Bu araştırma Ulusal Sağlık Enstitüleri (K99MH104595) ve T.J.P. NICHD intramural araştırma programı tarafından desteklenen Gord Fishell, kimin Laboratuarı'nda bu yaklaşım başlangıçta kurulmuş teşekkür ediyoruz.

<ol>
	<li>Bozzi, Y., Casarosa, S., Caleo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epilepsy+as+a+neurodevelopmental+disorder.">Epilepsy as a neurodevelopmental disorder.</a> Front Psychiatry. 3 (19), (2012).</li><li>Takano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+Dysfunction+in+Syndromic+Autism:+Recent+Advances.">Interneuron Dysfunction in Syndromic Autism: Recent Advances.</a> Dev Neurosci. , (2015).</li><li>Inan, M., Petros, T. J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Losing+your+inhibition:+linking+cortical+GABAergic+interneurons+to+schizophrenia.">Losing your inhibition: linking cortical GABAergic interneurons to schizophrenia.</a> Neurobiol Dis. 53, 36-48 (2013).</li><li>Batista-Brito, R., Machold, R., Klein, C., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+cortical+interneuron+precursors+is+prescient+of+their+mature+function.">Gene expression in cortical interneuron precursors is prescient of their mature function.</a> Cereb Cortex. 18 (10), 2306-2317 (2008).</li><li>Flames, 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=Delineation+of+multiple+subpallial+progenitor+domains+by+the+combinatorial+expression+of+transcriptional+codes.">Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes.</a> J Neurosci. 27 (36), 9682-9695 (2007).</li><li>Wonders, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+bias+for+the+origins+of+interneuron+subgroups+within+the+medial+ganglionic+eminence.">A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence.</a> Dev Biol. 314 (1), 127-136 (2008).</li><li>Inan, M., Welagen, J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+bias+in+the+mitotic+origins+of+somatostatin-+and+parvalbumin-expressing+interneuron+subgroups+and+the+chandelier+subtype+in+the+medial+ganglionic+eminence.">Spatial and temporal bias in the mitotic origins of somatostatin- and parvalbumin-expressing interneuron subgroups and the chandelier subtype in the medial ganglionic eminence.</a> Cereb Cortex. 22 (4), 820-827 (2012).</li><li>Petros, T. J., Bultje, R. S., Ross, M. E., Fishell, G., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+versus+Basal+Neurogenesis+Directs+Cortical+Interneuron+Subclass+Fate.">Apical versus Basal Neurogenesis Directs Cortical Interneuron Subclass Fate.</a> Cell Rep. 13 (6), 1090-1095 (2015).</li><li>Taniguchi, H., Lu, J., Huang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+and+temporal+origin+of+chandelier+cells+in+mouse+neocortex.">The spatial and temporal origin of chandelier cells in mouse neocortex.</a> Science. 339 (6115), 70-74 (2013).</li><li>Bandler, R. C., Mayer, C., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+interneuron+specification:+the+juncture+of+genes,+time+and+geometry.">Cortical interneuron specification: the juncture of genes, time and geometry.</a> Curr Opin Neurobiol. 42, 17-24 (2017).</li><li>Kepecs, A., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+cell+types+are+fit+to+function.">Interneuron cell types are fit to function.</a> Nature. 505 (7483), 318-326 (2014).</li><li>Mayer, 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=Developmental+diversification+of+cortical+inhibitory+interneurons.">Developmental diversification of cortical inhibitory interneurons.</a> Nature. 555 (7697), 457-462 (2018).