eğitim antikor alımını SV geri dönüşüm miktarını ölçmenin bir yöntem sağlar. Syt1 Ca2 + sensörü davranır ve nörotransmitter1,2exocytotic serbest bırakılması için gereklidir, SV ilişkili bir proteindir. Bir C-terminal sitoplazmik etki alanı dışında ZF ve SV3içinde N-terminal luminal etki alanı ile bir transmembran proteindir. Ekzositozu sırasında Syt1 luminal etki dış ortamına maruz olur. Bu dış orta, endositoz sırasında içselleştirilmiş olur sitoplazmik etki karşı antikorlar ekleyin. Bu antikorlar da önceden fluorophores veya immunostained ile ikincil antikorlar4,5,6,7ile Birleşik olabilir. Elde edilen immunosignal floresan yoğunluğu SV geri dönüşüm miktarı orantılıdır. Bu yaklaşım, kurucu ve depolarizasyon kaynaklı ZF6,8geri dönüşüm belirlemek için kullanılabilir.
Syt1 alımını deneyleri seyrek transfection hücreleri küçük bir dizi ya da virüs-aracılı gen transferi tabak içinde hemen hemen tüm hücrelere sonra gerçekleştirilebilir. Bizim Yöntem tahlil kalsiyum fosfat9kullanarak birincil hipokampal nöronlar seyrek Çift Kişilik transfection ile birleştirir. Presynapses biriktirmek için bilinen bir rekombinant marker protein fluorescently etiketli Synaptophysin, presynaptic terminalleri bulun ve bizim protein ilgi, Rogdi overexpress için kullanıyoruz. Bu Rogdi hedefleri fonksiyonel synapses ve geri dönüşüm SV etkiler olup olmadığını test bize sağlar. Rogdi kodlama gen aslında Drosophila mutantlar Engelliler bellek10tarafından karakterize bir ekran tespit edilmiştir. İnsanlarda, Kohlschütter-Tönz sendromu denen nadir ve yıkıcı bir hastalık Rogdi gen mutasyonlar neden. Hastalar diş mine malformasyonlar, pharmacoresistant epilepsi ve psikomotor gecikmeler muzdarip; Ancak, gen ürünü hücre altı lokalizasyonu zor11kaldı. Böylece, Syt1 alımını tahlil GFP öğesini Rogdi fonksiyonel sinapslarda9lokalizasyonu için anahtar kanıt sağladı.
Bu alımı teknik birçok faydası vardır. İlk olarak, SV geri dönüşüm hem gerçek zamanlı canlı görüntüleme7,12yaparak ve sonra fiksasyon6,9 Syt1 floresan etiket floresan yoğunluğu ölçme tarafından görülebilir. Ayrıca, çeşitli Syt1 antikor türevleri geliştirilmiştir. Standart immunostaining protokol fiksasyon sonra sonrası ikincil bir antikor ile etiketli etiketsiz türevleri ve önceden eklenmiş bir floresan etiketi önceden konjuge türevleri vardır. Son olarak, floresan antikor tabanlı kullanılan piyasada bulunan ikincil veya konjuge boyalar geniş yelpazesi nedeniyle avantajlıdır.
Ne zaman tamir ve immunostaining nöronlar, ayrıca ek proteinler için leke ve colocalization analiz gerçekleştirmek mümkündür. Bu geri dönüşüm SVs ile ilgili olarak bulundukları belirlemenize yardımcı olabilir. Floresans etiket yoğunluğunu geri dönüşüm SV miktarını doğrudan ölçüsüdür. Buna ek olarak, antikorlar seçmeli olarak yüksek özgüllük ve küçük arka plan floresans4ile sonuçlanan yapıları, Syt1 içeren etiket. Farklı stimulasyon protokolleri de, depolarizasyon arabellekleri veya elektrik stimulasyon protokolleri9,12,13,14gibi kullanılabilir. Ancak, bazal SV geri dönüşüm nöronal kültürler15uyarıcı olmadan ölçülebilir.
Bizim yöntem özellikle fiksasyon sonra Syt1 antikor Anlamazdın çift transfected nöronlar ile ikincil antikor immunolabeling giderir. Ancak, tüm rutin olarak kullanılan türevleri tahlil için izleyiciler Protokolü ihtiyaçlarınıza uyum için size şans vermek amacıyla tartışma bizim bakın.

