Bu çalışma, hayat sigortası şirketi &quot;AG2R-La Mondiale&quot; Ecole des Nörobilim de Paris (AKP), Agence Nationale de la Recherche &quot;ANR-09-NEUR-004&quot;, &quot;ERA-NET NÖRON çerçevesinde tarafından desteklenen &quot;Avrupa Komisyonu, ve Pasteur Vakfı tarafından FP7 programı. Sebastien Wagner Letten Vakfı tarafından desteklenmiştir.

1. Optik Kalibrasyon: Ölçme LED Güç <ol><li> LED dizisi aktif için bir fan ve yapıştırmayın bu LED / soğutucu aparat collimating objektif ile soğutulan bir soğutucu takın. </li><li> Aydınlık aydınlatma lambası kullanılan LED / soğutucu / fan / lens cihazları ile değiştirin. Bu cihaz dikkatle paralelleştirilmiş LED ışını düz bir optik yoğunlaştırıcı lens doğru yol boyunca hareket şekilde yerleştirilmesi gerekir. Soğutucu / fan sisteminin ortak bir zemin düzgün topraklanmış olduğundan emin olun. </li><li> Sürücü akım hızlı ve kare bakliyat verebilir bir güç kaynağı ile LED dizisi. Bu güç kaynağı bir darbe jeneratörü kaynaklanan bir 5V TTL darbe tarafından kontrol edilebilir. </li><li> Merkezi paralelleştirilmiş ışın alan diyafram ve kondenserin objektif arasında tanımlanan ışık yolu boyunca. İdeal olarak, LED ışın tamamen açılmış alanında diyafram biraz aşırı doldurmayın. Tipik olarak, daha sıkı collimated bir LED ışını bu diyafram daha az dolduracak, ve mor üretecektekdüzelik pahasına e güç. Kurulumu consenser diyafram konjuge düzlemde LED dizi biraz daha genişletilmiş bir görüntü yansıtılır collimating objektif seçerek ışık tekdüzelik arttı. </li><li> Kohler aydınlatma alanında diyafram (ışık kaynağının en yakın diyafram) görüntü dilim odası (Şekil 1) odaklanmış böylece kondansatör odaklanarak elde edin. Mercek kağıdı ince bir doku diğer derinliklerde alan diyafram odaklı görüntü görselleştirmek için bir projeksiyon perdesi olarak hareket edebilir. </li><li> Opak bir madde olarak bilinen bir çapa iğne deliği bir dizi delin. Bu küçük iğne deliği, optik bir güç metre sensör üzerinde tek bir yerde. Alan diyaframın maksimum okuma verir kadar sadece güç metre hareket ettirerek odaklı resmin üzerine numune aşamasında ve merkezi güç ölçer güç ölçer yerleştirin. Bu pozisyonda yapıştırmayın güç ölçer. </li><li> Tüm açıklıkları (diyafram açıklıkları diyafram ve tam olarak açılmasınaalanında diyafram). Sistematik optik lightpath göre ekli dilim haznesi / güç metre taşımak ve optik güç tekdüzelik ışıklı alanı içinde hesaplayabilirsiniz. Sisteminiz için tekdüzelik arsa oluşturun. Mikroskop objektif odak merkezli Kohler aydınlatma ile kurulum doğru ise, maksimum güç doğrudan hedefi altında olmalıdır ve bu odak dışında kalan bölgelerde artık bilinen bir miktar tekdüzelik arsa göre güç almalıdır. </li><li> Iğne deliği her boyutu için optik güç vs iğne deliği yüzey alanı, standart bir eğri oluşturmak. LED diziye giriş akımı ayarlayarak, birden çok güç düzeyinde bu eğri üretmek ve her bir eğri mm2 başına gücünü hesaplamak. LED dizi yama optikler için kullanılacak ise, ışık ne kadar yama aydınlatma altında iletilen bilmek için gerekli yama (kondansatörler, gözeneklerin ve filtreler) optik unsurlarını tanıtmak için emin olun. </li><li> Güç ölçer Flippingcıva lambası açıkken amacı yüz, 470Nm aydınlatma yoğunluğunu hesaplamak. </li><li> Odasının canlı bir dilim ekleyin ve saçılma beyin dokusu üzerinden iletilen ışık gücü belirlemek üzere standart eğrileri yeniden. </li></ol> 2. Dilimleme Usul ve Elektrofizyoloji Bölüm A: Dilim Hazırlık <ol><li> Fare anestezisi (60 mg / kg Ketamin ve 2mg/kg Xylazine) ve başını kesmek. ; Karışımı ile kabarmış ~ 310mOsm, pH 7.4 NaCl 124, 3 KCl, 1.3 MgSO 4, 26 NaHCO 3, 1,25 NaHPO 4, 20 glikoz, 2 CaCl 2: yapay beyin omurilik sıvısı beyin teşrih (ACSF mM % 95 O2 ve% 5, koku ampuller zarar vermemeye özen, 2 5,1) CO . Hatta bir kenarı (yatay bölümler için) ile ventral yüzeyi ile agar iki yarımküresinin ve yer ayırın. </li><li> Tutkal vibr agar ve her