Biz Dr teşekkür etmek istiyorum. Bize önceden çalışma ve Özge Fairless ve Shiyuan Liu nöronal rekonstrüksiyon ile yardım için bir parçası olarak hazırlanan doku kullanmak için izin Ed Callaway ve Marty Usrey. ve Whitehall Vakfı: Bu çalışma NIH (EY018683 HAYIR) tarafından finanse edildi.

<ol>
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Yazarlar ifşa hiçbir şey yok.

Nöroanatomik çalışmalar nörobilim ve connectomics ve yapı-işlev ilişkilerinin son bir ilgi ayağı seçici nöronal popülasyonlarının detaylı morfolojik karakterizasyonu için coşku yeniledi kalmıştır. Geleneksel olarak, nöroanatomik çalışmalar uzman neuroanatomists tarafından tanımlanan nöronların morfolojik farklı sınıflara nöronların nitel sınıflandırmaları yararlanmıştır. nöronlar yeniden ve morfolojik veri ayıklamak için tekniklerindeki gelişmeler sayesinde, tarafsız bir şe...

Nöroanatomi &quot;connectomics&quot; in nörobilim 1 ve son faiz temellerinden biri nöronal popülasyonlarının morfolojik çeşitlilik ve belirli nöronlar 2 arasındaki bağlantıları anlamak için coşku yeniledi olduğunu. Etiketleme yöntemleri ve yeniden nöronlar büyük ölçüde genetik ve virüs aracılı devre izleme de dahil olmak üzere son yenilikler ile iyileştirilmiştir nöronal nüfus 5 daha kapsamlı morfolojik araştırmalar sağlayan, 3, 4 yaklaşır. Bireysel nöronlar etiketleme gelişmelere ek olarak, nicel veri analiz teknikleri de bu morfolojik verilerle 5, 6 dayalı farklı alt popülasyonlar halinde nöronların bağımsız ve tarafsız sınıflandırma etkinleştirmek ortaya çıkmıştır. Bu tarafsız teknikler daha Traditiona üzerine bir gelişme vardırBir asrı aşkın süredir alanında standart olmuştur l nitel sınıflandırma yöntemleri. Bu çalışmanın amacı, ana hatlarıyla adım-adım olduğunu, bir seçici nüfus içindeki nöronların virüs aracılı etiketleme kombinasyonu, bir bu nöronların kapsamlı numune ve nicel veri analizi büyük ölçekli rekonstrüksiyon ile bağımsız kümeleme dayalı İstatistiksel değerlendirme. Bu yöntemleri birleştirerek, bir selektif nöronal nüfus içinde kapsamlı örnekleme ve morfolojik eşsiz nöronal türleri tarafsız sınıflandırma kolaylaştırmak için toplama ve nöroanatomik verilerin analizi doğru yeni bir yaklaşım özetlemektedir. Bu yöntemlerin bir örneği olarak, makak maymunu talamik retiküler nükleus (TRN) tek bir sektörü içinde nöronların büyük bir nüfusun analizimizi açıklar. Bu veriler daha önceki çalışmada 7 vardır. seçici dorsal Latera projelendirme TRN nöronların etiketleme YöntemleriEGFP 4, 8 kodlayan modifiye kuduz virüsünün cerrahi enjeksiyon kullanılarak talamus (dLGN) l Dirsekli çekirdeği özetlenen (Özel Malzemeler / Ekipman, satır 2 Tablo). Bu modifiye kuduz virüsü geni, bir esas kat proteinini kodlayan virüs trans-sinaptik hareketi ortadan yoksundur. Virüs enjeksiyon yerinde akson