Destek Tıp Pittsburgh Üniversitesi Okulu&#39;nda Farmakoloji ve Kimya Biyoloji Bölümü&#39;nden başlangıç ​​fonlar tarafından sağlanmıştır. Nicholas Graff: video gönderme katkıda Jacob laboratuvar üyeleri tanınması.

Aşağıda açıklanan tüm protokoller IACUC ve Tıp Pittsburgh Üniversitesi Fakültesi KİK kurumsal inceleme kurulları ile uyumludur. 1.. Doku Kültürü Hood Hipokampal Nöronal Kültürler hazırlanması Not: Protokol 1 genelinde Kullanım steril tekniği ve reaktifler. <ol><li> Nöronal kültürü için bir alt-tabaka olarak poli-D-lisin (0.1 mg / ml olarak H 2 O) kaplı cam lameller hazırlayın. <ol><li> Her 3,5 cm doku kültürü çanak içinde 4-5 yuvarlak cam lamelleri yerleştirin. </li><li> Her biri 12 mm örtücü kısım üzerine Noktası 70 ul poli-D-lizin. Not: canlı görüntüleme için, bir cam 3,5 cm&#39;lik bir doku kültürü kabı (14 mm gömülü cam lamel üzerine 200 ul poli-D-lisin nokta) kullanılabilir dipli. </li><li> Doku kültürü kaputu O / N. hazırlanan yemekleri bırakın Poli-D-lizin buharlaşmasını en aza indirmek ve doku kültürü kaputu steril yüzeyleri tutmak, pencere kanat kapatılması ve inci kapatmak içine blower O / N Not: UV lambaları O / N inkübasyon sırasında kullanılmazlar. </li><li> Ertesi sabah, yıkama yemekler H2O 2 ml ile 3 kez </li><li> 2 O yıkama son H çıkardıktan sonra, her 3.5 cm çanak medya 2 ml ekleyin ve adım 1.4 nöronlar plaka hazır olana kadar inkübatör yemekleri bırakın. </li></ol></li><li> Hipokampal nöron embriyonik gün 18-19 (Goslin 22 değiştirilmiş) (E18-19) sıçanları hazırlanır. </li><li> Maxiprepped yapısı, 1-4 ug DNA ile kültür gününde taze ayrışmış nöronlar transfekte. Not: Genellikle DNA 3 ug 50% bir canlılığı ve 2.000.000 nöronlar başlayarak% 60 bir transfeksiyon verimliliği (örn, 1-2.000.000 nöron transfekte edilir: ((2 x 10 x 6 nöronlar 0.5) 0.6) içerir Yaklaşık 1 milyon dolar nöronlar.. potansiyel sorunlarını giderirken 1 transfekte olan 600,000 olan yapı DNA&#39;nın Farklı miktarlarda transfekte edilebiliryapı, lokalizasyonu ve fonksiyon uygun karakterizasyonu, ardından aşırı ifade,. </li><li> 3.5 cm tabak başına yaklaşık 200,000 nöronların nihai yoğunlukta poli-D-lisin kaplı lamelleri transfekte nöronlar Plate. Not: Bir cam dipli çanak için, 14 mm cam alanı üzerinde 40,000-50,000 nöronlar plaka. </li><li> Medya nöronal kültürlerin hazırlanmasından sonra 4-24 saat değiştirin, sonra nöronlar in vitro (DIV) veya istenilen aşamada 14-17 gün kadar inkübatör içinde geliştirmesine olanak sağlar. </li></ol> 2. Endositoz Deneyi DİKKAT: α-bungarotoksinin ve tubokurarin atık ve kullanım