We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

Bu protokol açıklanan tüm deneysel prosedürleri İbrani Üniversitesi Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. Dilimle için Çözümler ve Araçlar hazırlama <ol><li> Tablo 1 &#39;de tarif edilen iyonu içeren standart bir fizyolojik çözelti (SPS) 1 L hazırlayın. <ol><li> Iyonu giderilmiş su (0.055 uS / cm&#39;lik iletkenlik) 1000 ml ile dolu bir depolama cam şişe içinde önceden 10 kat nihai konsantrasyonda tuzlarını ihtiva eden bir stok çözelti hazırlayın. Tuzlar (NaCI, KCI, KH 2 PO 4 ve MgSO 4) ekleyin ve tümüyle çözülene kadar karıştırın. 4 ° C &#39;de stok solüsyonu saklayın. </li><li> Deney gününde, nihai SPS çözeltisi olun. Manyetik bir karıştırma çubuğu kullanılarak glikoz (3.6 g) ve NaHCO3 (2.18 g) çözülür, saflaştınlmış su, 700 ml stok çözeltisi, 100 ml ilave edilir ve daha sonra 1 L üzere saflaştırılmış kadar su ekle </li><li> Soluti dengeleyin~ üzerinde 20 dakika süreyle% 95 O 2 /% 5 CO 2 ile bunu soluyarak. Bundan sonra, 1 M CaCI2 çözeltisi 2 ml ilave ediniz. </li></ol></li><li> Aşağıdaki araçları (Şekil 1) birleştirin. <ol><li> Kafatasını açmak için büyük makas veya başları kesilerek başka bir araç, küçük cerrahi makas hazırlayın, beyin maruz beynin istenilen bölümünü diseksiyon için bıçak ile neşter kafatası kaldırılması için ince uçlu forseps dilimlenmiş ve işlenmesi için küçük bir spatula edilecek disseke beyin parçaları. </li><li> Buna ek olarak, beyin fazla sıvıyı çıkarmak için filtre kağıdı küçük parçalar halinde hazırlanması, beyin için iki küçük Petri kapları için beyne yapıştırma ihtiva eden ısıtılmış saf su ve SPS ve siyanoakrilat yapıştırıcı için iki cam, 500 ml&#39;lik çanaklar içinde disseke dilimleme sahne. </li><li> Dilimleme ve kurtarma cha için dilimleme banyodan dilimleri hareket için geniş ağızlı cam Pasteur pipet sırasında dilimleri işlemek için küçük bir fırça hazırlayınmber. Sıcak dilim banyolarında bakteri üremesini engellemek için araçlarını dezenfekte edin. Ayrıca, temiz laboratuvar eldiven kullanın. </li></ol></li><li> Gibbs ve Edwards 15 tarafından tarif edildiği gibi bir yarı-kesit geri bölmesini hazırlayın. Sürekli% 95 O 2 /% 5 CO 2 ile o SPS gaz ve 36 ° C&#39;de tutmak. Gazlaştırma dilimleri zarar verebilir banyosu içinde sıkışıp küçük kabarcıklar ile sonuçlanmaz emin olun. </li><li> Sıcak ~ Manyetik karıştırıcı ile ya da bir su banyosu içinde bir ısıtıcı plaka üzerinde 36 ° C&#39;ye kadar, SPS ve 300 mi. Bir ısıtıcı plaka kullanılırsa, iyon tortulaşması neden olabilir aşırı ısınmaya karşı çözüm önlemek için dikkat edin. Durumda, yerine eski bir soğutma sıcak taze SPS olur. </li><li> Bir elektrikli su ısıtıcısı ile kaynatmak için saflaştırılmış su yaklaşık 500 ml getirmek ve bir beher içinde, dilimleme sıcaklığından biraz daha sıcak su elde etmek için soğuk suyla, 300 ml birleştirir. Bu, başı kesilerek sırasında saf su ısıtıldı kullanın remai kullanımıDaha sonra dilimleme banyonun fizyolojik sıcaklığı korumak için kaynamış su vaziyette. </li><li> Intraperitoneal pentobarbital 0.1 ml (60 mg / ml) enjekte edilerek fare anestezi uygulayın. Birkaç dakika sonra hayvan duyu eksikliği tespit etmek için güçlü parmak veya kuyruk tutam yanıt vermez emin olun. </li></ol> 2. Beyin Diseksiyon <ol><li> Kafatasının arka yanında büyük makas ile boyun kesme hızlı bir şekilde fare başını kesmek ve kafa aşırı kan durulayın ısıtıldı saflaştırılmış su ile beher içine akmasına izin verdi. </li><li> Belki küçük bir makas ile boyun daha kaldırarak, boyun kasları ve cilt altında kafatasında foramen magnum Açığa. </li><li> Açıkça kafatasının açılmasına rehberlik etmek amacıyla kafatası sütür görmek mümkün kafatasının üstündeki deriyi çekin. </li><li> Foramen magnum içerisinden küçük makas alt ucu takarak ve hemen yönünde çevirerek açık kafatası kestilateral yan. Yavaşça gözleri ve frontoparyetal sütür arkasında bir yere parietal kemik yukarı lateral kenarı boyunca kesilir; Daha sonra, (Şekil 2A yeşil kesikli çizgi bakınız) kafatasının merkezine doğru çevirin ve kafatasının diğer tarafına orta hat boyunca kesilir. </li><li> Ince uçlu forseps kullanarak, (Şekil 2A sarı kesikli oka bakınız) beyin maruz yana frontoparie- köşesinden kafatasını kaldırın ve çapraz yukarı çekin ve. Kafatası kaldırıldığında