terminalleri girdikten sonra, enfekte nöronlar 5, 9, 10, tam dendritik arborization boyunca EGFP ifade sürüş önemli yararı ile geleneksel retrograd tracer gibi davranır. Bu duruma göre, bu G-silinmiş kuduz virüs seçici bulaşabilir ve enjeksiyon ve retrograd taşıma sonraki herhangi bir nöronal popülasyonu etiket kullanılabilir. Belirli bir nöronal nüfusun geniş kapsamlı bir analizi gerçekleştirmek için, örnek için önemlidirnüfus içinde nöronların geniş bir dağılımı. Virüs-aracılı etiketleme tekniği tam hücre içi üretimi için, &quot;Golgi gibi&quot; virüs enjeksiyon yerinde akson birçok nöron doldurur, bir beyin yapısı tam kapsam dahilinde nöronların çok büyük bir örnek yeniden oluşturmak mümkündür. değiştirilmiş kuduz virüsü enfekte edilmesi ve nöron çok sayıda etiketleme de çok etkili olduğu için, ayrıca, hayvan başına nöronların yüzlerce yeniden oluşturmak mümkündür. DLGN verme TRN nöronların kapsamlı bir örnek oluşturmak için TRN 11 görsel sektör genelinde 160 nöronlar örnekleme için prosedürler özetlenmektedir. Bir mikroskop, kamera ve yeniden yazılımı içeren bir nöron rekonstrüksiyon sistemini kullanarak bireysel nöronlar yeniden süreci açıklanmıştır. Ayrıca tarif edilen (TRN içinde bu durumda) beyin yapısı içinde tek tek nöronların konumlarını belirlemek için ve virüs, enjeksiyon sit doğrulamak için yöntemlerhacimsel kontur rekonstrüksiyon kullanarak (dLGN içinde bu durumda) bir yapı içinde e hacmi ve konumu. Her bir nöron için ölçülen morfolojik ölçümlerini özetlenen morfolojik verileri dışa ve bağımsız küme gerçekleştirmek için adımlar dayalı analizleri. kümeleme yöntemleri sınırlamalar vardır ve mevcut farklı kümelenme algoritmaları çeşitli de vardır. Bu duruma göre, bu seçenekler ve daha yaygın olarak kullanılan algoritmalar bazı avantajları açıklanmaktadır. kümeleme analizi kümeleri tekliği istatistiksel doğrulama sağlamaz. Bu nedenle, ek adımlar optimum kümelenme yanı sıra içinde ve gruplar arasında morfolojik veriler arasındaki ilişkileri doğrulamak için özetlenmiştir. Bu TRN nöronlar onaylamak için TRN veri kümesi için kümeleri değerlendirmek için istatistiksel yöntemler tarif edilmiştir 10 bağımsız morfolojik metriklere dayalı üç benzersiz kümeler halinde gruplandırılmış. Böylece, seçici etiketleme için adımları özetleyerek, Yeniden ve belirli bir nöronal nüfus morfolojik verilerini analiz, bir popülasyon içindeki nöronlar arasındaki morfolojik farklılıklar ölçülmesi için yöntemler açıklanmaktadır. makak maymunu TRN&#39;nin görsel sektöründe farklı nöron tiplerinin önceki bulguları ayrı istatistiksel değerlendirme yöntemleri ile teyit edilir. Birlikte, bu teknikler, nöroanatomik veri kümelerine geniş uygulanabilir ve beyin üzerinden nöronal nüfus çeşitliliği kantitatif sınıflandırma kurmak yardımcı olacağını umuyoruz.

