Yazarlar, uzman teknik yardım için C. Roderigo teşekkür etmek istiyorum. Biz video üretimi için yardım S. Köhler (İleri Görüntüleme Merkezi, Heinrich-Heine Üniversitesi Duesseldorf) teşekkür ederim. (MC) R01NS032123 hibe: (Ro 2327 / 8-1 CRR için DFG), (nh) Heinrich-Heine Üniversitesi Duesseldorf ve Ulusal Sağlık Enstitüleri tarafından yazarın laboratuvarında araştırma Alman Araştırma Birliği tarafından finanse edilmiştir.

<ol>
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The authors declare no competing interests.

Sıvı taşıyıcı bazlı, iyon seçici elektrotlar on yıllardır başarıyla birçok iyonlar için kullanılmıştır, son derece spesifik sensörleri 22-26 mevcuttur. ECS genişliği sadece yaklaşık 20-50 nm, seçici iyon çapı ise: Omurgalı beyin hazırlıkları dışı boşluk (ECS) kullanıldığında, kimse bu oldukça invaziv bir tekniktir ki, akılda tutmak gerekir Mikroelektronlar yaklaşık 1 mikron (çift namlulu elektrotlar) veya daha büyük (konsantrik elektrotlar) &#39;dir. Iyon seçici Mikroelektronlar uçları bu nedenle, sadece doku kendi kazığa oturtma sırasında dokulara zarar değil, aynı zamanda iyon geçici bir azımsanmasını lehine ECS büyütmek olacaktır. Bu tuzaklara rağmen, nöronal aktivite cevaben dışı iyon geçici bu yöntemin güvenilirliği doğrulayan farklı laboratuvarlarda 7,8 arasında belirgin tutarlıdır. Iyon seçici elektrot performansı ve uygunluğukullanılan sensor kokteyl (&quot;sıvı membran iyonofor &#39;) tarafından tanımlanan duyarlılık ve seçicilik, bağlıdır. Sensör kokteyller potasyum 27 için yüksek bir seçicilik gösterir K + -seçici mikro elektrod Valinomycin örneğin özel bir taşıyıcı molekül içerir. Rağmen, diğer iyonlar ile çapraz reaksiyon oluşabilir ve test edilmelidir. Valinomycin sonuçları değerlendirilirken göz önüne alınmalıdır amonyum için önemli çapraz reaktivite (örneğin 11,12) gösterir. Iyonoforlar gerilim-tepkisi Nernst davranışı (bakınız denklemi 1) takip ettiği başka, sinyal-gürültü oranı ve saptama eşiği ölçülecek iyonunun konsantrasyonuna bağlıdır. Böylece, ise küçük [K +] o geçici geçici çok daha zor hi karşı algılamak için vardır o alçak taban [K +] o, küçük [Na +] karşı büyük voltaj değişikliklerini uyandırmakGH bazal [Na +] o (bakınız Şekil 5 ve 6). Iyon seçici elektrot performansı büyük ölçüde elektriksel zaman sabiti tarafından yönetilir zamansal çözünürlüğü ile belirlenir. Ikinci esas olarak sensör eksenel direnci ile belirlenir ve iç çözeltiler ve dış sıvısı arasındaki pipet uzunluğu boyunca dağıtılmış kapasitans kullanılarak verilir. Iki namlulu bir yapılandırmada, direnç backfilled iyon sensörünün uzunlukta sütun nedeniyle yüksektir. (Bu durumda borosilikat cam), belirli bir yalıtkan dielektrik için, kapasite dielektrik kalınlığı ile yönetilir. Çift namlulu elektrotlar ise, dielektrik genişlik pipet cam duvarın tutarlar. Cam ucu yakın incelir gibi, dielektrik genişlik düşer ve kapasitans artar. Bu faktörler birkaç yüz milisaniye birkaç değişir tepki süreleri ile elektrotlar üretmek için bir arayasaniye olarak bu faktörler çeşitlidir. Konsantrik tasarımın en büyük avantajı eksenel direnci ve banyo kapasite hem büyük ölçüde azaltılmış olmasıdır. İpucu önce son birkaç mikrometre sadece bir kalıntısı bırakarak backfilled iyon değiştirici direnişin en konsantrik pipet şantlar. Buna ek olarak, konsantrik pipet içindeki dolgu çözümü fiziksel ölçüde kapasitansını azaltarak, iki cam duvar kalınlığı ile ayrılmış, banyo mesafeli edilir. 