Yazarlar aşağıdaki kuruluşlar teşekkür etmek istiyorum: Ulusal Doğa Bilimleri Vakfı Çin (31171018, 31171355), bilim ve teknoloji bölümü, Guangdong (2013KJCX0054), Guangdong Eyaleti, Doğa Bilimleri Vakfı ( 2014A030313418, 2014A030313440) ve Guangzhou bilim ve teknoloji Bürosu (201607010320).

Hayvan barınma ve tüm hayvan deneyleri Etik Komitesi tarafından Ulusal Enstitüsü kurulan hayvan bakımı için yönergelere göre Güney Çin Normal Üniversitesi'nde hayvan araştırma tarafından onaylanmış prosedürler uyarınca yapılmıştır Sağlık.
 1. hazırlık çözümler <ol><li>hazırlamak 500 mL standart yapay serebrospinal Tablo 1 ' de listelenen gibi bizim eseri 7 ' açıklanan bileşenleri içeren sıvı (ACSF).
	Taze bir ACSF çözüm hazırlamak için deiyonize su ile <ol><li>kullanımı temiz cam şişeler. NaCl, KCl, NaH eklemek için kullanmadan önce kuru ve temiz spatula deiyonize su ile 2 PO 4, NaHCO 3, MgSO 4, D-glikoz ve çözüm / % 5 ile % 95'i O 2 bubbled sonra CaCl 2 ekleyin CO 2 ~ 20 dk (olarak ayrıntılı olarak açıklandığı tablo 1) 500 mL deiyonize su karışımı. Tamamen eriyene kadar karıştırın.</li>
		<li>Tamamen ACSF olduğunu emin olun herhangi bir yağış temizleyin. Sonra pH 7.2 7.4 için ayarlayın. Kabarcık % 95'i O 2 ve %5 CO 2 ~ 20 dakika süreyle oluşan bir gaz karışımı ile</li>
	</ol></li> <li>250 mL içeren Tablo 2 ' de açıklanan kimyasallar 7 dilim kesme çözeltisi hazırlamak.
	<ol><li>KCl, MgCl 2, NaH 2 PO 4, NaHCO 3, choline·Cl, D-glikoz Ekle ve CaCl 2 son 1.1.1, 250 ml deiyonize su gösterildiği gibi ekleyin ve 1.1.1 ve 1.1.2 anlatılan geçiyoruz.</li>
		Köpüren sonra % 95'i O 2 / %5 <li>CO 2, kesme çözüm olur, böylece çözüm için 15-20 dk bir dondurucu buz gibi ve kısmen donmuş yerleştirin. 
		Not: Biz ne zaman dilimleri 1-2 ay yapım bu kesme çözüm kullanın-eski fareler; formül 2 aydan daha büyük fareler için farklı olabilir.</li>
		<li>Steril endotoksin-Alerjik izotonik salin eriterek tarafından hazırlamak LPS çözüm (% 0.9 NaCl). LPS intraperitoneally yönetilen (0.5 mg/kg) ve 2 h sonra kaydını başlatmak.</li>
	</ol></li></ol> 2. Prefrontal korteks dilim hazırlanması <ol><li>kesme çözüm dondurucudan almak ve bir rüşvet almak için lunaparkçı. Çözüm buz koyun ve sürekli kabarcık / % 5 ile % 95'i O 2 CO 2.</li>
	<li>Büyük, keskin makas kullanarak, hızlı bir fare olarak açıklanan 9 başını kesmek ve hemen kafa ile dolu bir ölçek buz gibi koymak içine (0-4 ° C) kesme çözüm.</li>
	<li>Orta çizgi boyunca kafatası tepesine bir burun Kaudal yönde iyi makasla kesme ve yanal kafatası kısımları kaldırmak. Buz gibi soğuk kesme çözüm bir güzel spatula ve damla ile beyin kaldırın. 
	Not: Adım 2.2 2.3 hızla yapılmalıdır (ideal olarak &#60; 1dk).</li>
	<li>Beyin buz dolu bir 9 cm Petri kapağını yerleştirin. Buz gibi soğuk kesme çözüm beyinle durulayın. Serebellum ve olfaktör ampul bir tıraş bıçağı ile kesti.</li>
	<li>Uygula cyanoacrylate tutkal numune için sahibi bir vibratome. Dikkatle tutkal, rostral tarafı yukarı, damla üzerinde beyin blok yer ve hemen buz gibi kesme çözümde bırakın.</li>
