Bu çalışma NINDS ve S.Ge. Amerikan Kalp Derneği tarafından finanse edildi Bu yaklaşım başlangıçta Johns Hopkins Üniversitesi&#39;nden Dr. Hongjun Song laboratuvarda yazar tarafından kurulmuş ve geliştirilmiştir. Dr Hongjun Song Biz onun rehberlik ve destek için teşekkür etmek istiyorum.

1. Virüs Enjeksiyon Yüksek titrede mühendislik retrovirüs (1 × 10 9 birim / ml) co-retroviral vektörlerin transfeksiyon tarafından üretilen ve viral süpernatant Ultrasantrifügasyon tarafından takip HEK293T hücreleri içine VSVG. Kaynakları ve üretim yöntemleri, mükemmel vallahi gösteri (3) bkz. Not: Genç yetişkin (4-6 hafta) kadın C57BL / 6 fareler (Charles Nehri) standart şartlar altında muhafaza edilir. Tüm işlemler, Ulusal Araştırma Konseyi, Stony Brook Üniversitesi IACUC tarafından onaylanmış bir protokol kapsamında laboratuvar hayvanlarının bakımı ve kullanımı Kılavuzu izleyin. <ol><li> Buz üzerinde hayvan başına dondurulmuş retrovirüs çözülme 2ul. </li><li> Anesteziyoloji (100 mg ketamin + gram vücut ağırlığı başına 10 mg ksilizin) ve hayvan stereotaksik bir çerçeve (Steolting) monte edilir. Kafasına saç çıkarın ve% 70 etanol ile cilt silin. </li><li> Açığa kafatası ve diş aşağıdaki koordinatları bir matkap kullanarak (0.6mm matkap ucu) dört sığ delik: <ul><li> anterioposterior = -2 mm bregma; yanal = ± 1.6 mm; ventral = 2.5 mm; anterioposterior bregma = -3 mm; yanal = ± 2.6 mm; ventral = 3.2 mm. </li><li> anterioposterior = -2 mm bregma; yanal = ± 1.6 mm; ventral = 2.5 mm; anterioposterior bregma = -3 mm; yanal = ± 2.6 mm; ventral = 3.2 mm. </li></ul></li><li> Mount 1μl Hamilton, düz ucu şırınga ve sitenin yüzde 0,5 &#39;ul retrovirüs 0.25 ul / dak hızında dentat girus içine enjekte edilir. Yavaş yavaş sıvı akışı önlemek için şırınga çekilmeden önce her enjeksiyondan sonra (ventral derinliği için yukarıdaki koordinatları bakınız.) Pause 2 dakika. Enjeksiyonlar arasındaki kısaca steril PBS ile şırınga ucu ve kan izlerini kaldırmak için bir aplikatör dış yüzeyi durulayın. </li><li> , Steril cerrahi sütür malzeme ile yara (P-1 Tipi Boyutu 6-0) kapatın ve enjeksiyondan sonra istenilen zaman aşamalarına kadar standart konut koşulları hayvan . </li></ol> 2. Dilim Hazırlık Erişkin farelerde yüksek kaliteli akut dilimler elde etmek için, (aşağıya bakın) dilim hazırlanması için modifiye edilmiş bir kesim çözüm. <ol><li> 0-4 ° C,% 95 ile buz ve kabarcık O 2 -5% CO 2 için en az 30 dakika ön-chill kesim çözüm . </li><li> Anesteziyoloji ve buz gibi oksijen doymuş kesim çözüm ile bir hayvan transcardially serpmek. </li><li> Hızlı soğuk, oksijenli kesim çözüm filtre kağıdı ile önceden ıslatılmış bir petri yemek beyin kaldırmak. Beyincik kesiniz ve en az 1mm anterior montaj yüzeyine (görünür) enjeksiyonu koordinatları dilim. Tutkal beyin