10 Daha önce gösterildiği gibi, daha az direnç ve kapasitans birleşik etkisi, iki büyük kertesine zamansal çözünürlüğü bir gelişmedir. Eş-Ca 2 + ve pH Mikroelektronlar halinde,% 90 tepki süreleri gibi düşük msn 10-20 olarak 10 idi. Eş-tasarımın bir avantajı, ilgili düşük ses seviyesi (bakınız Şekil 8). Herhangi bir Ambien gelen, büyük ölçüde azaltılmış direnci gerilim transientler sayesindet gürültü en aza indirilmiştir. Ayrıca, bu tür geçici kurtarma çünkü hızlı zaman sabitinin, hızlıdır. Bu tür eserler, bu nedenle küçük ve hızlı, ve fizyolojik kayıtları (bakınız Şekil 8) üzerinde daha az yıkıcı bir etkiye sahiptir. Eş-tekniğin dezavantajları da vardır. İlk olarak, bunların montajı daha karmaşık ve zaman alıcı bir iştir. İkinci bir dezavantaj, ayrı bir mikromanipülatör veya özel bir çift manipülatör ya kullanımını gerektirmeyen, kendi ucu ile ayrı bir referans mikroelektrot yer ihtiyacıdır. Son olarak, iki namlulu Mikroelektronlar konsantrik elektrotlar için mümkün değildir, aynı zamanda 28, iki farklı iyon türü tespitine olanak sağlayan, üçlü namlulu tasarım uzatılabilir. En yaygın tuzaklar Verimsiz silanizasyon. Herhangi bir sıvı-sens imalatında en önemli adım, ve asıl engelya göre iyon seçici mikroelektrot silanizasyon işlemdir. Elektrotlar spesifik iyon konsantrasyonuna değişikliklere yanıt, ya da bir alt Nernst cevabı (on kat konsantrasyon farkı başına, yani iyi az 58 mV) ile yanıt başarısız olduğunda, silanizasyon kötü etkinliği genellikle nedenidir. Atmosferik nem yaz veya kış yükseklikte koşulları tipik, çok yüksek veya çok düşükse Bizim tecrübelerimize göre, bu sırasıyla oluşabilir. Oda nem üzerinde bir miktar kontrol uygulamak için mümkün değilse, bu sorunlar aşılabilir. Elektrot direnci çok yüksektir. Gerekirse, iyona duyarlı bir kovanın direnci Kaynak ağzı azaltılabilir. Bu amaçla, bir kaç saniye için su içinde süspansiyon haline getirilmiş, bir aşındırıcı maddenin kuvvetli jet olan ucu maruz. Bu, upmost ucu kırmak ve istenen değere daha düşük direnci neden olur. Tuz köprüler. Tuziyon ve referans varil arasında köprüler zayıf ya da hiç tepki vermeyen elektrotlar neden ve dolayısıyla da büyük ölçüde kalibrasyon performanslarını karıştırabilir. (Nokta 1.6 bkz.) Yukarıda da belirttiğimiz gibi, bu çift namlulu teta cam seçilir bir sorun esas olmakla birlikte, burada açıklanan ofset, bükülmüş varil tekniğini kullanırken nadir görülen bir durumdur. Akılda imalat kolaylığı sayesinde, Lux 29 orijinal çift namlulu tasarım genellikle karlı kullanılabilir. Bu yöntem aynı zamanda ucu ile, iyon değiştirici katılmasıyla, aşağıdaki, tuz çözeltileri, ucu olan emme ve çıkarma ile silan çözeltisine hızlı bir pozlama ile iyonu ve referans varil ön doldurma kullanır (30,31 bakınız) . Bu elektrotlar yaklaşık 10 dakika içinde imal, ama onların uç boyutu genellikle 4 mm veya daha fazla ve onlar bir deney sırasında başarısız daha yatkındır edilebilir. Bunun aksine, bu silanizasyon yöntemleri silan buhar ve ısı maruz kalınmasınıing küçük günler süren ipuçları ve bazen haftalık elektrotları üretebilir. Birlikte ele alındığında, birçok protokoller vardır ve iyon seçici Mikroelektronlar hazırlamak için nasıl yaklaşır. Burada, yakın% 100 genel başarı oranı ile, bizim laboratuarlarımızda iyi ve güvenilir çalışması bükümlü namlulu çift olarak konsantrik Mikroelektronlar imalatı için iki ana süreçleri de tarif etmiştik. Önemli olarak, bu teknikler, pH veya kalsiyum iyonları da dahil olmak üzere, diğer türler, ölçülmesi de aktarılabilir, ve aynı zamanda, genel olarak sıvı ile dolu boşlukların veya sıvıları da dahil olmak üzere beyinde dışında preparatlar için de geçerli olacaktır. Son fakat en az değil, iyon seçici Mikroelektronlar hücrelerin içinde iyon konsantrasyonlarının tespitine imkan. Nispeten büyük bir uç boyutu (~ 1 mm) arasında olduğundan, bu, bununla birlikte, omurgasız preparasyonlar 28,32 bulunan gibi yalnızca örneğin büyük bir hücre gövdesi, hücrelerde mümkün.