</li><li>Mi, 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=Early+emergence+of+cortical+interneuron+diversity+in+the+mouse+embryo.">Early emergence of cortical interneuron diversity in the mouse embryo.</a> Science. 360 (6384), 81-85 (2018).</li><li>Alvarez-Dolado, 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=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J Neurosci. 26 (28), 7380-7389 (2006).</li><li>Baraban, S. 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=Reduction+of+seizures+by+transplantation+of+cortical+GABAergic+interneuron+precursors+into+Kv1.1+mutant+mice.">Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.</a> Proc Natl Acad Sci U S A. 106 (36), 15472-15477 (2009).</li><li>De la Cruz, E., 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=Interneuron+progenitors+attenuate+the+power+of+acute+focal+ictal+discharges.">Interneuron progenitors attenuate the power of acute focal ictal discharges.</a> Neurotherapeutics. 8 (4), 763-773 (2011).</li><li>Gilani, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+precursor+transplants+in+adult+hippocampus+reverse+psychosis-relevant+features+in+a+mouse+model+of+hippocampal+disinhibition.">Interneuron precursor transplants in adult hippocampus reverse psychosis-relevant features in a mouse model of hippocampal disinhibition.</a> Proc Natl Acad Sci U S A. 111 (20), 7450-7455 (2014).</li><li>Larimer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caudal+Ganglionic+Eminence+Precursor+Transplants+Disperse+and+Integrate+as+Lineage-Specific+Interneurons+but+Do+Not+Induce+Cortical+Plasticity.">Caudal Ganglionic Eminence Precursor Transplants Disperse and Integrate as Lineage-Specific Interneurons but Do Not Induce Cortical Plasticity.</a> Cell Rep. 16 (5), 1391-1404 (2016).</li><li>Martinez-Cerdeno, V., 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=Embryonic+MGE+precursor+cells+grafted+into+adult+rat+striatum+integrate+and+ameliorate+motor+symptoms+in+6-OHDA-lesioned+rats.">Embryonic MGE precursor cells grafted into adult rat striatum integrate and ameliorate motor symptoms in 6-OHDA-lesioned rats.</a> Cell Stem Cell. 6 (3), 238-250 (2010).</li><li>Quattrocolo, G., Fishell, G., Petros, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+Transplantations+Reveal+Environmental+Influences+on+Interneuron+Diversity+and+Maturation.">Heterotopic Transplantations Reveal Environmental Influences on Interneuron Diversity and Maturation.</a> Cell Rep. 21 (3), 721-731 (2017).</li><li>Xu, Q., Tam, M., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+Nkx2.1-lineage+cells+in+the+mouse+telencephalon.">Fate mapping Nkx2.1-lineage cells in the mouse telencephalon.</a> J Comp Neurol. 506 (1), 16-29 (2008).</li><li>Thompson, L., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+differentiation,+and+connectivity+of+ventral+mesencephalic+dopamine+neurons+following+transplantation.">Survival, differentiation, and connectivity of ventral mesencephalic dopamine neurons following transplantation.</a> Prog Brain Res. 200, 61-95 (2012).</li><li>Liang, Y., Agren, L., Lyczek, A., Walczak, P., Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cell+survival+in+mouse+brain+can+be+improved+by+co-transplantation+of+helper+cells+expressing+bFGF+under+doxycycline+control.">Neural progenitor cell survival in mouse brain can be improved by co-transplantation of helper cells expressing bFGF under doxycycline control.</a> Exp Neurol. 247, 73-79 (2013).</li></ol>