kortikal yarımkürenin dorsal yüzeyatome ayna, ve banyo yavaş yavaş buz ACSF ile doldurun. Dilim, ventral yüzeyinden 300 mikron bölümlerde ısıtılmış (34-36 º C) ve her bölümün transfer oksijenli ACSF onları kurtarmak için 30-45 dakika izin verir. </li></ol> Bölüm B: Gevşek Patch Ölçüm Spike Eşik <ol start="3"><li> 30 dakika oda sıcaklığında dilim getirerek sonra, yavaşça oksijenli ACSF sürekli perfüzyon altında mikroskop odası kayıt odasında bir dilim koyun. Aşırı ChR2 enfekte olmuş nöronların uyarıcı riski, EYFP-ChR2 varlığı Epifloresans (şekil 2a) altında teyit edilebilir </li><li> Bir pipet çektirmesi (Sutter P-97) cam elektrotlar çekin. Bu elektrot ACSF doldurun. ACSF banyosuna konulduğunda ucu direnci 7-10 Mohms arasında olmalıdır. </li><li> Floresan aydınlatma altında, olgun morfolojisi ile dilim sağlıklı bir ChR2 EYFP nöron bulun. Ayrıca bu yama optik altında nöronun soma bulun. </li><li> Elektrot ucu geçirilir hafif pozitif basınç ile tespit floresan nöron doğru yama elektrot düşük. Membran temas yapıldığında, hızlı bir şekilde bırakın ve pozitif basınç emme ucu ile küçük ve kısa bir miktarda uygulayın. Giga-ohm mühür plazma membran ve yama elektrot duvarları arasında yapılmalıdır. </li><li> Giga-mühür, ölçülebilir bir yerel alan potansiyeli üretmek elektrot floresan nöron aktivitesi spike yeterince yakınsa oluşur. Olsa bile Farklı dozlarda ışık yanıp sönen bu nöron ChR2 etkinleştirin. Hafif doz LED gücü ve süresi, hem de bir fonksiyonu olduğundan, çok fazla ışık nasıl bir aksiyon potansiyeli, birden fazla yetki ve süreleri (şekil 2b) uyandırmak için gerekli hesaplar. Spike cıva lambası aydınlatma altında oluşur ne kadar gözlemliyoruz. </li></ol> 3. Temsilcisi Sonuçlar: Mikroskopta (Olympus BX51WI), LED i2 menfez ve yoğunlaştırıcı mercek n hattı, böylece orijinal fabrika yüklü ark lambası lightpath sürdürmek. Alan diyafram ve diyafram açıklıkları diyafram kapatarak, yama kelepçe kayıtları (Şekil 1b) yeterli aydınlık kontrast elde edebilirsiniz. Tüm diyaframlar ile tam channelrhodopsin aktivasyonu (şekil 1a) için maksimum ışık gücü dilim maruz açın. Bizim mikroskop, bu yama yapılandırma (4.1 μW / mm 2 karşı 6.88 mW / mm 2), maksimum alan yoğunluğu daha büyüklüğü yaklaşık üç siparişlerin düşük ışık yoğunluğu üretir . Biz tek bir yetişkin doğumlu ChR2 EYFP granül hücresi ifade belirtir rostral göçmen akımı (şekil 2a) Gevşek bir yama kayıt neuroblasts göç lentiviral enfeksiyonu sonra yetişkin doğumlu koku alma ampul granül ve periglomerular nöronlar hafta sağlam bir etiket maxim 5 ms stimülasyonum güç (6.88 mW / mm 2) spike (şekil 2b) uyandırmak için yeterli olacaktır. Ekspresyon seviyesi hücreleri arasındaki farklılık gösterdiğinden, başak eşiği geçen ışığın miktarı değişebilir ve ilgi her hücre türü için istatistiksel olarak tarif edilmelidir. <img src="/files/ftp_upload/3379/3379fig1.jpg" alt="Şekil 1" /> Şekil 1 tam saha photostimulation ve patch-kelepçe dilim elektrofizyoloji dizi kurulum LED. Channelrhodopsin etkinleştirmek için (ChR2) açık arka diyafram açıklığı ve kondenser optikler (a) ile bir paralel ışın proje. Bu yapılandırma, tam alanında diyafram kapatma ve modüle alan diyafram genişliği (b), yüksek kontrastlı yama optik içine değiştirilebilir. Kısaltmalar: a. fan, b. soğutucu, c LED dizisi, d. collimating lens, e. ayna, f. alanında diyafram, g. diyafram, h. yoğunlaştırıcı lens, i. sample sahne, j. objektif. <img src="/files/ftp_upload/3379/3379fig2.jpg" alt="Şekil 2" /> Şekil 2. resmi yama kelepçe ve tüm alan photostimulation için koku alma ampul 300μm yatay dilim (a). Yayılan koku ampul çekirdek ChR2-EYFP ifade Lentivirally enfekte yetişkin doğumlu granül hücreleri görülebilir. Spike uyandırmak için gerekli olan ışık doz, LED flaş, (b) süresi artırılarak bulunabilir. Bu granül hücre için eşik tam LED yoğunluğu (2.43mW/mm 2) 5ms oldu. Ölçeği (a), (b) = 50ms ölçek = 500μm.