<table border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">SAD&#916;G-EGFP</td>
			<td style="width:171px;">E.M. Callaway Laboratory, Salk Institute</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Prepared by Dr. F. Osakada. G-deleted rabies virus available through the Salk Institute Viral Core</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Recording electrode: platinum/iridium or tungsten</td>
			<td style="width:171px;">FHC</td>
			<td style="width:133px;">UEPSGGSE1N2M</td>
			<td style="width:281px;">Visit website (www.fh-co.com) for alternative order specifications</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:233px;">Nanoject II</td>
			<td style="width:171px;">Drummod Scientific</td>
			<td style="width:133px;">3-000-204, 110V</td>
			<td style="width:281px;">Alternatives: picospritzer, Hamilton syringe</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:233px;">Freezing microtome</td>
			<td style="width:171px;">Thermo Scientific</td>
			<td style="width:133px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">DAB</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:133px;">D5905-50TAB</td>
			<td style="width:281px;">3,3&#39;-Diaminobenzidine tetrahydrochloride, tablet, 10 mg substrate per tablet. Caution: carcinogen - must be bleached before discarding</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:233px;">Cytochrome C</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:133px;">C2037-100MG</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:233px;">Catalase</td>
			<td style="width:171px;">Sigma-Aldich</td>
			<td style="width:133px;">C9322-5G</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-GFP</td>
			<td style="width:171px;">Life Technologies/Thermo Fisher</td>
			<td style="width:133px;">#A-11122&#160;</td>
			<td style="width:281px;">Primary antibody</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:233px;">Biotinylated goat anti-rabbit</td>
			<td style="width:171px;">Vector Laboratories</td>
			<td style="width:133px;">#BA-1000</td>
			<td style="width:281px;">Secondary antibody</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Neurolucida System&#160;</td>
			<td style="width:171px;">MicroBrightField</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Software for neuron tracing and analysis. http://www.mbfbioscience.com/neurolucida</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:233px;">Neurolucida Explorer</td>
			<td style="width:171px;">MicroBrightField</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Data export software</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Microfire Camera&#160;</td>
			<td style="width:171px;">Optronics</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">2-Megapixel true color microscope camera. http://www.simicroscopes.com/pdfs/microfire.pdf</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Nikon E800 Microscope</td>
			<td style="width:171px;">Nikon Instruments Inc.</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Biological research microscope. http://www.microscopyu.com/museum/eclipseE800.html</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Matlab</td>
			<td style="width:171px;">The MathWorks Inc.&#160;</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Matrix-based computational mathematics software. http://www.mathworks.com</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Microsoft Office Excel</td>
			<td style="width:171px;">Microsoft</td>
			<td style="width:133px;"></td>
			<td>Spreadsheet program</td>
		</tr>
	</tbody>
</table>

large-scale reconstructions and independent, unbiased clustering based on morphological metrics to classify neurons in selective populations

Bu protokol, bağımsız ve tarafsız kümelenme kullanımı ile birlikte nöronların büyük ölçekli rekonstrüksiyon seçici nöronal nüfus arasında gözlenen morfolojik özelliklerinin kapsamlı bir anket oluşturmak için analiz özetliyor. Bu tekniklerin kombinasyonu Nöroanatomik verilerin toplanması ve analiz için yeni bir yaklaşım oluşturmaktadır. Birlikte, bu teknikler büyük ölçekli etkinleştirmek ve selektif nöronal popülasyonlarının dolayısıyla daha kapsamlı, örnekleme ve bir nüfus içinde morfolojik benzersiz nöronal sınıfları tanımlamak için tarafsız nicel yöntemler kurmak. protokol seçici nöronlar etiketlemek için modifiye kuduz virüsünün kullanımını özetliyor. ilgi hedef beyin yapısına stereotaksik enjeksiyonu takiben retrograd tracer gibi kuduz virüsü eylemleri G-silinmiş ve nöronlarda EGFP teslimat ve ifade için bir araç olarak hizmet vermektedir. nöronların çok sayıda bu kullanarak enfekteteknik ve bireysel nöronların &quot;Golgi gibi&quot; tam doldurur üreten, kendi dendritler boyunca GFP ifade eder. Buna göre, virüs aracılı retrograd izleme yöntemi tam hücre içi dolgular üreterek geleneksel boya bazlı retrograd izleme teknikleri geliştirir. çalışılan beyin bölgesi tüm bölgelerini kapsayan bireysel iyi izole edilmiş nöronları temsili bir örneği elde etmek için yeniden için seçilir. protokol hücre gövdeleri yeniden ve çoklu doku bölümleri kapsayan etiketli nöronların dendritik arborization kalıplarını tamamlamak için prosedürler sıralanıyor. Beyin yapısı içinde her bir nöronun pozisyonları da dahil olmak üzere morfolojik veriler, daha fazla analiz için ayıklanır. Standart programlama fonksiyonları bağımsız küme analizleri ve morfolojik metriklere dayanan küme değerlendirmelerini gerçekleştirmek için kullanılmıştır. Bir kümeleme analizi perfo bu analizlerin programı, istatistiksel değerlendirme doğrulamak içinmakak maymunu talamus (TRN) talamik retiküler çekirdeğinde yeniden 160 nöronlar üzerinde rmed yapıldı. Orijinal kümeleme analizi ve burada yapılan istatistiksel değerlendirmeler, hem TRN nöronlar üç alt popülasyonlar, benzersiz morfolojik özelliklere sahip her ayrılır olduğunu göstermektedir.