An author's middle initial was omitted from the publication, <a href="http://www.jove.com/video/53058/double-barreled-concentric-microelectrodes-for-measurement">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue</a>. The author&#39;s name has been updated to:
Christine R. Rose
from:
Christine Rose

A correction was made to Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.

Erratum: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical signaling in the brain is based on the flux of ions across plasma membranes. Major ion movements into and from the extracellular space are not only mediated by passage through voltage-gated ion channels, but also by postsynaptic ionotropic receptors as well as ion transporters. Neuronal activity is thus accompanied by complex changes in the extracellular concentration of all major ions 1. For example, influx of sodium into neurons during excitatory activity has been shown to result in a decrease in the extracellular sodium concentration ([Na+]o) 2. The same holds true for the extracellular calcium concentration because calcium ions rapidly enter both pre- and postsynaptic structures 3. At the same time, potassium moves the opposite way and this mediates an increase in the extracellular potassium concentration ([K+]o) in the low mM range 4,5. Synaptic activity also causes changes in extracellular pH that are partly mitigated by concomitant glial membrane fluxes that change intraglial pH 6,7. These activity-related changes in extracellular ion concentrations have significant functional consequences. For example, even small increases in [K+]o depolarize neurons as well as glial cells thereby altering neuronal excitability, and several mechanisms exist to remove excess potassium 8. Failure of these may result in epileptiform activity of neurons or phenomena like spreading depression 1. 
Because of their critical importance, quantitative determination of extracellular ion concentrations is often necessary and required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. For decades, double-barreled ion-selective microelectrodes have proven to be excellent tools for such measurements in brain tissue 9. For many ions, highly specific sensors with low cross-reactivity for other ions are available. In addition to the classical double-barreled electrodes, so-called concentric electrodes were recently introduced. The latter provide a superior time resolution, but take a little more time and effort to construct 10. 
In the following, we will describe the preparation and calibration of these two types of ion-selective microelectrodes. We then show how these electrodes can be employed in brain slice preparations for measurement of changes in [K+]o or [Na+]o induced by excitatory activity following different stimulation paradigms including bath and pressure application of drugs.