	<li>Vibratome 15 ° yatay düzlemde referansla açısı için bıçak sahibi ayarlamak ve koronal beyin dilimleri 350 µm hazırlamak (bıçak titreşim frekansı 85 Hz, hız = 0.1 mm/s =).</li>
	<li>Bir pipet kullanarak, kurtarma odası dilimlere dolu ACSF ile transfer kuluçkaya 1 sürekli köpüren % 95'i O 2 / %5 ile h (RT) için CO 2.</li>
</ol> 3. Bütün hücre yama kayıt <ol><li>yapmak cam borosilikat MİKROPİPETLER için elektrotlar kayıt olarak kullanın veya çektirme işlemi el kitabında MİKROPİPETLER belirli esaslarına göre puf. Elektrotlar kayıt için ipucu Dirençleri aralığında 4-6 M olan &#8486;, puf MİKROPİPETLER aralığında 2-5 µm uç çapı varken.</li>
	<li>Blok hızlı Na + kanal ve böylece aksiyon potansiyelleri, ACSF Na + kanal engelleyici (TTX, 1 µM), ekleme ve Peristaltik Pompa Odası ile 2-3 mL/dk kayıt odasında bu çözümün bir sabit akış hızı sürdürmek ve bunun dışında aspirasyon. ACSF % 95'i O 2 / %5 kabarcık sürekli ve yaşayabilirliği dilimleri sağlamak için CO 2.</li>
	<li>Bir beyin dilim kimin iyi ipucu dilim boyutuna sığacak şekilde kesildi değiştirilmiş bir Pasteur pipet kullanarak kayıt odasına aktar. Dilim bir platin dilim çapa ile aralıklı paralel naylon konu ile kapak &#8805; dilimi platform üzerinde tutmak için 2 mm ayrı.</li>
	<li>Kayıt micropipette iç çözüm 7 ile (Tablo 3) doldurun ve puf MİKROPİPETLER 10 µM, 50 µM ve 100 µM (ACSF içinde çözünmüş) veya araç kontrol olarak ACSF GABA ile doldurmak.</li>
	Enkaz ile tıkanma önlemek için <li>ACSF kayıt elektrot çeker önce 1 mL şırınga kullanarak ışık pozitif basınç uygulayın. Bir micromanipulator (4 x objektif lens), mikroskop altında kullanarak kayıt elektrot ve puf micropipette ipuçları monitör ( şekil 1 A) video görüntüsündeki ortasına görünmesini sağlayacak şekilde getirin.</li>
	<li>Yavaş yavaş puf micropipette düşürürken mikroskop odak (40 x objektif lens) ayarlamak ve 45 ° açılı kayıt nöron yukarıda yerleştirin. Puf micropipette ucu ve 20-40 µm ( şekil 1 B) arasında kaydedilen nöron arasındaki mesafe tutun. Yavaş yavaş ve dikkatli bir şekilde kayıt elektrot ile nöron yaklaşım ve pozitif basınç düğmesini bırakın. Zayıf ve kısa emme giga-ohm mühür oluşturmak için elektrot tutucu bağlı boru aracılığıyla uygulamak.</li>
	<li>Koru gerilim 0, mV. Giga-ohm mühür oluşumu sonra hızlı için telafi etmek ve kapasite el ile veya otomatik olarak yavaş. Yukarıda belirtilen bütün hücreli moduna almak için boru aracılığıyla kısa ve güçlü bir emici uygulamak.</li>
	<li>Kayıt eIPSC gerilim tipi kelepçe içinde modu. Düşük gaz basıncı (azot, 4-6 PSI) tek veya eşleştirilmiş GABA puffs ( Şekil 2) sunmak için bir Master 8 gerilim adım jeneratör tarafından kontrollü bir Picospritzer kullanarak puf micropipette uygulamak.</li>
	<li>En iyi eIPSC sonuçları ( şekil 3) elde etmek ve LPS kaynaklı depresyon modeli ( şekil 4) eIPSC ölçmek için puf süresini değiştirmek.</li>
</ol>