üzerine bir vibrotome aşamada soğuk ve oksijenli kesim çözümü ile dolu kesme odasına monte. </li><li> Koronal veya yatay dilim (300-350μm) hazırlayın ve oda sıcaklığında ACSF içeren bir kuluçka odasına, büyük çaplı bir pipet yardımıyla transfer% 95 O 2 /% 5 CO 2 ile kabarmış. </li><li> Dilimleri, sürekli köpürme, 30-60 dakika süreyle 32 ° C&#39;de inkübe edin. Kayıt süresi boyunca oda sıcaklığında odasına dönün. </li></ol> 3. Elektrofizyolojik deneyler <ol><li> 34 ° C - 32 ° C&#39;de sürekli oksijen doymuş ACSF ile perfüze kayıt odasına Dilimleri aktarılır </li><li> Bipolar tungsten stimülasyon elektrot, düşük bir büyütme mikroskop objektif (10X) kullanarak dentat girus moleküler tabaka yerleştirilir. </li><li> Alt-granüler bölge / granüler hücre katmanı Yenidoğan KEGM GFP veya dTomato ifade tarafından tespit edilir. Tüm hücre patchclamp kayıt altında yeni doğmuş nöronlar yüksek büyütme (40X) yapılır. </li><li> Kaydedilen hücreleri Ateşleme özellikleri akım kelepçe modunda altında akımların bir dizi enjeksiyon yoluyla elde edilir. </li><li> Elektrik uyaranlara kayıt yazılımı tarafından kontrol edilen bir stimülasyon izolatör üzerinden uyarılması elektrot tarafından teslim ve kaydedilir yenidoğan KEGM postsinaptik akımların uyarılmış. </li></ol> 4. Veri Analizi Yetişkin doğumlu nöronların farklı gelişim evreleri, uyarılmış postsinaptik yanıtlar genlikleri analiz edilmektedir. 5. Temsilcisi Sonuçlar Yukarıdaki protokolünü kullanarak, Şekil 1 &#39;e benzer yenidoğan dentat girus nöronlarda GFP ifade yaygındır. Yeni doğan nöronlar kolayca görüntülenebilmekte ve hem dendrit ve aksonlar sağlam etiketli olduğunu unutmayın. Kalite ve virüs titresi, kullanılan organizatörü ve in vivo ifade uzunluğu, ince DGC aksonlar ve küçük dikenler İfade bağlıdır. Bizim ellerde, yenidoğan hücreleri ve alt hücresel organelleri genellikle enjeksiyonu takiben bir kaç gün içinde görünür ve omurga ifade gelişiminin erken aşamalarında takip edilebilir. Wfarklı zaman aşamasında yeni doğmuş nöronlar delik hücre kayıtları benzersiz yeni doğan hücre özelliklerini, örneğin aksiyon potansiyelleri (Şekil 2a), sinaptik entegrasyon sürecinin yanı sıra olgunlaşma (Şekil 2b) sırasında mevcut nöral devrelerin çalışma izin verir. <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="Şekil 1" /> Şekil 1 yetişkin bir farenin yatay bir dentat girus bölümünde yeni doğan nöronlar 2 foton konfokal görüntü. Yeşil hücrelerin 21dpi (gün enjeksiyon) GFP ifade pUX-GFP retrovirüs ile etiketli yeni doğan KEGM. <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="Şekil 2" /> Şekil 2) Aksiyon potansiyeli, 21, yeni doğan bir DGC özellikleri ateş dpi b) temsilcisi, 21 dpi çözünürlükte yeni doğmuş bir DGC kaydedilmiş eksitatör postsinaptik akımlar (EPSCs ) uyandırdı.