<table border="0" cellpadding="0" cellspacing="0">
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		<tr>
			<td>Abrasive&#160;&#160;</td>
			<td>MicroPolish&#160;</td>
			<td>Buehler GmbH</td>
			<td>Dissolved in A.dest</td>
		</tr>
		<tr>
			<td>Borosilicate-glass capillaries</td>
			<td>1405059</td>
			<td>Hilgenberg</td>
			<td>Application pipette; 75 mm x 2 mm, wall thickness 0.3 mm</td>
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		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC 150 F-15&#160;</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the sensor of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC100-F-15</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the reference of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-200TF-15&#160;</td>
			<td>Science Products</td>
			<td>Concentric, outer channel. o.d. 2.0 mm&#160;&#160;</td>
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		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-120TF-10</td>
			<td>Science Products</td>
			<td>Concentric, inner channel. o.d. 1.2 mm</td>
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		<tr>
			<td>Digidata</td>
			<td>1322A</td>
			<td>Axon Instruments</td>
			<td></td>
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			<td>Electrometer amplifier with headstage</td>
			<td></td>
			<td>Custom-made</td>
			<td>Rin = 10T&#937; and Ibias=50fA-1pA (commercially available&#160; alternatives: e. g. Dagan IX2-700, with headstage (10 Gig feedback resistor) or EPMS-07, NPI, Tamm, Germany)</td>
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		<tr>
			<td>Experimental chamber</td>
			<td></td>
			<td>Custom-made</td>
			<td>Commercially available from e.g.&#160; Warner Instruments,USA;&#160; Scientifica, UK</td>
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		<tr>
			<td>Furnace</td>
			<td></td>
			<td>Heraeus</td>
			<td>Must stay constant at 200&#176;C</td>
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			<td>Hard sticky wax / dental wax&#160;</td>
			<td>Deiberit 502&#160;</td>
			<td>Siladent Dr. Boehme &#38; Schoeps GmbH</td>
			<td></td>
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		<tr>
			<td>Hot plate</td>
			<td></td>
			<td>Custom-made</td>
			<td>Must stay constant at 40&#176;C</td>
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		<tr>
			<td>Microelectrodes holder made of plexiglas</td>
			<td></td>
			<td>Custom-made</td>
			<td>Double-barreled: o.d.&#160; capillaries 1.5 mm, concentric: o.d.&#160; capillaries 2 mm</td>
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		<tr>
			<td>Micromanipulator</td>
			<td></td>
			<td>Leitz</td>
			<td></td>
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		<tr>
			<td>Micromanipulator</td>
			<td>MD4R</td>
			<td>Leica</td>
			<td></td>
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			<td>Stereo microscope</td>
			<td>M205C</td>
			<td>Leica</td>
			<td></td>
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			<td>Objective</td>
			<td>Plan 0.8xLWD</td>
			<td>Leica</td>
			<td></td>
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		<tr>
			<td>Pipette puller</td>
			<td>Model PP-830</td>
			<td>Narishige</td>
			<td>Concentric microelectrodes&#160;</td>
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		<tr>
			<td>Pipette puller</td>
			<td>Model P-97</td>
			<td>Sutter Instruments&#160;</td>
			<td>Sensor of concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic drug ejection system</td>
			<td>Picospritzter Type II</td>
			<td>General Valve TM Corporation</td>
			<td></td>
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		<tr>
			<td>Travel dovetail stage</td>
			<td>DT 25/M</td>
			<td>Thorlabs</td>
			<td></td>
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		<tr>
			<td>Two-component glue</td>
			<td>&#160;Araldite</td>
			<td>Huntsman advanced materials GmbH</td>
			<td>One may also use a small stripe of aluminum foil to stick the capillaries together&#160;</td>
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		<tr>
			<td>Silverwire</td>
			<td>99.9%&#160;</td>
			<td>Wieland Edelmetalle</td>
			<td></td>
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			<td>Slicer / Vibratome</td>
			<td>Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>AxoScope 8.1&#160;</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical puller&#160;</td>
			<td>Type PE-2</td>
			<td>Narishige Scientific Instruments</td>
			<td>With a revolvable chuck for&#160; double-barreled microelectrodes</td>
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		<tr>
			<td>x/y translational stage</td>
			<td></td>
			<td>Custom-made</td>
			<td></td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Compound</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>1(S),9(R)-(&#8722;)-Bicuculline methiodide</td>
			<td>Sigma aldrich</td>
			<td>14343</td>
			<td>Competitive antagonist of GABAA receptors (light-sensitive); CAUTION toxic</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma aldrich</td>
			<td>C-127</td>
			<td>AMPA/kainate receptor antagonist; CAUTION toxic</td>
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		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma aldrich</td>
			<td>D5879</td>
			<td></td>
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		<tr>
			<td>DL-AP5</td>
			<td>Alfa Aesar</td>
			<td>J64210</td>
			<td>NMDA receptor antagonist; CAUTION toxic</td>
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		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td>CAUTION: Flammable, acute toxicity (oral, dermal, inhalation) and corrosive to metals and skin</td>
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		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma aldrich</td>
			<td>A9256</td>
			<td>Activates NMDA and non-NMDA and EAATs</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma aldrich</td>
			<td>G1626</td>
			<td>Activates NMDA-R, AMPA-R, QA-R and KA-R),&#160; mGluRs and EAATs</td>
		</tr>
		<tr>
			<td>Potassium ionophore I - cocktail B</td>
			<td>Fluka&#160;</td>
			<td>60403</td>
			<td>Based on valinomycin; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Sodium ionophore II - cocktail A</td>
			<td>Fluka&#160;&#160;</td>
			<td>71178</td>
			<td>Based on ETH 157</td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Ascent Scientific</td>
			<td>Asc-055</td>
			<td>Inhibitor of voltage-dependent Na+ channels; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Water, ultra pure</td>
			<td>Sigma aldrich</td>
			<td>W3500&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