<table><tbody><tr><td>B27</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7030-50g</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C3306-100g</td><td></td></tr><tr><td>CoolSNAP HQ2</td><td>Photometrics</td><td></td><td></td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Invitrogen</td><td>15230</td><td></td></tr><tr><td>DABCO</td><td>Merck</td><td>8.03456.0100</td><td></td></tr><tr><td>donkey anti mouse Alexa 647</td><td>Jackson-Immunoresearch</td><td>715605151</td><td>antibody</td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>41966</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>Eppendorf tubes</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>multiwell 24 well</td><td>Fisher Scientific</td><td>087721H</td><td></td></tr><tr><td>tube (50 mL)</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>FBS superior</td><td>BiochromAG</td><td>S0615</td><td></td></tr><tr><td>Glucose</td><td>Merck</td><td>1,083,421,000</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14170</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034-500g</td><td></td></tr><tr><td>Hera Cell 150 (Inkubator)</td><td>ThermoElectron Corporation</td><td></td><td></td></tr><tr><td>KCL</td><td>Sigma-Aldrich</td><td>P9541-500g</td><td></td></tr><tr><td>L-Glutamin</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Honeywell</td><td>M0250-500g</td><td></td></tr><tr><td>microscope slides</td><td>Fisher Scientific</td><td>10144633CF</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Mowiol4-88</td><td>Calbiochem</td><td>475904</td><td></td></tr><tr><td>NaCl</td><td>BioFroxx</td><td>1394KG001</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>BioFroxx</td><td>5155KG001</td><td></td></tr><tr><td>Neurobasal</td><td>Invitrogen</td><td>21103049</td><td></td></tr><tr><td>OpenView Experiment Analysis Application</td><td>Free software, see comments</td><td></td><td>written by Noam E. Ziv, Technion &amp;ndash; Israel Institute of Technology, Haifa, Israel</td></tr><tr><td>PBS (10x)</td><td>Roche</td><td>11666789001</td><td></td></tr><tr><td>Optimem</td><td>Invitrogen</td><td>31985</td><td></td></tr><tr><td>Penstrep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>P6148-1kg</td><td></td></tr><tr><td>safety hood</td><td>ThermoElectron</td><td></td><td>Serial No. 40649111</td></tr><tr><td>Sucrose</td><td>neoFroxx</td><td>1104kg001</td><td></td></tr><tr><td>Synaptotagmin1</td><td>Synaptic Systems</td><td>105311</td><td>mouse monoclonal; clone 604.2</td></tr><tr><td>Triton X-100</td><td>Merck</td><td>1,086,031,000</td><td></td></tr><tr><td>Vortex Genius 3&amp;nbsp;</td><td>IKA</td><td>3340001</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>1004</td><td></td></tr><tr><td>Zeiss Observer. Z1&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins

Active presynaptic sinir terminallerine sinaptik veziküller exo - ve endositoz döngüleri tabi. Geri dönüşüm sırasında luminal etki alanları SV transmembran proteinler, hücre yüzeyi maruz olur. Bu proteinler biridir Synaptotagmin-1 (Syt1). Syt1, bir kez kültür ortamına eklenen luminal etki karşı bir antikor exo-endocytotic döngüsü sırasında alınır. Bu alımı SV geri dönüşüm miktarı ile doğru orantılıdır ve ayirt sayılabilir. Burada, Syt1 antikor alımını kültürlü hipokampal nöronlar Çift Kişilik transfection ile birleştirmek. Bu bize (1) yerelleştirmek rekombinant presynaptic işaretçisi Synaptophysin ifade üzerinde temel alan presynaptic siteleri (2) Syt1 alımını kullanarak onların işlevlerini belirleyin ve karakterize (3) hedefleme ve etkileri ilgi, GFP-Rogdi bir protein sağlar.