ile ilgili Not: <ol><li> Tüm peptid Nörotoksinlerin taşınması Biyogüvenlik Düzeyi-2 (BL-2) araştırma protokollerine kurallar içinde tavsiye edilir. Tüm uygun kişisel koruyucu donanım giyilmelidir. Liyofilize tozlar toksin üretici firmanın talimatlarına uygun olarak yeniden olmalıdır. Toksin atık should düzenleyen kurumun Çevre Sağlığı ve Güvenliği kurallarına uygun olarak imha edilmelidir. </li><li> Depolama sırasında oluşabilen olası peptid agrega kaldırmak için, α-bungarotoksin tam bölünen miktarları, kullanımdan önce kısa bir süre için santrifüj edilmelidir, ve süpernatant deney için kullanılmalıdır. </li><li> α-bungarotoksin (Bgt) stokları, -20 ° C&#39;de steril H2O içinde 1 mg / ml &#39;lik bir konsantrasyona kadar yeniden süspansiyona alınmış ve 10 ul alikolar içinde saklanır Floresan bağlanmış Bgt stokları 3 ug / ml &#39;de kullanılmıştır. Tubokurarin, 150 uM değerinde bir nihai konsantrasyonda kullanılmıştır. </li></ol><ol><li> Üstüne ince alüminyum veya paslanmaz çelik levha ile soğuk bir odada 16 ° C&#39;ye ayarlayınız tezgah üst ısıtıcı blok. Mevcut Alternatif olarak, bir tezgah üstü soğutma / ısıtma cihazı 16 ° C gerektiren her adım için kullanılabilir </li><li> MM olarak ihtiva eden hücre dışı serin Hepes-tamponlu tuz çözeltisi (HBS: 135 NaCl, 4.7 KCl, 10 HEPES, 11 glukoz, 1,2 MgCl2, ve 2.5CaCl2, bir su banyosu içerisinde 16 ° C&#39;ye pH 7.4) eklenmiştir. </li><li> 16 ° C &#39;de alüminyum levha transfer nöron yemekleri ve 5 dakika boyunca soğuk. </li><li> Ortamı çıkarın (adım 2.8, endositoz deney zaman noktaları için kuluçka makinesi içinde koşullu ortam ve rezerv tutmak) ve 2 dakika boyunca 16 ° C HBS + tubokurarin (150 uM), 1 ml ile değiştirin. </li><li> Etiketli bir atık kabına HBS + tubokurarin çıkarın ve Alexa 594 [3 ug / ml], 15 dakika boyunca 16 ° C&#39;de inkübe 1 ml 16 ° C HBS + tubokurarin (150 uM) + α-bungarotoksinin ile değiştirin. </li><li> Kaldır HBS + tubokurarin + α-bungarotoksin Alexa 594 (yıkar% 50 çamaşır suyu 50 ml içeren bir vakum şişesine aspirasyon yoluyla tahsil edilebilir), atık konteyneri, etiketlenmiş ve yemekler 16 ° C HBS 2 ml 3x yıkayın. </li><li> Bir bardak kullanarak reseptör endositozu aşağıdaki canlı görüntüleme zaman serisi deneyleri 3,5 cm kültür dipli çanak için, adımları 2.1-2.6 gerçekleştirin, daha sonra ısıtılmış sahneye çanak transferi(Peltier cihaz) mikroskop ve konfokal veya TIRF mikroskop kurulumu ile görüntüleme başlar. </li><li> 2.9 adım diğer lamelleri ile devam eden, 20 dakika boyunca RT% 4 paraformaldehid /% 4 sukroz bir çanak içine T = 0 