<html> Izlemek için [K +] o ve oluşan değişimler uyarıcı aktiviteye karşılık olarak, iki namlulu bir potasyum seçici mikroelektrot CA1 stratum radiatum sokulmuştur. Birkaç dakika Impalement sonra elektrot iyon seçici varil yaklaşık 2,8 mM, kullanılan ACSF [K +] yakın bir değere (o kadar [K +] tekabül eden, kararlı bir taban çizgisi (Şekil 7A) ulaşıldığında 2,5 mM). 10 sn için 0,5 mM glutamata Banyo uygulaması ~ 0.7 mM (Şekil 7A) tutarında taban altında undershoot, sonra, 4 mM o [K +] bir döner artışa neden. 0.4, 0.3, glutamat konsantrasyonunun azaltılması, ve 0.2 mM geçici bir artış ve [K +] o (Şekil 7A) içinde undershoot iki genliği karşılık gelen bir azalmaya yol açtı. OAD / 53058 / 53058fig7.jpg &quot;/&gt; Şekil uyarıcı aktiviteye karşılık olarak 7. Hücre dışı potasyum geçici. (A): [K +] o geçici, gösterildiği gibi 10 sn için glutamat farklı konsantrasyonlarda banyo uygulaması ile uyarılan taban altında undershoot ardından bir artış, aşağıdakilerden oluşan. (B) glutamat reseptör blokerleri ile perfüzyon Etkisi (CNQX, 100 uM; APV, 100 uM) ile tetrodotoksine (TTX 0.5 uM) [K +] o geçici 10 sn için 0,5 mM glutamat banyo uygulaması ile uyarılan. 0mg 2 + / BIC varlığında (C) kendiliğinden [K +] o geçişler. Ac gösterilen deneyler çift namlulu elektrotlar kullanılarak yapıldı. Gösterim amacıyla, izleri Sawitzky-Golay filtresi (genişlik 20) ile düzeltti bulundu. <a href="https://www.jove.com/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">Cl Lütfen</a>Bu rakamın büyük halini görmek için buraya ick. Daha önce deneyler glutamat kaynaklı [K +] o sinyalleri belirgin TTX uygulama sırasında değişmiş olmadığını göstermiştir ve dolayısıyla voltaj kapılı sodyum kanalları ve nöronal aksiyon potansiyeli nesil 15,16 açılması büyük ölçüde bağımsız olması. Glutamata karşılık olarak gözlenen [K +] o değişikliklerin altında yatan mekanizmaları incelemek için, glutamat reseptör blokerleri, yani CNKX (100 uM; AMPA alıcılarının engelleyici) uygulanmış ve APV (100 uM; NMDA reseptörlerinin bloke) varlığında, TTX (0.5 uM). (Şekil 7B, örneğin 17), bu bloker banyo perfüzyon üzerine, glutamat kaynaklı [K +] değişikliklerden o önce bildirildiği gibi iyonotropik glutamat reseptör kanallarının açılması bağımlılığını teyit neredeyse mülga idi. Bundan başka t göstermek içinhücre-dışı üretimi için glutamaterjik uyarma o ilgisi [K +] sinyalleri, dilimler nominal Mg2 + içermeyen salin 10 uM bicuculline metiyodid (0mg 2 + / BIC) içeren perfüze edildi. Bu NMDA reseptörlerinin voltaja bağımlı Mg2 + -blok azaltır ve (örneğin 18,19) ağ spontan tekrarlayan epileptiform activity neden GABA A reseptörleri bloke ederek inhibe azaltır. Beklendiği gibi 20, kendiliğinden, tekrarlayan [K +] o geçici, yaklaşık 1.5 mM tutarında 0mg 2 + / BIC salin (Şekil 7C) &#39;de tespit edilmiştir. 