Beklenen sonuç bu yaklaşımın coverslip başına yaklaşık 50 nöronlar çift transfected bir yoğunluk iyi başına 50.000 nöronların, yerini. Her nöron axon birikimi kümeleri ofSVs gösteren fluorescently öğesini Synaptophysin, birden çok etkin noktaları göstermek için bekleniyor. Fonksiyonel presynaptic siteler, rekombinant Synaptophysin sinyal punctate Syt1 floresan ile colocalizes. Çift Kişilik transfection, GFP-Rogdi ilgi (Şekil 1) prot...

Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Biz optik bir tahlil sinaptik vezikül (SV) kültürlü nöronlarda geri dönüşüm için açıklar. Bu iletişim kuralı bir presynaptic işaret ve protein ilgi ifade etmek için çift transfection ile birleştirerek presynaptic siteleri, onların sinaptik vezikül kapasitesi geri dönüşüm bulun ve faiz protein rolünü belirlemek için bize izin verir.

Sinaptik vezikül Presynaptic protein Overexpressing kültürlü nöronlarda geri dönüşüm için optik bir tahlil

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

an-optical-assay-for-synaptic-vesicle-recycling-cultured-neurons

Research

JoVE Journal

Neuroscience

7.4K Görüntüleme.  University Medical Centre Göttingen. Biz optik bir tahlil sinaptik vezikül (SV) kültürlü nöronlarda geri dönüşüm için açıklar. Bu iletişim kuralı bir presynaptic işaret ve protein ilgi ifade etmek için çift transfection ile birleştirerek presynaptic siteleri, onların sinaptik vezikül kapasitesi geri dönüşüm bulun ve faiz protein rolünü belirlemek için bize izin verir.

Video: An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Sinapslar miktarının: Synapse Numarası ölçmek için bir İmmünositokimya tabanlı Testi

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Nörobilim

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Kemirgen Beyin Dilimleri Nöronlar Vibrodissociation Sinaptik İletim ve Görüntü Presinaptik Terminalleri Eğitim

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM Boyalar Kültür Serebellar Granül Nöronlar Synaptic veziküllü Havuzu Yenileme Kantitatif Analiz

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

Uyarılmış, Spontan ve Minyatür Synaptic Faaliyetleri Sırasında FM boyalar kullanarak sinaptik vezikül Geri Dönüşüm Sınavı

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM ve pH-duyarlı GFP-sondalar SH-SY5Y Nöroblastom Hücrelerinde nörotransmitter Vesikül Dynamics değerlendirin: Hücre Görüntüleme ve Veri Analizi

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

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 ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

AMPA izlemek için bir yüksek-içerik tahlil reseptör ticareti

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

Presynapse oluşum assay kullanarak Presynapse Organizatör boncuk ve "neuron Ball" kültür

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Amiyotrofik Lateral Skleroz Modellerinde Sinaptik Fonksiyonların Gerçek Zamanlı Floresan Ölçümü

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) in response to an action potential (AP). Since SVs are recycled locally at the presynaptic terminal, coordination of SV exocytosis and endocytosis is important for sustained synaptic transmission. A pH-sensitive green fluorescent protein, called pHluorin, provides a powerful tool to monitor SV exo/endocytosis by targeting it to the SV lumen. However, tracking AP-driven SV recycling with the pHluorin-based probes is still largely limited to in vitro culture preparations because the introduction of genetically encoded probes and subsequent optical imaging is technically challenging in general for in vivo animal models or tissue preparations. Zebrafish is a model system offering valuable features, including ease of genetic manipulation, optical clarity, and rapid external development. We recently generated a transgenic zebrafish that highly expresses a pHluorin-labeled probe at motor neuron terminals and developed a protocol to monitor AP-driven SV exo/endocytosis at the neuromuscular junction (NMJ), a well-established synapse model that forms in vivo. In this article, we show how to prepare larval zebrafish NMJ preparation suitable for pHluorin imaging. We also show that the preparation allows time-lapse imaging under conventional upright epifluorescence microscope, providing a cost-effective platform for analyzing NMJ function.

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

Diseke Larva Zebra Balığının Nöromüsküler Kavşağında Sinaptik Vezikül Geri Dönüşümünün Canlı Görüntülenmesi

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) ...