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

Yetişkin memelilerin beyinlerinin hippocampus bölgesinde Sürekli nöron olgun beyindeki nöronal gelişim ve entegrasyon çalışmak için benzersiz bir deneysel model sistemi sağlar. Burada sunulan protokol retroviral etiketleme ve elektrofizyolojik yöntemler, yetişkin farelerin beyinlerinde yenidoğan dentat granül nöronların entegrasyonu çalışma birleştirir. Deneylerde başarılı olmasını sağlamak için, prosedürün kritik adımlar sırasında aşağıdaki öneriyor: Istenmeyen enfeksiyon ve inflamasyon önlemek için ameliyat öncesi tüm araçlar (otoklav, sterilizatör herhangi bir tür veya% 70 etanol) sterilize edilmelidir. Enjeksiyondan önce şırınga iğne ucu steril PBS yıkamak için kullanın. Virüs enjeksiyon için, hayvanların iyi stereotaksik cihaz monte edilmesi gerekir ve enjeksiyon sırasında gereksiz hareketinin kaynakları minimize edilmelidir. Kafası iyi sabit ve sağlam olduğundan emin olun ve seviyesi bregma ve lambda hesaplanırken enjeksiyon koordinatları önce burun farenin konumunu ayarlamak. Oda sıcaklığında maruz kalma en aza indirmek için virüs hızla şırıngaya Extract - retrovirüsler özellikle ısıya duyarlı. Basınç hasarı en aza indirmek için tavsiye edilen oranda yavaş virüs enjekte edilir. Önerilen düz uçlu bir enjektör (vs ortak Eğimli ucu) eşit olarak dağıtılmış bir dentat girus enfeksiyon alan sağlayacaktır. Hazırlamadan önce dilim, çözüm kesme ve ACSF kuluçka oksijen çözümleri doyurmak için izin vermek için en az 30 dakika süreyle% 95 O 2 -5% CO 2 ile kabarmış olmalıdır . Hayvanlar hızla perfüze ve doku soğuk, en iyi dilim kalitesi için oksijen doymuş çözüm sağlanmalıdır. Postsinaptik bir yanıt gelene kadar kayıt için, yavaş yavaş uyaran yoğunluğunu artırır. Dentat girus moleküler katman içinde gerektiğinde uyarı elektrot yerini değiştirin. Özet olarak, önerilen yaklaşım, aktif, olgun devresi içine erken evre entegrasyonu sırasında farklı özellikleri ve yetişkin doğumlu nöronların olası fonksiyonel rolleri keşfetmek için bir yol sunar.

<ul><li> Kesme: 110 kolin klorür, 2.5 KCl, 1.3 KH 2 PO 4, 25.0 NaHCO 3, 0.5 CaCl 2, 7 MgCl 2, 10 dekstroz, 1.3 sodyum askorbat, 0.6 sodyum piruvat, 5.0 kynurenic asit çözeltisi (mM). </li><li> Yapay beyin-omurilik sıvısı (ACSF, mM): 125,0 NaCl, NaHCO 3 25.0, 2.5 KCl, CaCl 2, 2, 1.3 KH 2 PO 4, 1.3 MgCl 2, 1.3 sodyum askorbat, 0.6 sodyum piruvat, 10 dekstroz . </li><li> Tam hücreli patchclamp İç çözeltisi (mM): 120 Potasyum glukonat, 15 KCl, 4 MgCl 2, 0.1 EGTA, 10,0 Hepes, 4 MgATP, 0.3, Na 3 GTP, 7 phosphocreatine (pH 7.4, 300 mOsm). </li></ul>