beyin yerinde kalacak şekilde kafatası baş herhangi bir kemik veya deriye bağlı olmadığından emin olun. Beyin kafatası ile hareket halinde, kafatası ve beyin arasında herhangi bir bağ dokusunu kaldırmak için küçük bir makas kullanın. </li><li> Beyin maruz kaldığı zaman, beyin kuru olması için izin yoktur. Kafatası ve ısındı ile dolu bir Petri kabındaki beyin tutarken Böylece, bir sonraki adımları uygulayın, SPS gazlandı. </li><li> E için kullanılan beyin parçası ayırınbir neşter ve bir ıspatula kullanılarak beynin geri kalanından Xperiment. Beyincik kullanılması durumunda, (Şekil 2B-D kırmızı çizgilerde) midbrain ve pons aracılığıyla tek bir kesim tarafından ön beyin ayırmak ve taze içeren küçük bir petri taşımak, SPS gazlandı. </li><li> Yatay düzlemde beyincik dilimleme sahne kesme beyin yapıştırma için geniş ve düz bir taban oluşturmak için beyin sapı çıkarın. Önbeyinden kesildi tarafı aşağıya bakacak şekilde kısaca filtre kağıdı ıslak parçasının üzerine beyincik yerleştirin. <ol><li> (Şekil 2B mavi çizgi ile belirtildiği gibi) bazı oluşturmak için bir neşter ile beyin sapı kesilmiş ve SPS beyincik dönün. Kesme diğer uçaklar gerektiğinde, buna göre beyincik bloğu (Şekil 2C-D mavi çizgiler) kırpın. </li></ol></li></ol> 3. Beyin dilimleme <ol><li> O gün olduğu araştırarak kesme aşamasında hazırlayınry, sahnenin ortasında superglue küçük bir damla uygulamadan önce. </li><li> Doğru tarafı yukarıya gelecek şekilde bir spatula ile SPS&#39;den beyin blok kaldırın ve filtre küçük kâğıt parçaları kullanarak beynin herhangi bir aşırı sıvı kaldırmak. Sonra, tek bir hareketle, sahnede tutkal damla üzerine spatula beyni kaydırın. </li><li> Kurutmadan beyin ve doku yüzüne kadar çalışan tutkal önlemek için, bir Pasteur pipeti ile SPS birkaç damla uygulamak ve dilimleme odası içindeki kesme aşamasını yerleştirin. Çevrede bulunan bölgeler (örneğin, serebral korteks) bıçak bakacak şekilde beyin blok hizalama ve böylece dilimlenmeden önce, mekanik basınç, en az miktarda maruz kalacaktır. </li><li> Sürekli kesme odası içindeki SPS gazlanması olarak 34-37 ° C sıcaklık muhafaza edilirken, üreticinin talimatlarına göre her dilim. Extern doldurarak bu mesafeden kesme bölmenin sıcaklığını tutmakısındı saf su ve sıcaklığı çok düşük düştüğünde su değiştirme ile dilicisinin al odası. NOT: dilimleme parametreleri deneysel olarak incelenmiştir ediliyor doku ve nöronların her türü için bulunması gerekir. Seramik bıçaklar ve serebellar Golgi hücreleri ile Campden SMZ 700 dilicisinin durumunda, 0.75 mm titreşim genliği 65 Hz ve 0,05 mm / sn hıza ilerleyen kullanın; serebellar çekirdek nöronlar daha yüksek genlik ve frekans (1 mm ile 90 Hz, sırasıyla), ve daha yavaş ilerleme hızı (0,01-0,02 mm / sn) için tavsiye edilir. Golgi ve serebellar nükleer nöronlar hem de, dilim kalınlığı 300 mikron olmalıdır. </li><li> Dilimleme sırasında dilimler kendi üzerine katlanmasına izin vermez. Yavaşça tercihen sadece deney için ilgi bölgelerinde dilim dokunmadan, yumuşak bir fırça ile destekleyerek bu önleyin. Vibratome bıçak dokunmadan gelen fırça önlemek için özen; Özellikle seramik bıçakları durumunda, hatta hafif bir temasbıçak zarar verebilir. </li><li> Geniş ağızlı, yangın parlatılmış cam Pasteur pipeti kullanarak bölmeyi tutan / bir toparlanma dilimleme odasından her dilim taşıyın. </li><li> Dilimler, deney başlamadan önce en az 1 saat boyunca oda içinde bekletin. Bu, hasar görmüş veya ölmekte doku ve hücrelerinin bozulması sağlar ve böylece temiz bir dilim yüzeyinin sonuçlanır. İlk saat boyunca, 34-36 ° C aralığı içinde oda içindeki SPS sıcaklığını tutmak; dilimleri dilimleri zarar ya da onları yüzer yapabilirsiniz hava kabarcıkları tarafından dokunulmaz değildir emin olun. </li></ol> 4. Deney <ol><li> 1 saat sonra, kurtarma bölmesi sıcaklığı RT (17-25 ° C) kadar soğumasını bekleyin. Not: Bu dilimler bakteri büyümesini hem de hücresel metabolizmayı yavaşlatarak kullanılabilecek sırasında süreyi uzatabilir. Bu süre, beyin bölgesi ve hücre tipine bağlıdır. Örneğin, golgi yer alan bazı hücre türleri dilimleri sağlıklı olduğu bulunabilir8 saat diğer türleri ise kesim sonrası sadece 6 saat sürer. </li><li> 1 saat dilimleme geçtikten sonra çeşitli elektrofizyolojik deneyler için dilimleri kullanarak. </li></ol>