0.2 bir ortalama amplitüdü olan, bu tepkiler arasında, daha küçük bir [K +] o geçici olarak mM (Şekil 7C) oluştu. Deneylerin ikinci setinde, biz uygulanması ile uyarılmış [Na +] o değişiklikleri belirlemek için [Na +] -duyarlı Mikroelektronlar kullanılanglutamat agonistleri. Burada, biz de iki farklı uygulama paradigmaları kullanan çift namlulu ve konsantrik Mikroelektronlar tepki özelliklerini karşılaştırdık. [Na +] -duyarlı Mikroelektronlar yaklaşık 50 uM&#39;lik bir derinlikte stratum radiatum içine yerleştirilmiştir. Stabil bir temel çizgisi elde edildikten sonra, glutamat agonistinin L-aspartat (2,5 ml / dakika ile 10 mM, 120 saniye, Banyo perfüzyonu) Banyo perfüzyonu başına uygulanmıştır. Daha önce 21 gözlendiği gibi, aspartat uygulama yaklaşık 5 dakika sürmüş ve daha sonra taban çizgisine geri (Şekil 8A) kurtarmak için başladı yaklaşık 15 mM ile o [Na +] yavaş bir düşüşe neden olmuştur. Her iki elektrot ile belirlenir: [Na +] o sinyalleri zirve amplitüdleri ve kinetiği bu koşullar altında hemen hemen aynı ise Özellikle, konsantrik elektrotlar daha stabil bir taban çizgisi ve gürültü seviyesini azaltır (Şekil 8A) arzetmiştir. R test etmek için<html> Daha hızlı bir uygulama paradigma altında farklı elektrot özelliklerini esponse biz iyon seçici mikroelektrot ucundan 20-40 um&#39;lik bir mesafe stratum radiatum yerleştirilmiş ince bir cam pipet ile glutamat uygulanan basınç. 21 Beklendiği gibi, 200 ms için glutamat uygulama (10 mM) [Na +] o (Şekil 8B) bir düşüşe neden oldu. [Na +] o düşüş zirve genlikleri elektrotların her iki tip için de aynı aralıkta idi (:;: 19.1 mM, n = 15 - - 2,0 konsantrik 13.5 mM, n = 14 4.5 çift namlulu). Ancak yavaş Banyo perfüzyonu (yukarıya bakınız), sinyal-gürültü oranı değil, aynı zamanda iki elektrot tip tespit [Na +] o sinyallerinin zaman tabii sadece elde edilen sonuçların aksine önemli ölçüde farklılık göstermiştir. Zirveye ortalama süre konsantrik Mikroelektronlar için namlulu çift ve sadece 1,3 saniye 3.5 saniye idi. ve_content &quot;&gt; Bu nedenle, bu sonuçlar, ve genel eş çift namlulu Na + -seçici Mikroelektronlar hızlı tepki kinetikleri onaylamak (bakınız Şekil 6C ve 8B), aynı zamanda Ca + 2 ve not edildi pH-seçici muadilleri 10 . Kısa patlama sinaptik stimülasyonu hızlı sinaptik kaynaklı iyon transientleri uyandırmak için yapıldığı