Yazarlar, üstün teknik yardım için Mayumi Sakuraba ve Rebecca Foehring teşekkür etmek istiyorum. Buna ek olarak, Dr teşekkür etmek istiyorum. Rodrigo Andrade transfeksiyon için cDNA yapıları bize sağlamak için Organotipik dilim kültür ve Biyolistik transfeksiyon protokolleri ve Dr. Jeanne Nerbonne uygulanmasında yardım için. NS044163 NINDS (RCF): Bu çalışma NIH hibe tarafından desteklenmiştir.

<ol>
	<li>Beique, J. C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex.">Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex.</a> Proc Natl Acad Sci U S A. 104, 9870-9875 (2007).</li><li>Buonomano, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+neural+responses+in+cortical+organotypic+slices.">Timing of neural responses in cortical organotypic slices.</a> Proc Natl Acad Sci U S A. 100, 4897-4902 (2003).</li><li>Caeser, M., Bonhoeffer, T., Bolz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+organization+and+development+of+slice+cultures+from+rat+visual+cortex.">Cellular organization and development of slice cultures from rat visual cortex.</a> Exp Brain Res. 77, 234-244 (1989).</li><li>Foehring, R. C., Toleman, T., Higgs, M., Guan, D., Spain, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actions+of+Kv2.1+channels+in+rat+neocortical+pyramidal+neurons.">Actions of Kv2.1 channels in rat neocortical pyramidal neurons.</a> Soc Neurosci Abstr. 34, (2009).</li><li>G&#228;hwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> J Neurosci Methods. 4, 329-342 (1981).</li><li>G&#228;hwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+of+neural+tissue.">Organotypic cultures of neural tissue.</a> Trends Neurosci. 11, 484-489 (1988).</li><li>G&#228;hwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures:+a+technique+has+come+of+age.">Organotypic slice cultures: a technique has come of age.</a> Trends Neurosci. 20, 471-477 (1997).</li><li>G&#228;hwiler, B. H., Thompson, S. M., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Maintenance+of+Organotypic+Slice+Cultures+of+CNS+Tissue.">Preparation and Maintenance of Organotypic Slice Cultures of CNS Tissue.</a> Current Protocols in Neuroscience. , 6.11.1-6.11.11 (2001).</li><li>Guan, D., Lee, J. C., Tkatch, T., Surmeier, D. J., Armstrong, W. E., Foehring, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+biophysical+properties+of+Kv1+channels+in+supragranular+neocortical+pyramidal+neurones.">Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones.</a> J Physiol. 571, 371-389 (2006).</li><li>Guan, D., Lee, J. C. F., Higgs, M., Spain, W. J., Armstrong, W. E., Foehring, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+Kv1+containing+channels+in+neocortical+pyramidal+neurons.">Functional roles of Kv1 containing channels in neocortical pyramidal neurons.</a> J. Neurophysiol. 97, 1931-1940 (2007).</li><li>Guan, D., Tkatch, T., Surmeier, D. J., Armstrong, W. E., Foehring, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv2+subunits+underlie+slowly+inactivating+potassium+current+in+rat+neocortical+pyramidal+neurons.">Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons.</a> J Physiol. 581, 941-960 (2007).</li><li>Johnson, H. A., Buonomano, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+chronic+stimulation+of+cortical+organotypic+cultures+using+implanted+electrodes.">A method for chronic stimulation of cortical organotypic cultures using implanted electrodes.</a> Neurosci Methods. 176, 136-143 (2009).</li><li>Johnson, H. A., Buonomano, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+plasticity+of+spontaneous+activity+and+Up+states+in+cortical+organotypic+slices.">Development and plasticity of spontaneous activity and Up states in cortical organotypic slices.</a> J Neurosci. 27, 5915-5925 (2007).</li><li>Malin, S. A., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+rectifier+K++currents,+IK,+are+encoded+by+Kv2+alpha-subunits+and+regulate+tonic+firing+in+mammalian+sympathetic+neurons.">Delayed rectifier K+ currents, IK, are encoded by Kv2 alpha-subunits and regulate tonic firing in mammalian sympathetic neurons.</a> J Neurosci. 22, 10094-10105 (2002).</li><li>O'Brien, J. A., Holt, M., Whiteside, G., Lummis, S. C., Hastings, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+to+the+hand-held+Gene+Gun:+improvements+for+in+vitro+biolistic+transfection+of+organotypic+neuronal+tissue.">Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue.</a> J Neurosci Methods. 112, 57-64 (2001).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Villalobos, C., Shakkottai, V. G., Chandy, K. G., Michelhaugh, S. K., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKCa+channels+mediate+the+medium+but+not+the+slow+calcium-activated+afterhyperpolarization+in+cortical+neurons.">SKCa channels mediate the medium but not the slow calcium-activated afterhyperpolarization in cortical neurons.</a> J Neurosci. 24, 3537-3542 (2004).</li><li>Walker, P. D., Andrade, R., Quinn, J. P., Bannon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+preprotachykinin+promoter+activity+in+single+cortical+neurons.">Real-time analysis of preprotachykinin promoter activity in single cortical neurons.</a> J Neurochem. 75, 882-885 (2000).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li><li>O'Brien, J. A., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biolistic+transfection+of+neuronal+cultures+using+a+hand-held+gene+gun.">Biolistic transfection of neuronal cultures using a hand-held gene gun.</a> Nat Proc. 1, 977-981 (2006).</li><li>Joshi, P., Dunaevsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+gun+transfection+of+hippocampal+neurons.">Gene gun transfection of hippocampal neurons.</a> J Vis Exp. , (2006).</li><li>Biewanga, J. E., Destree, O. H., Scharma, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Neurosci Met. 71, 67-75 (1997).</li></ol>