Bir seçici nüfus içinde nöronların büyük ölçekli rekonstrüksiyonlar dLGN 5 modifiye kuduz virüsünün mümkün enjeksiyonu takiben olduğunu göstermiştik. Son zamanlarda, aynı doku TRN görsel sektörde 160 nöronların yeniden kullanılmıştır (Bragg ve arkadaşları, yorumda,. Şekil 2A-B), yukarıda tarif edilen ayrıntılı metodolojik aşağıdaki basamakları. TRN çalışmasında, TRN nöron üç benzersiz kümeleri bağımsız ...

Watch this Scientific Journal Video about Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations at JoVE.com

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

Bu protokol değiştirilmiş kuduz virüsü floresan işaretlerini ifade ve bağımsız, tarafsız küme farklı nöronal alt sınıfları arasında morfolojik metrik kapsamlı karakterizasyonu olanak analizleri ile retrograd enfeksiyonu takiben etiketli seçici nöronal popülasyonlarının büyük ölçekli rekonstrüksiyon açıklar.

Morfolojik Ölçümler dayanarak büyük ölçekli yeniden düzenlemesi ve Bağımsız, Unbiased Kümelenme Seçici Populasyonlarında Nöronlar Sınıflandıran

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

large-scale-reconstructions-independent-unbiased-clustering-based-on

Research

JoVE Journal

Neuroscience

6.8K Görüntüleme.  Geisel School of Medicine at Dartmouth.  Bu protokol değiştirilmiş kuduz virüsü floresan işaretlerini ifade ve bağımsız, tarafsız küme farklı nöronal alt sınıfları arasında morfolojik metrik kapsamlı karakterizasyonu olanak analizleri ile retrograd enfeksiyonu takiben etiketli seçici nöronal popülasyonlarının büyük ölçekli rekonstrüksiyon açıklar. 

Video: Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

Kültür Hipokampal Nöronlar Dendritik Spine Floresan Takip edilen Proteinler Photobleaching sonra Floresan Kurtarma (sıkı bağlamak)

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Nörobilim

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Hareketi tarafından Modülasyonlu Nöronlar Fizyolojik, Morfolojik ve nörokimyasal Karakterizasyonu

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

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 ...

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Anestezi Rat Silikon Probları Büyük ölçekli Nöronal Toplulukları Kaydı

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

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 ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

Kemirgenler davranmak yılında Hareketli Silikon Problar tarafından Nöronlar Büyük ölçekli Kaydı

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Kortikal Nöronlar In Vivo İki foton Of Görüntüleme Deneyimi bağımlı Moleküler Değişiklikler

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

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

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

Eşzamanlı Elektrofizyolojik Kayıt ve Suprakiazmatik Nucleus Nöronlar Kalsiyum Görüntüleme

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

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Frenik Motor Nöronlar tarafından Diyafram İNNERVASYONU Fonksiyonel ve Morfolojik Değerlendirmesi

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.