eski çalışmada, aksine, sadece bir eğilim, ama bizim uygulama ile konsantrik ve çift namlulu elektrotlar arasındaki ortalama pik amplitüdlerinde anlamlı bir fark yoktu paradigması. Bu nedeni muhtemelen sonuç olarak iki (20-40 um) &#39;in bir faktör ile farklılık iyon seçici mikroelektrot ucu ve uygulama pipet mesafe, hedef bölgede maksimum glutamat konsantrasyonu zirve amplitüdlerinde varolan herhangi bir fark tıkayan, farklı deneylerde aynı değildi. (A) Üst izleri: volta DeğişimBir çift namlulu elektrodun (siyah noktalı iz) ve banyo Na + konsantrasyonu değişikliklere tepki olarak konsantrik elektrot (gri iz) referans varil (V ref) ve ge ([Na +] b) gösterildiği gibi. Her iki elektrot voltaj tepkileri hemen hemen aynı olduğuna dikkat edin. Alt izleri: iki namlulu bir elektrot (siyah iz) ve [Na +]; b değişikliklere karşılık olarak bir eş-elektrot (gri iz) Na + -potansiyel (V Na +) gerilim değişiklikler. Her iki elektrot V Na + karşısında B (b) [Na +], yan-logaritmik çizimleri. Verilerin Lineer araziler her iki elektrot için yaklaşık 48 mV eğim ortaya koymaktadır. 70 mM 152 mM ila b çift namlulu (siyah iz) V Na + (C) Müdahale ve [Na +] hızlı bir değişime konsantrik elektrot (gri iz). Konsantrik elektrodun tepki süresi önemli ölçüde fas olduğunu unutmayınter. Gösterim amacıyla, izleri Sawitzky-Golay filtre ile düzeltti edildi (genişlik 20). <img alt="Şekil 8" src="/files/ftp_upload/53058/53058fig8.jpg" /> Şekil 8: çift namlulu ve konsantrik elektrotlar ile tespit edilen hücre dışı sodyum geçici. (A) Geçici V ref değişiklikler ve [Na +] bar belirtildiği gibi o 120 saniye boyunca 10 mM aspartat ile banyo perfüzyon tarafından uyarılan. Üst izleri iki namlulu bir elektrot ile gerçekleştirilen bir kayıt, düşük değerli izleri eşmerkezli elektrot kullanılarak kaydedildi göstermektedir. (B) ok ile gösterilen şekilde 0.2 sn için 10 mM glutamat yerel basınç uygulaması ile uyarılan o V ref ve [Na +] Geçici değişir. Üst izleri iki namlulu bir elektrot ile gerçekleştirilen bir kayıt, düşük değerli izleri eşmerkezli elektrot kullanılarak kaydedildi göstermektedir.Noktalı kırmızı çizgiler onun maksimum sinyal başlangıcı arasındaki dönemde doğrusal uyuyor temsil etmektedir. Eşmerkezli elektrot yanıt süresi bu koşullar altında çok daha hızlı olduğunu not edin. Gösterim amacıyla, izleri Sawitzky-Golay filtresi (genişlik 20) ile düzeltti bulundu. <a href="https://www.jove.com/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Beyin Dokusu Ölçme Ekstraselüler Ion Sinyalleri için Çift namlulu ve Konsantrik Mikroelektronlar