Çıkar çatışması ilan etti.

Biz sıçan neokorteks (4, 9-11) piramidal nöronlar voltaj bağımlı potasyum kanalları ifade ve işlevsel roller eğitim görüyor. Çünkü bu kanallar için özel farmakolojik ajanların eksikliği, kanal ifade (1,14,15,17-19) işlemek için genetik bir yaklaşım kullanır. Hücre morfolojisi ve korteks laminer düzenini korumak için Stoppini ve ark (16) yaklaşım değiştirilmiş. Organotipik kültür hazırlanması (12,13,15-22; 5-8 2,3) kullanır. Biz 6-17 postnatal gün akut neokortikal dilim izole ve 2-7 gün boyunca kültür dilimleri korumak. Bu bize benzer yaşlı nöronların bu akut dilimleri ile çalışma sağlar ve dilim coşkulu eksitatör bağlantıları gelişimini en aza indirir. Biz kızılötesi aydınlatma (IR) ve akım veya voltaj-klemp tüm hücre yama kelepçe ile diferansiyel girişim kontrast mikroskobu (DIC) kullanarak katmanları II / III veya V görsel olarak belirlenen piramidal nöronlar kaydı. Biyolistik yabani türü veya mutant potasyum kanal DNA (Gen tabancası) transfeksiyon fonksiyonu (1,14,15,17) çalışma kanalları ifade işlemek için kullanılır. GFP için cDNA ile birlikte transfeksiyon, transfekte hücreler Epifloresans mikroskobunda kolayca tespit edilir. Biz transfekte hücreler bitişik, aynı dilim (1,17) aynı katmanda untransfected nöronlar kayıtları karşılaştırın. Yok burada ele prosedürlerinin zor sonuçları büyük ölçüde artırabilir, ancak birkaç ayrıntıya dikkat. Bizim ellerde, hücre canlılığı, kültürleri ~ P8-P10 hayvanlar dilim yapıldığında en iyisidir. Dilim dilimleme ve kültür sırasında yarı steril koşullarda korumak için önemlidir. Bakteriyel kontaminasyonu önlemek için, biz aletler otoklav ve UV ışığı ve EtOH, filtre çözümleri, eldiven, beyin kaldırma ve dilimleri arasındaki değişim eldiven ve cerrahi alanını temiz. Bakteriyel kontaminasyon sonuçları kötü doku canlılığı ve inkübatörler arındırma gerekliliği içerir. Tüm beyin dilimleri ile gibi, canlılığı, hayvanın kurban arasındaki zamanı en aza indirmek ve dilimleme tarafından geliştirilmiştir. Beyin konulmalıdır ve dilimleme Kesme Çözüm buz gibi bir bulamaç batmış beyin ile yapılmalıdır. Bıçak düşük genlik yatay titreşim ile, yavaş hızda dilimleyin. Bir diğer potansiyel sorun inkübatör dilimleri kuruma. Sınırlı ilgi korteks ve striatum (oryantasyon amaçlar için) küçük bir parça: Kurutma küçük dilimleri hazırlanması ile minimize edilebilir. Büyük dilim inkübatör kurumasına eğilimindedir ve büyük dilimleri ile istikrarlı bir kayıt koşullarını elde etmek için daha zordur. Dilimler, 250-300 mikron kalınlığında kesilir. Bulduğunuz ince dilim (250 mikron tercih) daha az kurutma sonucu. Inkübatör kesim çözüm dilim aktarırken, sonra yıkayın Medya Kültür Medya ilk dilim birkaç kez yıkayın ve. Membran (dilim örgü eklemek için izin) transferinden sonra aşırı sıvı kaldırmak için önemlidir. Kurumasını önlemek için kalın dilim (örneğin, 300 mikron), Medya dilim üst tempolu Kültür tek bir damla (membran üzerine oturmuş iken) yardımcı olur. Bu prosedür, dilimin üst yüzeyi nemli bir medya hareketi prime görünür. Her gün Kültür Medya Değiştirilmesi kurumayı önler ve canlılığı teşvik.