<ol>
	<li>Nissant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+promotes+synaptic+plasticity+in+the+olfactory+bulb.">Adult neurogenesis promotes synaptic plasticity in the olfactory bulb.</a> Nature Neuroscience. 12, 728-730 (2009).</li><li>Apicella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+Cells+in+Piriform+Cortex+Receive+Convergent+Input+from+Distinct+Olfactory+Bulb+Glomeruli.">Pyramidal Cells in Piriform Cortex Receive Convergent Input from Distinct Olfactory Bulb Glomeruli.</a> Journal of Neuroscience. 30, 14255-14260 (2010).</li><li>Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=genetically+targeted+optical+control+of+neural+activity.">genetically targeted optical control of neural activity.</a> Nature. 8, 1263-1263 (2005).</li><li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb.">where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> The Journal of Neuroscience. 30, 17023-17034 (2010).</li><li>Grubb, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+the+first+synapse+in+olfaction:+development+and+adult+neurogenesis.">Functional maturation of the first synapse in olfaction: development and adult neurogenesis.</a> The Journal of neuroscience. 28, 2919-2932 (2008).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit-breakers:+optical+technologies+for+probing+neural+signals+and+systems.">Circuit-breakers: optical technologies for probing neural signals and systems.</a> Nature reviews. Neuroscience. 8, 577-581 (2007).</li><li>Grossman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-site+optical+excitation+using+ChR2+and+micro-LED+array.">Multi-site optical excitation using ChR2 and micro-LED array.</a> Journal of neural engineering. 7, 16004-16004 (2010).</li><li>Dhawale, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-redundant+odor+coding+by+sister+mitral+cells+revealed+by+light+addressable+glomeruli+in+the+mouse.">Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse.</a> Nature neuroscience. 13, 1404-1412 (2010).</li><li>Weick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+control+of+transplantable+human+ESC-derived+neurons+via+optogenetic+targeting.">Functional control of transplantable human ESC-derived neurons via optogenetic targeting.</a> Stem cells. 28, 2008-2016 (2010).</li><li>Toni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+born+in+the+adult+dentate+gyrus+form+functional+synapses+with+target+cells.">Neurons born in the adult dentate gyrus form functional synapses with target cells.</a> Nature Neuroscience. 11, 901-907 (2008).</li><li>Grubb, M. S., Burrone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2+Localised+to+the+Axon+Initial+Segment.">Channelrhodopsin-2 Localised to the Axon Initial Segment.</a> PLoS ONE. 5, e13761-e13761 .</li><li>Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdala+circuitry+mediating+reversible+and+bidirectional+control+of+anxiety.">Amygdala circuitry mediating reversible and bidirectional control of anxiety.</a> Nature. 471, 358-362 (2011).</li></ol>

Çıkar çatışması ilan etti.

Son yıllarda popülaritesi sinirbilim araştırması, 6 optogenetic araçları bir patlama gördük. Sonuç olarak, bu yeni araçları kullanmaya başlamak isteyen laboratuarlar için giriş bariyer düşürmek için giderek daha önemli. İşte biz bu channelrhodopsin ifade nöronlar tam alan optik stimülasyonu yapabilirsiniz böylece geleneksel bir yama klemp kulesi basit ve düşük maliyetli güçlendirme ve kalibrasyon yapmak için nasıl açıklar. Özellikle, biz, özellikle ışık ile yeni doğan n...