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The authors declare that they have no competing financial interests.

Bu fizyolojik yerine buz soğukluğunda sıcaklık farelerden akut beyin dilimleri hazırlamak için bir yöntem göstermektedir. Bu, soğuk koşullar ile hazırlananlar ile karşılaştırıldığında, sıcak koşullarda elde edilen dilimlerin kalitesi yüksek olduğu 11 gösterilen dilimleyici bıçak düşük dikey titreşimi sahip olması koşuluyla edilmiştir. Fizyolojik sıcaklık dilimleme gibi tek hücreli anormalliklerin yanı sıra ağ davranış tezahür edebilir meta...

Yöntemin amacı, özellikle yetişkin hatta yaşlı hayvanları kullanarak, in vitro elektrofizyolojik deneyler için yüksek kaliteli akut beyin dilimleri elde etmektir. İki şık cümle içinde Skrede ve Westgaard 1 ile anlatıldığı gibi akut beyin dilimleme yöntemi, modern sinirbilim araştırma temellerinden biri haline gelmiştir ve dünya çapında sayısız varyasyonları kullanılmaktadır. Dilimlerinin kalitesi dilim başına canlı nöronlar arasında sayısı tarafından yansıtılır, zaman süresi sırasında, hücreler dokularının bütünlüğünün yanı sıra bunların elektrofizyolojik ve morfolojik özelliklerini korurlar. Ayrıca, sabit bir kayıt için maksimum süresi dilimlerin kalitesine bağlıdır. Bu nedenle, on boyunca, orijinal dilimleme yöntem ayrıca kesme bileşimin kompleksi modifikasyonları ile, genellikle 2-10, kesme sonrası bir dilim iyileşmeyi geliştirmek için tek tek grup tarafından geliştirilen O edilmiştirgibi soğutulmuş fizyolojik çözelti ile hayvanın kalp içi ön-perfüzyon (örneğin askorbat, thiourea ya da H2O 2 ekleme gibi) r-kurtarma çözümleri. Son zamanlarda 11 Görüldüğü gibi, dilimleme sırasında fizyolojik sıcaklık nöronal sağlığı için soğutma daha yararlı gibi görünüyor; yetişkin (2-8 ay) kemirgenler ile çalışırken gelişme en çarpıcı. Büyük ısı değişiklikleri kaçınma nedeni, plastiklik 13 ve iyon kanalları kinetiği 13,14 gibi hücrelerde sıcaklığa bağlı süreçler, eserler önler. Bu tür değişiklikler membran voltaj ve hücre içi kalsiyum sinyalizasyon, başak eşiğini etkileyen ve şekil artabilir. Burada sunulan &quot;sıcak&quot; akut dilim hazırlama yöntemi, herhangi bir beyin bölgesinden yüksek kaliteli akut beyin dilimleri elde beyincik, korteks ve hipokampus, beyin sapı 16 çekirdeklerin dahil olmak üzere genel bir prosedürdür &lt;/ Sup&gt; yanı koku ampul gibi, hem sıçan ve farelerde. Özellikle, fizyolojik sıcaklık dilimleme işlemi kesme bıçağı neredeyse mükemmel yatay titreşir ve herhangi bir yapısal kusurları olmaksızın olmasını gerektirir. Böyle hassas eski dilimleme makinesi modelleri ile ulaşılabilir olmayabilir; düşük sıcaklık metabolik sapmaları pahasına olsa bile, mekanik hasarlara karşı doku daha dirençli hale getirmek için görünüyor gibi durumlarda, biz Dondurucu soğuk koşullarda dilim hazırlık yapmak öneririz.