<ol><li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260 (5554), 799-802 (1976).</li>
<li>Sakmann, B., Edwards, F., Konnerth, A., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patch+clamp+techniques+used+for+studying+synaptic+transmission+in+slices+of+mammalian+brain%5BTitle%5D)+AND+%22Q+J+Exp+Physiol%22%5BJournal%5D)">Patch clamp techniques used for studying synaptic transmission in slices of mammalian brain.</a> Q J Exp Physiol. 74 (7), 1107-1118 (1989).</li>
<li>Eyo, U. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+hyperactivity+recruits+microglial+processes+via+neuronal+NMDA+receptors+and+microglial+P2Y12+receptors+after+status+epilepticus%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.</a> J Neurosci. 34 (32), 10528-10540 (2014).</li>
<li>Dickinson, G. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+determining+the+recruitment+of+inositol+trisphosphate+receptor+channels+during+calcium+puffs%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs.</a> Biophys J. 105 (11), 2474-2484 (2013).</li>
<li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Columnar+distribution+of+activity+dependent+gabaergic+depolarization+in+sensorimotor+cortical+neurons%5BTitle%5D)+AND+%22Mol+Brain%22%5BJournal%5D)">Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.</a> Mol Brain. 5, 33(2012).</li>
<li>Glykys, J., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+main+source+of+ambient+GABA+responsible+for+tonic+inhibition+in+the+mouse+hippocampus%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus.</a> J Physiol. 582, (Pt 3) 1163-1178 (2007).</li>
<li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((APP+modulates+KCC2+expression+and+function+in+hippocampal+GABAergic+inhibition%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.</a> Elife. 6, (2017).</li>
<li>Dunn, A. J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li>
<li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. (117), (2016).</li>
<li>Wondolowski, J., Frerking, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subunit-dependent+postsynaptic+expression+of+kainate+receptors+on+hippocampal+interneurons+in+area+CA1%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.</a> J Neurosci. 29 (2), 563-574 (2009).</li>
<li>Yang, L., Wang, Z., Wang, B., Justice, N. J., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+precursor+protein+regulates+Cav1.2+L-type+calcium+channel+levels+and+function+to+influence+GABAergic+short-term+plasticity%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.</a> J Neurosci. 29 (50), 15660-15668 (2009).</li>
<li>Huo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prefrontal+Cortical+GABAergic+Dysfunction+Contributes+to+Aberrant+UP-State+Duration+in+APP+Knockout+Mice%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.</a> Cereb Cortex. , (2016).</li>
<li>Zhou, W. L., Gao, X. B., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acetylcholine+Acts+through+Nicotinic+Receptors+to+Enhance+the+Firing+Rate+of+a+Subset+of+Hypocretin+Neurons+in+the+Mouse+Hypothalamus+through+Distinct+Presynaptic+and+Postsynaptic+Mechanisms%5BTitle%5D)+AND+%22eNeuro%22%5BJournal%5D)">Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms.</a> eNeuro. 2 (1), (2015).</li>
</ol>

Yazarlar ifşa gerek yok.

Puf uygulama postsinaptik reseptör işlevi3,4,7değerlendirmek için yaygın olarak kullanılır, ancak her deney da hassas doğrulukta denetim gerektirir. Biz burada GABA-puf prefrontal kortikal beyin dilimler halinde IPSCs (yani eIPSCs) indüklenen gösteren bütün hücreli yama sıkma, içeren bir açıklayınız. Kayıt elektrot dayanıklılığını yaklaşık 5 MΩ, puf MİKROPİPETLER uç çapı hakkında ise 2-5 µm. P...