<table border="1">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue No.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>100 mg/ml</td>
			<td>Imalg&egrave;ne 1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>2%</td>
			<td>Rompun</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbps;</td>
			<td>Sigma </td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>P5405</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>M1880</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5011</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>C7902</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>A9539</td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>P-97</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>1.5 mm O.D./1.17 mm I.D.</td>
			<td>Harvard Apparatus</td>
			<td>GC150T-10</td>
		</tr>
		<tr>
			<td>LED array</td>
			<td>&nbsp;</td>
			<td>Bridgelux</td>
			<td>BXRA-C2000</td>
		</tr>
		<tr>
			<td>Collimating lens</td>
			<td>40 mm beam diameter</td>
			<td>Thor Labs</td>
			<td>LEDC1</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>2.8 amp</td>
			<td>A1W Electronik</td>
			<td>HKO2800</td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>&nbsp;</td>
			<td>Thor Labs</td>
			<td>PM 100</td>
		</tr>
		<tr>
			<td>Heatsink </td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Fan</td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>&nbsp;</td>
			<td>Leica</td>
			<td>VT1200S</td>
		</tr>
	</tbody>
</table>

using affordable led arrays for photo-stimulation of neurons

Standart dilim elektrofizyoloji araştırmacılar, elektrik veya farmakolojik manipülasyonlar 1,2 yanıt olarak tek hücre elektriksel yanıtlar kayıt sinir devresi bileşenleri tek tek prob izin verdi . Genetik hedef nöronların (optogenetics) optik kontrol etmek için yöntemler icat sayesinde, araştırmacılar standart bir dilim hazırlık nöronların belirli gruplar üzerinde kontrol görülmemiş bir düzeye sahiptir. Özellikle, araştırmacılar ışık 3,4 nöron aktif hale getirmek için channelrhodopsin-2 (ChR2) ışığa sağlar. LED tabanlı standart dilim elektrofizyoloji ChR2 photostimulation dikkatli kalibrasyon birleştirerek, daha fazla ayrıntı, koku alma ampul, koku alma sisteminin ilk merkezi röle yetişkin doğumlu internöronlar rolü prob. Özellikle yetişkin doğumlu nöronlar ChR2-YFP viral bir ifade kullanarak, biz seçici eski bir ortamda genç bir yetişkin doğumlu nöronlar kontrol edebilirsinizd olgun nöronlar. Optik kontrol, basit ve ucuz bir LED sistemi kullanır ve bu sistem çok fazla ışık, tek nöronların faaliyet spike uyandırmak için gerekli olan anlamak için nasıl kalibre göstermektedir. Bu nedenle, kısa mavi ışık yanıp söner uzaktan ChR2 transduced yeni doğan hücrelerin ateşleme düzenini kontrol edebilirsiniz.

Watch this Scientific Journal Video about Using Affordable LED Arrays for Photo-Stimulation of Neurons at JoVE.com

Using Affordable LED Arrays for Photo-Stimulation of Neurons

ChR2 ifade Yetişkin doğmuş nöronlar, koku alma nöral devrelerin işlevini doğru katkıları incelemek amacıyla dilim elektrofizyolojik hazırlıkları manipüle edilebilir.

Nöronlar Foto-Stimülasyon için uygun fiyatlı LED Diziler

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

using-affordable-led-arrays-for-photo-stimulation-of-neurons

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.5K Görüntüleme.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). ChR2 ifade Yetişkin doğmuş nöronlar, koku alma nöral devrelerin işlevini doğru katkıları incelemek amacıyla dilim elektrofizyolojik hazırlıkları manipüle edilebilir.

Video: Using Affordable LED Arrays for Photo-Stimulation of Neurons

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Bozulmamış 3-D Beyin Devrelerin Hedefleme Viral için Ölçeklenebilir akışkan Enjektör Diziler

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Nörobilim

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

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

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

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

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

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Chef-tdTomato kullanarak Zebrafish Somatosensoriyel Nöron Optogenetic Etkinleştirme

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

Hayvanlar davranmak, Awake Tek-birim Recordings Microwires Organize Dizileri implantasyon için Usulü

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Yapısal Aydınlatma Mikroskobu kullanılarak Rat İlköğretim hipokampalinden Dentritik Spine görüntüleme

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Biyokimyasal Analiz için Duyu Nöronlar gelen üretim ve Aksonlar izolasyonu gözenekli Filtre Kullanma

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Nöronlar bir Genetiği kodlanmış doğal olmayan Amino Asit kullanarak Nöronal Protein Optik Kontrol

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

GLUT4 protein Deconvolution mikroskobu kullanarak fare birincil hipotalamik nöronlarda ticareti canlı görüntüleri

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

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). ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Çoklu Esnek Polimer Elektrot Dizilerinin Kronik İmplantasyonu

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...