Cerrahi / transfeksiyon / kültür: <ol><li> Beyin Dilimleme: Chipping Vibroslice # MA572 Dünya Hassas Aletler, Sarasota, FL, Amerika Birleşik Devletleri </li><li> Gene Silah Sistemi: Bio-Rad, Helios # 165-2431 (Bio-Rad Laboratories, 1000 Alfred Nobel Sürücü, Hercules, CA 94.547) <ul><li> Gen tabancası, regülatör, tüp hazırlık istasyonu (165-2418), şırınga kiti, Tefzel tüp, boru kesici, optimizasyon kiti (165-2424), boru kesici ile helyum hortum montaj dahil </li><li> Bio-Rad Helyum Regülatörü (165-2413) </li><li> tek kullanımlık malzemeler Bio-Rad Helios: <ul><li> 1.6 mikron altın Microcarriers: # 165-2264 </li><li> Tefzel Boru: # 165-2441 </li></ul></li></ul></li><li> Kuluçka: Forma Bilimsel model # 3110 (Termo-Bilimsel: (866) 984 - 3766). </li></ol> Medya: <ol><li> At Serum: Hyclone donör at # SH 30.074. (HyClone, 925 Batı 1800 Güney, Logan, UT 84.321) </li><li> HMEM (Minimal Essential Medya artı HBSS ve Hepes, glutamin: Lonza BioWhittaker Katalog # 12-137F): Gibco / Invitrogen, (800) 955 - 6288, 1. Seçenek. </li><li> HBSS (Gibco Hanks, # 24.020-117 tamponlu tuz): Gibco / Invitrogen, (800) 955 - 6288, Seçenek 1. </li><li> MEM (Gibco asgari temel, orta, 12.360-038), Gibco / Invitrogen, (800) 955 - 6288, Seçenek 1. </li><li> 250 ml Millipore 0.2 mikron filtre: # SC6PU02RE </li><li> Plastik Transfer pipet: Fisher # 13-711-20. </li><li> 50 ml Millipore steriflip 0.22 mikron filtre (# SCGP00525) </li></ol> Elde edilen: Fisher Scientific, 1241 Büyükelçisi Blvd, PO Box 14.989, St Louis, MO 63.132 Öğeler 6-8. Kayıt: <ol><li> Pipetleyin cam: Harvard GC150TF-10: 84 Ekim Hill Road, Holliston, Harvard Apparatus, Massachusetts 01.746 </li><li> Sutter P-87 yatay elektrot çektirmenin: Sutter Instrument Company, Bir Dijital Sürücü, Novato, CA 94.949 </li><li> Axon Instruments Multiclamp 700B amplifikatör: Moleküler Devices, Inc 1311 Orleans Sürücü, Sunnyvale, CA 94.089-1136 </li><li> Moleküler Devices, Inc, 1311 Orleans Drive, Sunnyvale, CA 94.089-1136: 10 veri toplama yazılımı PClamp </li><li> Sutter Instrument Company, Bir Dijital Sürücü, Novato, CA 94.949 lectrode pozisyonu Sutter ROE-200 manipülatörler ve PC-200 denetleyici ile kontrol edilir. </li><li> Mikroskop: IR-DIC optik ile Olympus BX-50WI dik mikroskop </li><li> IR duyarlı kamera OLY-150 (Olympus) veya DAGE MTI (DAGE MTI, 46.360 IN 01 Kuzey Roeske Avenue, Michigan City). </li></ol>