Yazarlar ifşa gerek yok.

Önemli bir özelliği, bu iletişim kuralı hücrelerin Beka maksimize. Doku ve hücreler her zaman buz gibi carboxygenated sACSF sağlama hücre survival tanıtmak gereklidir. Bu bir verimli diseksiyon ve çeşitli çözüm ve beyin ortamı dışında hücreleri harcamak süreyi en aza indirmek için ayrılma strateji gerektirir. Disseke ve nakledilen beyin bölgeleri sayısına bağlı olarak, diseksiyon ve/veya nakli adımda deneme uzunluğu azaltmak için yardımcı bir arkadaşın olduğu yararlı olabilir. Sağl?...

Düzgün kortikal uyarıcı projeksiyon nöronlar ve inhibitör GABAergic interneurons, farklı türleri morfoloji, elektrofizyolojik özellikleri, connectivity ile son derece heterojen bir nüfus arasında bir denge gerektirir ve nörokimyasal işaretleri. Anormal gelişimi ve interneurons (ve belirli interneuron alt gruplar) fonksiyonu bağlı psikiyatrik bozukluklar şizofreni, otizm ve epilepsi1,2,3gibi pathobiology. Ayrıca, birçok genler bu beyin bozukluklarında karıştığı güçlü genç interneurons4' te zenginleştirilmiş. Böylece, interneuron kader tayini ve olgunlaşma düzenleyen mekanizmalar daha büyük bir anlayış normal gelişim ve potansiyel etiologies çok sayıda beyin hastalıkları anlamak için gereklidir.
Ön interneurons öncelikle iki geçici embriyonik yapılardan, medial ve kaudal ganglionic efendiler tarihi (MGE ve CGE, sırasıyla). Bu postmitotic hücreler (interneuron öncüleri) nerede onlar çok çeşitli devreleri entegre telencephalon dağıtmak için uzun süren teğet geçiş aşaması geçmesi. MGE kaynaklı interneurons oluşur üç büyük ölçüde örtüşen, neurochemically tanımlı alt gruplar: hızlı parvalbumin (PV+) interneurons, hızlı-somatostatin (SST+) interneurons, spiking yükseliyor ve geç yükseldi nöronal nitrik hipokampal neurogliaform ve Ivy hücreleri teşkil oksit sentaz (nNOS+) interneurons. Çok sayıda labs düzenleyen ilk kader kararlar PV+ veya SST + interneurons, morfojenlerdeki, interneuron öncüleri, Doğum tarihi ve nörojenik bölümü modu kayma degradeleri de dahil olmak üzere çeşitli mekanizmalar içinde MGE belirledik 5 , 6 , 7 , 8 , 9 , 10. interneurons başlangıçta 'Kardinal sınıfa' ayırt etmek ve onların çevre11ile etkileşimde bulunan 'kesin sınıfa' aşamalı olarak olgun önerilmiştir. Son kanıtlar bu hücreler ganglionic efendiler, postmitotic olmak gibi bazı olgun interneuron alt türlerinden genetik olarak kablolu erken tanımlanmış içsel genetik programlar daha önce daha büyük bir rol oynayabilir gösteren olabileceğini gösterir takdir12,13. Ancak, içsel genetik programlar için sürücü farklılaşma ayrı interneuron alt türlerini içine çevre ipuçları ile etkileşimini anahtar soru büyük ölçüde keşfedilmemiş kalır.
Çok sayıda çalışma embriyonik MGE hücreleri doğrudan beyin bölgeleri, hücreler olgun aşılı ve genellikle yerel endojen devresi14,15inhibe GABA bırakın uzlaşma sonuçları ile çeşitli içine nakledilen, 16,17,18,19. İnsan indüklenmiş pluripotent kök hücre (hIPSC) kullanarak önemli ilgi oluşturulan bu gözlemler umut verici-beyin hastalıkları çeşitli tedavi interneurons türetilmiş. Ancak, bu aşılı hücreler olgun interneurons beklenen türlerini olgun olmadığını çok az bu çalışmaların değerlendirmek, ne zaman bir kritik bir bileşeni translasyonel yaklaşımlar hakkında düşünüyor.
Nasıl interneuron farklılaşma ve olgunlaşma çevre etkileri gidermek amacıyla bir strateji aşılı interneurons şekil-in belgili tanımlık ev sahibi kabul olup olmadığını incelemek için yeni beyin ortamlara olgunlaşmamış interneuron öncüleri nakli için geliştirildi çevre veya özelliklerini donör çevre20korumak. MGE nakli MGE nüfus interneuron ve çok sayıda beyin bölgeleri21dağıtmak GABAergic projeksiyon hücreler içerdiğinden bu soruyu çözmek uygun değildir. Nerede bu MGE hücreler göç var bilmeden, bir tam olarak nasıl bu nakillerine beyin ortamı tarafından etkilenen değerlendirmek değil. İnterneuron öncüleri erken postnatal timepoints, hasat tarafından bu sorunu kendi geçiş tamamlayan ve hedeflerine ulaştı olgunlaşmamış hücreleri elde ederek hile beyin bölgesi ama çevre en az etkileşim yok. Differentially farklı beyin bölgeleri arasında ifade edilir interneurons özellikleri üzerinde odaklanarak, kimse o zaman nasıl interneuron özellikleri ana ortamı değiştirir belirleyebilirsiniz. Bu protokol için özetlenen genel yaklaşım herhangi bir araştırmacı için uygulanabilir olmalıdır meydan yeni bir ortamda ne kadar genç nöronlar incelemek istiyor ne zaman davranırlar.