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

double-barreled-concentric-microelectrodes-for-measurement

Research

JoVE Journal

Neuroscience

13.4K Görüntüleme.  Heinrich Heine University Düsseldorf. We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations. 

Video: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Fare Beyin Dokusu Immunoelectron Mikroskopi için hazırlanması

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Nörobilim

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

Odaklı İyon Işın Freze ve Beyin Dokusu Taramalı Elektron Mikroskobu

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Küçük Hayvanlar Kronik Sinir Kayıtlar için Ultra-hafif Motorlu Microdrive Tasarımı ve Montajı

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

Yapma, test etme ve yetişkin beyin doku dilimler halinde potasyum iyon seçici Microelectrodes kullanma

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Yerel Ca2 + sinyalleri membran hedefli Ca2 + göstergeleri tarafından kültürlü hücrelerdeki diseksiyon

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Doğal Polifenol Curcumin Kullanarak Beyin Dokusunda Amiloid Plakların Etiketlenmesi ve Görüntülenmesi

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

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 ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Sıçan Beyin Dokusu için Hidrofobik Doku Temizleme Yöntemi

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Measurement of the Directional Information Flow in fNIRS-Hyperscanning Data using the Partial Wavelet Transform Coherence Method

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural synchronization (INS) during social interactions, functional near-infrared spectroscopy (fNIRS) is one of the most popular techniques for hyperscanning naturalistic social interactions because of its relatively high spatial resolution, sound anatomical localization, and exceptionally high tolerance of motion artifacts. Previous fNIRS-based hyperscanning studies usually calculate a time-lagged INS using wavelet transform coherence (WTC) to describe the direction and temporal pattern of information flow between individuals. However, the results of this method might be confounded by the autocorrelation effect of the fNIRS signal of each individual. For addressing this issue, a method termed partial wavelet transform coherence (pWTC) was introduced, which aimed to remove the autocorrelation effect and maintain the high temporal-spectrum resolution of the fNIRS signal. In this study, a simulation experiment was performed first to show the effectiveness of the pWTC in removing the impact of autocorrelation on INS. Then, step-by-step guidance was offered on the operation of the pWTC based on the fNIRS dataset from a social interaction experiment. Additionally, a comparison between the pWTC method and the traditional WTC method and that between the pWTC method and the Granger causality (GC) method was drawn. The results showed that pWTC could be used to determine the INS difference between different experimental conditions and INS's directional and temporal pattern between individuals during naturalistic social interactions. Moreover, it provides better temporal and frequency resolution than the traditional WTC and better flexibility than the GC method. Thus, pWTC is a strong candidate for inferring the direction and temporal pattern of information flow between individuals during naturalistic social interactions.

This protocol describes partial wavelet transform coherence (pWTC) for calculating the time-lagged pattern of interpersonal neural synchronization (INS) to infer the direction and temporal pattern of information flow during social interaction. The effectiveness of pWTC in removing the confounds of signal autocorrelation on INS was proved by two experiments.

fNIRS-Hipertarama Verilerindeki Yönlü Bilgi Akışının Kısmi Dalgacık Dönüşümü Tutarlılık Yöntemi Kullanılarak Ölçülmesi

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

Fare Beyni Hipokampal Dokusunun Kombine Mekanik ve Enzimatik Ayrışması

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...