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			<td style="width:292px;">Comments</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For monitoring SPS temperature during dissection and slicing</td>
		</tr>
	</tbody>
</table>

slice it hot: acute adult brain slicing in physiological temperature

Burada akut beyin dilimleri hazırlanması için bir protokol mevcut. Bu prosedür büyük ölçüde sonuçların kalitesini belirler elektrofizyolojik yama-kelepçe deneyler için kritik bir unsurdur. Bu prosedür kesme sırasında soğutma aşamasının atlanması yetişkin hayvanlarda yüksek ölçüde miyeline beyin yapıları ile ilgili, özellikle, sağlıklı dilimleri ve hücreler elde etmek yararlı olduğu gösterilmiştir. Yüksek sıcaklık sadece üzerine speküle edilebilir sinir sağlığını destekler bu sayede kesin mekanizması, bu nedenle duruyor olsa da, mümkün, dilimleme gerçekleştirildiği sıcaklık sıcaklık ilgili eserler önlemek için fizyolojik koşullara yakın olmalıdır. Bu yöntemin bir diğer önemli avantajı, işlemin basitlik ve dolayısıyla kısa hazırlık zamanı. Gösterilen yöntemde, yetişkin fareler kullanılmaktadır ancak aynı prosedür genç farelerde hem de sıçanlarda ile uygulanabilir. Ayrıca, aşağıdaki yama clCo. deney yatay serebellar dilimler üzerinde gerçekleştirilir, fakat aynı prosedür, aynı zamanda, başka düzlemlerde gibi beynin diğer arka alanlarda kullanılabilir.

Tarif edilen şekilde hazırlandı dilimleri çeşitli elektrofizyolojik ve Optogenetic deneyleri için de kullanılabilir. Şekil 3A ve 3C olarak, sırası ile, diferansiyel girişim (DIC), optik altında kullanıldığında, yatay serebellar dilimi ve koronal beyin korteks dilim, temsili bir örneğini gösterir. Serebellar dilim, serebellar nöronların çeşitli türleri kolayca, kendi konum ve hücre vücut şekli kabul edilebilir elektrofizyolojik kayıtları hedefe yönelik. <str...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Fizyolojik Sıcaklık Akut Yetişkin Beyin Dilimleme

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Research

JoVE Journal

Neuroscience

26.1K Görüntüleme.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Video: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

TMS Devlet Bağımlılık Etkileri: Motive Phosphene Davranış Bir Bakış

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Nörobilim

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Görüntüleme Feromon Fare Vomeronasal Akut Doku Dilim Hazırlama içinde Algılama

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Yetişkin Fare önbeyin akut dilimleri içinde Neuroblast Göç Time-lapse Görüntüleme

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Yetişkin Rat spesifik Beyin Alanlarının eksitotoksik Lezyon için Stereotaksik Cerrahi

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Immünopresipitasyon ve Açil-Biotin Borsası (ABE) tarafından Yetiştirilmiş Hipokampal Nöronlar Protein Palmitoylation tespiti

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Beyin Dilim Biyotinilasyonu: An<em&gt; Ex Vivo</emYetişkin Nöronlar Bölge özgü Plazma Membran Protein Ticaretiyle Tedbir&gt; Yaklaşım

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Kaynaklı Nöronlar içine Yetişkin İnsan Beyninin perisit türetilmiş Hücreler Lineage yeniden programlama

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Fare Vomeronazal Organ Akut Doku Dilimleri Tanımlı hücre popülasyonlarının derinlemesine Fizyolojik Analizi

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Sinaptik plastisite akut Hipokampal dilimleri bir küçük hacimli geri dönüşüm-, perfüzyon- ve batma-tipi oda sistemi içinde tutulan kayıt

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Drosophila larva termal tercihlerini belirlemek için bir sıcaklık gradyanı tahlil

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...