zaman noktası lamelleri aktarın. </li><li> Diğer zaman noktaları için, ile HBS yerine 37 ° C ortam (adım 2.4 aittir) ve 37 ° C Not endositoz için inkübatör dönmek dengelenmiş klimalı: ek süre T = 15 puan, 30, ve 60 önerdi. </li><li> Zaman noktalarında gerekli, inkübatörden yemekler almak RT DPBS 2 ml hızla iki kez yıkayın (herhangi bir kalsiyum, magnezyum, DPBS), daha sonra 20 dakika süreyle% 4 paraformaldehid /% 4 sukroz içinde çözmek. </li><li> 20 dk Fiksasyon sonra, kabı atık ve 2 ml RT DPBS ile iki kez bulaşıkları yıkamak için% 4 paraformaldehid /% 4 sukroz kaldırın. </li><li> Immünofloresan blok çözeltisi içinde 10 dakika boyunca oda sıcaklığında inkübe lamelleri (blok çözeltisi = DPBS içinde% 5 at serumu,% 0.5 BSA) geçirgenliğini için% 0.2 Triton X-100 ihtiva edennöronlar ize ve hücre içi reseptör havuz ve / veya ilgi duyulan diğer proteinlerin etiketlenmesine anti-GFP immün sağlar. </li><li> Blok +% 0.2 Triton X-100 çıkarın ve 20-30 dakika boyunca Triton X-100 olmadan imüno blok çözeltisi içinde lamelleri inkübe edin. </li><li> 4 ° C&#39;de, oda sıcaklığında ya da O / N birkaç saat boyunca blok lamelleri birincil antikor inkübasyonlar yapın Not: 4 ° CO / N de Primer ile lamelleri kuluçka rutin geliştirilmiş immunofloresans sonuçlar üretir. </li><li> Yıkama DPBS (her bir yıkama adımı boyunca 5 dakika) ile 3 kez lamelleri. </li><li> Oda sıcaklığında 1 saat blok çözeltisi içinde sekonder antikor ile inkübe lamelleri. </li><li> Yıkama DPBS (her bir yıkama adımı boyunca 5 dakika) ile 3 kez lamelleri. </li><li> Tek tek her lamel taşıma Dağı lamelleri: laboratuvar doku ile lamel arkasından aşırı sıvı kaldırmak sonra bir cam slayt orta montaj 4 ul üzerine lamel ters. </li><li> Oda sıcaklığında kurumaya slaytlar st daha sonra 30 dakika boyunca kapalı izin ver4 ° C&#39;de cevher mikroskobu gerçekleştirmek için hazır olana kadar. </li><li> Görüntü edinimi ve konfokal mikroskopi (deneysel durumuna kör) ile deney analizi. 60X petrol NA aşağıdaki lazerler ile 1.49 daldırma hedefi: Argon gazı 488 nm, 561 diyot, 640 diyot. <ol><li> Aynı görüntü elde etme ayarlarını kullanarak, nöronal hücre gövdesi ve odak çeşitli dendritik süreçleri ile tek Z bölümünde görüntü kazanır. </li><li> Tüm endositoz verilerin analizi için aynı eşik kullanarak, nöron başına 3-4 proksimal dendritlerin 20 mikron birlikte α-bungarotoksin Alexa floresan sinyali ve GFP bağışıklık boyamasım ölçmek. </li><li> Birkaç bağımsız nöronal kültürler ile deney tekrar, her bir zaman noktası için 10-12 nöronlardan verileri analiz. </li></ol></li></ol>