studying the integration of adult-born neurons

Nöron lateral ventrikülün alt-ventriküler zon (SVZ) ve hayatı boyunca hipokampal dentat girus alt granüler bölgesi (SGZ) yetişkin memelilerin beyinlerinde oluşur. Önceki raporlar, epilepsi, şizofreni, depresyon ve anksiyete (1) de dahil olmak üzere çeşitli beyin bozuklukları, yetişkin hipokampal nöron ile ilişkili olduğunu göstermiştir. Normal ve anormal yetişkin doğumlu nöron entegrasyon süreci Deşifre bu hastalıkların etyolojisi ışık tutacak ve yeni tedavilerin geliştirilmesi bilgilendirebilir. SGZ yetişkin nöron kaderi şartname, göç, sinaptik bütünleşme ve olgunlaşma aşamaları da dahil olmak üzere, embriyonik ve post-natal nöronal gelişim aynalar. Ancak, tam entegrasyon, 6 haftalık uzun bir süre içinde oluşur. İlk sinaptik giriş yetişkin doğumlu SGZ dentat granül hücreleri (KEGM), 14 gün (2) glutamaterjik girişi takiben, GABAerjik . Dentat girus yetişkin doğumlu nöronların devre oluşumunu düzenleyen özgü faktörler henüz bilinmemektedir. Laboratuvarımız bu hızla çoğalan hücreleri floresan proteinleri ve hipotez düzenleyici genler teslim Moloney fare lösemi virüsü dayalı bir çoğaltma eksikliği retroviral vektör kullanır. Bu viral teknik hücre doğum tarihini, soy, morfoloji ve sinaptogenez analizi için yüksek özgüllük ve çözünürlük sağlar. Tipik bir deney, genellikle benzersiz bir etiket, transgen, ve in vivo olarak istenen bir gelişim sürecinin tek hücreli analiz için organizatörü unsurları içeren iki ya da üç virüsler kullanmaktadır. Aşağıdaki protokol fonksiyonel yeni doğan nöron entegrasyonu tek bir yeşil (GFP) veya kırmızı (dTomato) floresan proteini retrovirüs ve yama klemp elektrofizyoloji kullanarak analiz etmek için bir yöntem açıklanır.

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

Yetişkin hayvanlarda yenidoğan dentat granül hücrelerin entegrasyonu çalışma bir şekilde açıklanmıştır. Bu teknik, yeni doğan nöronlar, in vivo işlevsel entegrasyonu belirlemek için elektrofizyolojik kayıtlar takip etiketlemek için tasarlanmış bir retrovirüs kullanır.

Yetişkin doğmuş Nöronlar Entegrasyon incelenmesi

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

studying-the-integration-of-adult-born-neurons

Research

JoVE Journal

Neuroscience

13.7K Görüntüleme.  State University of New York at Stony Brook. Yetişkin hayvanlarda yenidoğan dentat granül hücrelerin entegrasyonu çalışma bir şekilde açıklanmıştır. Bu teknik, yeni doğan nöronlar, in vivo işlevsel entegrasyonu belirlemek için elektrofizyolojik kayıtlar takip etiketlemek için tasarlanmış bir retrovirüs kullanır.

Video: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elektroporasyon Kortikal nöronlar Altküme Gen Fonksiyonu Öğrenmenin İlköğretim Nöronal Kültür takip

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Nörobilim

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Kronik Yetişkin doğmuş Koku Nöronlar Seçici Viral İletimi In vivo Optogenetic Uyarım

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

İnsan Pluripotent Kök Hücreler Telencephalic glutamaterjik Nöron Şartnamesi

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Glutamat ve GABA hızlı Micro-iyontoforez: Synaptic Entegrasyon Araştırma yararlı bir araç

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Canlı Kültür Nöronlar Antikor Besleme sonra Hücre-yüzey ve İçselleştirilmiş Proteinlerin Diferansiyel Etiketleme

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Varisler oluşumu ve kurtarma merkezi nöron aksonlar içinde çalışmak için bir Microbiomechanical sistemi

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Tau Aggregation Lentiviral kullanarak eğitim için bir In vitro modeli-ınsan nöronların aracılı dönüştürücü

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Serbest Yüzme Balık hareketli Tetrodes tarafından Nöronların Kablosuz Elektrofizyolojik Kayıt

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

Primer Embriyonik Fare Orta Beyin Dopamin Nöronlarında Pre-formed Fibril Indüklenen α-Sinüklein Birikiminin İncelendirilmesi

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Zebra Balıklarında Yüzme Sırasında Posterior Lateral Line Afferent Neurons'un Aktivitesi

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...