Yama-klemp tekniği, nöronlar, elektrik sinyalleri soruşturma için birincil araç 1970'lerde1,2geliştirilmiştir. O nasıl spesifik tedaviler üzerinde bilgi sağlamak bu tekniği önemli bir avantajı olduğunu (örneğin, farmakolojik) nöronal işlevleri veya gerçek zamanlı3kanalları değiştirebilir. Nöronal fonksiyon farmakolojik değerlendirme bütün hücreli bir beyin dilimi kayıt sırasında kaydedilen nöronlar agonistler veya belirli reseptörlerinin antagonistleri gibi ilaçlar uygulamaya gerektirir. Bu yöntem uygulama böylece daha iyi nöronlar4fizyolojik ve patolojik özelliklerini anlamak için önde gelen özel bir ilaç takip meydana gelen nöronal değişiklikler tanımlamasını sağlar. Farmakolojik yönetim ya da perfüzyon5 veya puf6yapılabilir rağmen ikinci üstün bir tekniktir. Özellikle: (i) uygulama hızla puf artar ilaç konsantrasyonu bir seviyeye kaydedilen nöron etrafında öyle ki membran reseptörleri duyarsızlaştırma engellenir; (II) böylece bu nedenle yönetilen kimyasallar beyin dilimleri üzerinde herhangi bir istenmeyen etkileri azaltır banyo çözüm üzerinde küçük etkisi şişirilmiş uyuşturucu hacmi son derece düşüktür; (iii) puf Protokolü olabilir ayarlamak ve kaydedilmiş, deneme çok hassas tekrarlanabilir; yapma (iv) puf uygulama agonistler/antagonistleri, özellikle bu tür reaktifler pahalı veya elde etmek zor olduğu ekonomik kullanımını gösterir.
Burada, Bütün hücreli akımları GABA akut hazırlanan beyin dilimleri, nispeten iyi korunmuş beyin devreleri avantajına sahip bir hazırlık şişirme tarafından indüklenen kayıt üzerinde durulacak. Bu makalede nasıl inhibitör akımları puf kaynaklı7 kuralları açıklanacaktır. Sezyum (Cs+) kullanarak-tabanlı iç çözümler ve nöronlar 0 tutan mV, GABA-puf uyarılmış tanıtacak inhibitör postsinaptik akımları (eIPSCs) uygun teknik ayrıntılarla. Biz gösteriyor ki eIPSC lipopolysaccharide (LPS) enjeksiyon8tarafından indüklenen depresyon bir fare modeli kullanarak GABA puff tarafından uyarılmış genlik, LPS-Injected fareler araç kontrollere göre dilim içinde önemli ölçüde azalır. Bu makalede nasıl nefes tekniği yaygın olarak uygulanabilir herhangi bir kimyasal madde, bileşikleri veya uyuşturucu beyin dilimleri nöronal faaliyetleri üzerindeki etkisini değerlendirmek yönelik çalışmalar için göstermelidir bizim niyeti olduğunu.

<table><tbody><tr><td>Glass Borosilicate micropipettes</td><td>Shutter Instruments</td><td>BF150-86-10</td><td>1.50 mm outer diameter; 0.86 mm inner diameter</td></tr><tr><td>Micropipette Puller</td><td>Shutter Instruments</td><td>MODEL P-97&amp;nbsp;</td><td>Flaming/Brow Micropipette Puller</td></tr><tr><td>Micromanipulators</td><td>Shutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system&amp;nbsp;</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Imaging Camera</td><td>Nikon&amp;nbsp;</td><td>2115001</td><td>Inspection equipment</td></tr><tr><td>Microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Eclipse FN1&amp;nbsp;</td><td></td></tr><tr><td>Master 8</td><td>A.M.P.I.</td><td>Master-8 Pulse stimulator</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica</td><td>VT 1000S</td><td></td></tr><tr><td>Razor blade</td><td>Gillette</td><td>74-S</td><td>FLYING EAGLE</td></tr><tr><td>Video monitor</td><td>Panasonic</td><td>WV-BM 1410</td><td></td></tr><tr><td>502 Glue</td><td>Deli</td><td>7146</td><td>Cyanoacrylate Glue</td></tr><tr><td>Peristaltic pump</td><td>Shanghai JIA PENG Corporation</td><td>BT100-1F</td><td></td></tr><tr><td>Video Camera</td><td>Olympus America Medical</td><td>OLY-150</td><td></td></tr><tr><td>Transfer Pipets</td><td>Biologix</td><td>30-0138A1</td><td></td></tr></tbody></table>