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Yazarlar ifşa hiçbir şey yok.

Burada açıklanan BBS göre sabit ve canlı teknikleri reseptörü veya hücre hatları, nöronlar ve diğer primer hücrelerde diğer plazma zar proteini kaçakçılığı izlemek için kullanılabilir. Bu yöntem, başarılı bir zar yerleştirilmesi ve ligand-kapılı iyon kanalları ve GPCR çıkarılmasını çalışma nedeniyle reseptör agonistleri ve modülatörlerinin varlığında sirkülasyondan değişiklikleri değerlendirmek için kullanılmıştır. Anahtar açıdan uygun bir hücre dışı bir konuma et...

Floresan reseptör lokalizasyonu ve dinamiklerini incelemek için α-bungarotoksin birleştirilmiş kullanımı, nikotinik asetilkolin reseptörü 13-15 toksinin endojen hedef çalışmalarda öncüleridir. Daha sonra, en az Bgt bağlayıcı peptid (BBS) dahil edilmesi hem de uyarıcı ve inhibitör ligand-kapılı iyon kanalları ve G protein birleştirilmiş alıcıların 2,10,16-21 kaçakçılığı incelemek için kullanılmıştır. Bu BBS-temelli bir yöntem, yüzey biyotinilasyon yöntemler, hücre-dışı epitopuna antikor canlı hücrelerin antikor etiketleme ve (sıkı bağlamak) ışıkla ağartma sonra floresan kurtarma gibi ticareti çalışmalar için kullanılan diğer yaklaşımlara avantajlar sağlamaktadır. Hücre yüzeyinde sırasında biotinilasyon serbest aminler kovalent hücresel aktiviteyi etkileme potansiyeline sahip, modifiye edilir. Antikor bazlı çalışmalar genellikle kaçakçılığı olayları değiştirebilir yüzey antijeni kümeleme veya kapatması, engel olmuştur. Nedeniyle sıkı bağlamak s beyazlatma aşamasındantudies önemli bir endişe, altta yatan hücresel yapının zarar veriyor. Diğer bir avantaj BBS konstruktları da biotin ile birleştirilmiş eşek bungarotoksin reseptör ticareti biyokimyasal yöntemleri için kullanılabilir etiketli olmasıdır. Bu teknik, hücre hatları ve primer hücre kolayca uygulanabilir. Belirtildiği gibi nikotinik asetilkolin (nAChR) reseptörlerini eksprese eden hücrelerde kullanım için, nAChR antagonist tubokurarin protokol boyunca kullanılmalıdır. Tubokurarin yokluğunda transfekte edilmemiş hücrelerde (endositoz protokolü için T = 0 zaman noktası eşdeğer) basit bir yüzey Bgt etiketleme Sahne endojen nAChR&#39;nin kanıt sağlar. Bu ilgilenilen protein plazma zarının teslim edilir hücre dışı bir konumda mevcut olduğu için bu tekniği kullanarak için önemli bir faktör, BBS uygun yerleştirilmesini içerir. Örneğin, GABAAR alt birimlerin N-terminal etki alanları prose sırasında veziküler lümen ikametticaretine ve hücre yüzeyi reseptörlerine ve endositik olayların hücre yüzeyinden kendi kaldırma değerlendirmesi belli etiketleme sağlayan, plazma zarında reseptör yerleştirilmesi sonra hücre-dışı hale gelir. Daha önce GFP, myc veya BBS eklenmesinin GABAAR alt birimlerin bu etki için epitoplarını göstermiştir ki, işlevsel olarak sessizdir. Standart kontrol uygun şekilde lokalize olduğunu ve etiketli protein, bir etiketlenmemiş yapısına benzer seviyelerde ifade olduğundan emin olmak için gerçekleştirilmiştir ve bu reseptör fonksiyonunu etkilemez edilmelidir. Transfekte yapıları Bu karakterizasyonu da sorun overekspresyonu kaygıları yardımcı olacaktır.

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			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B35450</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
			<td style="width:196px;">cS703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:196px;">P6407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">640-0702</td>
			<td></td>
		</tr>
	</tbody>
</table>