<table><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6297</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S0751</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Brain Matrices</td><td>Roboz</td><td>SA-2165</td><td>Only needed if harvesting striatum</td></tr><tr><td>Fine point Dumont Forceps</td><td>Roboz</td><td>RS-4978</td><td></td></tr><tr><td>Microdissecting scissors</td><td>Roboz</td><td>RS-5940</td><td></td></tr><tr><td>Razor blades</td><td>ThermoFisher</td><td>12-640</td><td></td></tr><tr><td>Pasteur pipettes</td><td>ThermoFisher</td><td>1367820C</td><td></td></tr><tr><td>Nanoject III</td><td>Drummond</td><td>3-000-207</td><td></td></tr><tr><td>Manual Manipulator w/ stand</td><td>World Precision Instruments&amp;nbsp;</td><td>M3301R/M10</td><td></td></tr><tr><td>5 ml round bottom plastic tubes</td><td>ThermoFisher</td><td>149591A</td><td></td></tr><tr><td>60 mm Petri dishes</td><td>ThermoFisher</td><td>12556001</td><td></td></tr><tr><td>100 mm Petri dishes</td><td>ThermoFisher</td><td>12565100</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>10165921001</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher</td><td>16140063</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>4716728001</td><td></td></tr><tr><td>Celltrics 50um filters</td><td>Sysmex</td><td>04-0042327</td><td></td></tr><tr><td>Trypan blue</td><td>ThermoFisher</td><td>15-250-061</td><td></td></tr><tr><td>Hemocytometer</td><td>ThermoFisher</td><td>02-671-6</td><td></td></tr></tbody></table>

homochronic transplantation of interneuron precursors into early postnatal mouse brains

Nöronal kader tayini ve olgunlaşma genetik programlarla çevre sinyalleri arasında karmaşık bir etkileşim gerektirir. Ancak, bu Farklılaşma süreci düzenleyen dışsal mekanizmaları vs içsel rolleri disentangling tüm gelişimsel neurobiologists için bir bilmece olduğunu. Bu sorun GABAergic interneurons, geçici embriyonik yapılardan tarihi ve telencephalon dağıtmak için uzun süren bir göçmen faz tabi bir inanılmaz türdeş olmayan hücre nüfus için büyütülür. Ne kadar farklı beyin ortamları etkiler interneuron kader ve olgunlaşma keşfetmek için geliştirdiğimiz fluorescently etiketli olgunlaşmamış interneuron öncüleri (P0-P2) yeni doğan farelerde belirli beyin bölgelerinden hasat için bir iletişim kuralı. Bu yaşta interneuron geçiş neredeyse tamamlanmış ve bu hücreler nispeten küçük sinaptik entegrasyonu ile son istirahat ortamlarında bulunan. Toplama tek hücre çözümler akış sitometresi ile bu interneuron öncüleri P0-P2 wildtype Doğum sonrası pups nakledilen. Her iki Homotopik yaparak (örneğin, korteks korteks) veya heterotopik (örneğin, korteks hipokampus) nakillerine, bir can değerlendirmek yeni beyin ortamlarda olgunlaşmamış interneurons zor onların kaderi, olgunlaşma ve devre entegrasyon nasıl etkiler. Beyin yetişkin fareler hasat ve posthoc analiz aşılı hücrelerde immunohistokimyasal, dahil olmak üzere, çok çeşitli ile denetlesinler elektrofizyolojik ve transkripsiyon profil oluşturma. Müfettişler nasıl ayrı beyin ortam tahlil için bir strateji ile çok sayıda nöron gelişim yönlerini etkilemeye ve belirli nöronal özellikleri öncelikle kablolu genetik programlar tarafından tahrik edilmektedir tanımlamak bu genel yaklaşım sağlar veya çevre ipuçları.

Bu iletişim kuralı erken postnatal beyin (Şekil 1-2) belirli beyin bölgelerine hasat, tek hücre dissociations interneuron öncüleri, toplamak ve saf WT çeşitli beyin bölgeleri içine bu hücrelerin nakli gösterilmiştir postnatal pups (Şekil 3). Posthoc analiz için interneuron habercisi Greftler alınan beyin P30-35 arasında hücre morfolojisi, nörokimyasal işaretleri ve elektrofizyolojik özellikl...

Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Yeni beyin bölgelerindeki genç nöronlar zorlu nasıl çevre nöronal kader ve olgunlaşma sculpts içine önemli anlayışlar ortaya çıkarabilir. Bu iletişim kuralı interneuron öncüleri belirli beyin bölgelerinden hasat ve onları her iki homotopically nakli için bir yordam açıklanır veya beyin ve postnatal yavrularını içine heterotopically.

Erken Postnatal fare beyin içine Interneuron öncüleri Homochronic nakli

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

homochronic-transplantation-interneuron-precursors-into-early

Research

JoVE Journal

Neuroscience

7.8K Görüntüleme.  Norwegian University of Science and Technology. Yeni beyin bölgelerindeki genç nöronlar zorlu nasıl çevre nöronal kader ve olgunlaşma sculpts içine önemli anlayışlar ortaya çıkarabilir. Bu iletişim kuralı interneuron öncüleri belirli beyin bölgelerinden hasat ve onları her iki homotopically nakli için bir yordam açıklanır veya beyin ve postnatal yavrularını içine heterotopically.

Video: Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

In vivo Nöromimarlık çalışmaları için tasarlanan nöronal öncülerin intraventriküler nakli

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetik

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Yenidoğan subventricular Bölge Elektroporasyon

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Nörobilim

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Gelişmekte Fare Embriyonik Cortex mitoz Canlı Görüntüleme

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Fare embriyonik kök hücre farklılaşma içine kortikal Interneuron Kara filmin tarih öncesi

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

Embriyonik Neurogenesis çalışmak için bir Model olarak fare arka beyin

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Erken Doğum Sonrası Fare Beyinlerine Kemogenetik Mühendislik Kortikal İnkornöron Progenitorlarının Nakli

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

İntraserebral Transplantasyon ve Fare Beynindeki İnsan Nöral Progenitör Hücrelerinin İnVivo Biyolüminesans İzlenmesi

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

İnsan Kök Hücre Kaynaklı GABAerjik Nöronların Nörogelişimsel Bozuklukları Hafifletmek İçin Erken Postnatal Fare Hipokampüsüne Transplantasyonu

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

Gelişmekte Olan Fare Beyninde Nöronal ve Glial Hücre Dinamiğinin Görselleştirilmesi

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Yazar Spotlight: Gelişmekte Olan Beyinde Hücre Göçünü ve Gen Rollerini Keşfetmek

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

Embriyonik Fare Beyin Dilimlerinde Göç Eden Nöronların ve Glial Progenitörlerin Hızlandırılmış Görüntülenmesi

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Erken Postnatal fare beyin içine Interneuron öncüleri Homochronic nakli

Özet

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Bu videodaki bölümler

In vivo Nöromimarlık çalışmaları için tasarlanan nöronal öncülerin intraventriküler nakli

Yenidoğan subventricular Bölge Elektroporasyon

Gelişmekte Fare Embriyonik Cortex mitoz Canlı Görüntüleme

Fare embriyonik kök hücre farklılaşma içine kortikal Interneuron Kara filmin tarih öncesi

Embriyonik Neurogenesis çalışmak için bir Model olarak fare arka beyin

Erken Doğum Sonrası Fare Beyinlerine Kemogenetik Mühendislik Kortikal İnkornöron Progenitorlarının Nakli

İntraserebral Transplantasyon ve Fare Beynindeki İnsan Nöral Progenitör Hücrelerinin İnVivo Biyolüminesans İzlenmesi

İnsan Kök Hücre Kaynaklı GABAerjik Nöronların Nörogelişimsel Bozuklukları Hafifletmek İçin Erken Postnatal Fare Hipokampüsüne Transplantasyonu

Embriyonik Fare Beyin Dilimlerinde Göç Eden Nöronların ve Glial Progenitörlerin Hızlandırılmış Görüntülenmesi

Embriyonik Fare Beyin Dilimlerinde Göç Eden Nöronların ve Glial Progenitörlerin Hızlandırılmış Görüntülenmesi