whole-cell currents induced by puff application of gaba in brain slices

Farmakolojik yönetim yaygın olarak bütün hücreli yama-kelepçe beyin dilimler halinde kayıt yürütmek için kullanılır. Elektrofizyolojik kayıt sırasında uyuşturucu uygulamasının en iyi yöntemlerden birini farmakolojik reaktifler beyin dilimleri nöronal faaliyetleri üzerindeki etkisini incelemek için kullanılan puf tekniğidir. Puff uygulama en büyük avantajı ilaç konsantrasyonu kayıt site çevresinde hızlı, böylece membran reseptörleri duyarsızlaştırma engelleyen artırır olmasıdır. Puff uygulama başarılı kullanımı dikkat için aşağıdaki öğeleri içerir: ilaç konsantrasyonu, puf micropipette, puf micropipette ve kaydedilen nöron ucu ve süresi arasındaki mesafe parametreleri ve puf sürüş basınç (inç kare başına pounds PSI). Bu makalede gama - aminobütirikasit (GABA) bir nöron prefrontal kortikal dilimin üzerine şişirme tarafından indüklenen bütün hücreli akımları kaydetmek için adım adım bir yordam açıklanır. Özellikle, aynı yordamı küçük değişiklikler oluşur ve striatum gibi diğer beyin bölgeleri ve hücre kültürleri gibi farklı ürünleri ile uygulanabilir.

Puff tekniği araştırmacılar uyuşturucu etkisi belirli bir neuron yüzeyinde belirli reseptörleri üzerinde çalışmaya izin verir. Bu makalede, GABA reseptörleri tarafından aracılı akımları odaklanmak. Şekil 1 üfle ve kayıt MİKROPİPETLER gösterir. Fiziksel baskı kaydedilen nöronlar üzerinde herhangi bir etkisi ortadan kaldırmak için puf, ACSF tarafından üretilen ve sukroz puffs (Şekil 2A</strong...

Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

GABA beyin dilimleri içinde uygulanması Puff tarafından indüklenen bütün hücreli akımları

Biz hangi tarafından farmakolojik reaktifler bütün hücreli yama-kelepçe kayıt sırasında yönetilemez duruma gelir ve başarısı için çok önemli özellikleri çeşitli yönlerini vurgulamak puf tekniğini tanımlamak.

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

whole-cell-currents-induced-puff-application-gaba-brain

Research

JoVE Journal

Neuroscience

9.3K Görüntüleme.  South China Normal University. Biz hangi tarafından farmakolojik reaktifler bütün hücreli yama-kelepçe kayıt sırasında yönetilemez duruma gelir ve başarısı için çok önemli özellikleri çeşitli yönlerini vurgulamak puf tekniğini tanımlamak.

Video: Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

Rat Beyin Dilimleri GABA-aktive Tek kanallı ve Tonik Akımlar

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Nörobilim

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Akut Hipokampal Dilimler içinde Synaptically-uyarılmış astroglial ve Nöronal Cevaplarının İkili Elektrofizyolojik Kayıtlar

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- ve nörokimyasal tanımlanan hipokampal internöronlardan gelen tüm hücre Patch-kelepçe Kayıtlar

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

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 ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Beyin Dilimleri tüm hücre Patch-kelepçe Kayıtlar

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

Bir Neurotransmission roman bileşikleri bir gama - aminobütirikasit reseptör oransal keşfi için yöntemleri yazın

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Akut Ictal faaliyet kemirgen ve insan dokusu üretimi ve isteğe bağlı başlatma

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Fare Beyninin Yatay Hipokampal Dilimleri

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Source: Feng, Y. et al., <a href="https://app.jove.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

Bir Fare Beyin Diliminde GABA Puffing ile Tüm Hücre Akımlarının Kaydedilmesi

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions

	...