using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

Bu giderek daha belirgin olduğunu İyonotropik GABA A reseptörleri (GABAAR), sergi derece dinamik kaçakçılığı ve hücre yüzey hareketliliği 1-7 gibi nörotransmitter reseptörleri. Hücre yüzeyi reseptör lokalizasyonu ve endositoz çalışma yapmak için, burada açıklanan teknik hücreler, bir α-bungarotoksin (Bgt) bağlama bölgesi (BBS) içeren yapıları ifade eden floresan α-bungarotoksinin kullanımını birleştirmektedir. BBS (WRYYESSLEPYPD) yüksek afinite ile 8,9 Bgt bağlanan kas nikotinik asetilkolin reseptörü, bir α alt birimi dayanmaktadır. BBS site dahil edilmesi, daha önce GABAA ve GABAB metabotropik takibi 2,10 reseptörleri de tarif edildiği gibi, yüzey lokalizasyonu ve alıcı ekleme veya eksojen floresan Bgt uygulama ile çıkarılması ölçümleri sağlar. BBS sitesine ek olarak, amino asitlerden 4 ve standart m tarafından olgun GABAAR alt biriminin 5 arasında bir pH-duyarlı bir GFP (pHGFP 11) takılıolecular biyoloji ve PCR klonlama stratejileri 12 (bkz. Şekil 1). BBS, bir 13-amino asit alanin / prolin bağlayıcı tarafından ayrılan, pH duyarlı GFP raportör, 3 &#39;dir. Toplam bir muhabiri Bgt normalleşme toplam reseptör nüfusa reseptör nüfusu etiketli izin GABAAR altbirim düzeyde protein etiketlenmiş olarak sabit örnekleri dayalı bu yayında açıklanan çalışmalar kaçakçılığı için, pHGFP hizmet vermektedir. Bu etiketli GABAAR alt birimlerinin yüksek veya daha düşük bazal ifade kaynaklanan Bgt lekeleme sinyal değişkenliği hücre hücre en aza indirir. Ayrıca pHGFP etiketi canlı veya sabit görüntüleme deneyleri için yapı sentezleyen hücrelerin kolayca tanınmasını sağlar.

BBS etiketli yapı karakterizasyonu gibi ifade edilen protein (özellikle birden fazla alt-birimden oluşan alıcıları ile) uygun bir şekilde bir araya saptanması önemlidir kontrolleri içeren, hücre yüzeyine trafikleri ve uygun şekilde lokalize eder. İnhibitör sinaps önleyici iskele protein gephyrin ile colocalize ve sinaptik vesiküller (VIAAT / VGAT) içine ve GABA glisin yükler veziküler önleyici amino asit taşıyıcı ile tanımlanır presinaptik inhibitör terminalleri, apposed olan bir reseptör y?...

Watch this Scientific Journal Video about Using an Î±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Burada GABA hipokampal nöronlar bir reseptör yüzey lokalizasyonu ve, endositoz ölçmek için α-bungarotoksin bağlanmış flüoresan Alexa boya kullanımını göstermektedir. Α-bungarotoksin, plazma zar proteini endositik ticaretinin analiz elde edilebilir bağlanan kısa bir hücre dışı bir etiket taşıyan konstruktların kullanımı yoluyla.

GABA A reseptör Membran Yerelleşme ve Ticareti Eğitim için bir α-bungarotoksin Bağlama Sitesi Tag Kullanımı

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

Research

JoVE Journal

Neuroscience

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

15.7K Görüntüleme.  University of Pittsburgh School of Medicine.  Burada GABA hipokampal nöronlar bir reseptör yüzey lokalizasyonu ve, endositoz ölçmek için α-bungarotoksin bağlanmış flüoresan Alexa boya kullanımını göstermektedir. Α-bungarotoksin, plazma zar proteini endositik ticaretinin analiz elde edilebilir bağlanan kısa bir hücre dışı bir etiket taşıyan konstruktların kullanımı yoluyla. 

Video: Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

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 ...

Nörobilim

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

Mekansal Navigasyon Öğrenme Eğitim için Infrared Spektroskopisi civarında MazeSuite ve Fonksiyonel kullanma

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Görüntüleme İlköğretim Kültürlü Fare Nöronlar içinde Plazma Membran Reseptör Yerleştirme pHluorin etiketli

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Fare Beyin dilimleri içinde Nikotinik Asetilkolin Reseptör Fonksiyon Okumak Uyuşturucu Yerel Uygulama

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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

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

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

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

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

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Membran Yetişkin farelerin ventromedial Hipotalamus Nöronlar Potansiyel Boya Görüntüleme Glikoz Algılama Çalışması için

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

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 ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Locust Antenlerinde Odorant Reseptör Genlerinin RNA ile Lokalizasyonu<em&gt; Yerinde</em&gt; Hibridizasyon

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

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 Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ateş Ex Vivo sıçan beyin dilimleri kullanarak birincil olayları incelemek için alternatif bir yaklaşım

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...