whole cell recording from an organotypic slice preparation of neocortex

Biz sıçan neokorteks piramidal nöronlar gerilim-kapılı potasyum kanalları ifade ve işlevsel roller eğitim görüyor. Çünkü bu kanallar için özel farmakolojik ajanların eksikliği, kanal ifade işlemek için genetik bir yaklaşım almıştır. Biz hücre morfolojisi ve korteks laminer desen korumak için bir Organotipik kültür hazırlama (16) kullanın. Biz genellikle doğum sonrası gün 8-10 akut neokortikal dilim izole ve 3-7 gün boyunca kültür dilimleri korumak. Bu, bize benzer bir yaşta akut dilimleri ile çalışmalarımızı bu nöronların çalışma sağlar ve dilim coşkulu eksitatör bağlantılarının gelişimi en aza indirir. Biz akım veya voltaj-klemp tüm hücre yama kelepçe ile kızılötesi aydınlatma (IR) ve diferansiyel girişim kontrast mikroskobu (DIC) kullanarak katmanları II / III veya V görsel tespit piramidal nöronlar kaydı. Biz onların fonksiyonu çalışma kanalları ifade işlemek için yabani türü veya potasyum kanal DNA mutant Biyolistik (Gen tabancası) transfeksiyon kullanın. Transfekte hücreleri kolayca yeşil flüoresan protein (GFP) için cDNA ile birlikte transfeksiyon sonra Epifloresans mikroskobu ile tanımlanır. Biz transfekte hücreler bitişik, aynı dilim aynı katmanda untransfected nöronlar kayıtları karşılaştırın.

Watch this Scientific Journal Video about Whole Cell Recording from an Organotypic Slice Preparation of Neocortex at JoVE.com

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Bu piramidal nöronların elektriksel kayıtlar yapma amaçlı bir Organotipik kültür neokortikal dilim hazırlama hazırlamak ve sürdürmek için bir protokoldür.

Neokorteks bir Organotipik Dilim Hazırlık Tüm Hücre Kaydı

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

whole-cell-recording-from-an-organotypic-slice-preparation

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.1K Görüntüleme.  University of Tennessee Health Science Center. Bu piramidal nöronların elektriksel kayıtlar yapma amaçlı bir Organotipik kültür neokortikal dilim hazırlama hazırlamak ve sürdürmek için bir protokoldür.

Video: Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Nöronal Sakatlık incelenmesi için Organotipik Hipokampal Dilim Kültür Modeli

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Nörobilim

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

Organotipik Dilim Kültür GFP-Periferik Sinir akıbet Gerçek zamanlı Görüntüleme Fare Embriyolar ifade

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

Dilim hazırlanması, Organotipik Doku ekimi ve suprakiazmatik nükleus Saat Gen Faaliyet Lusiferaz Kayıt

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotipik Hipokampal Dilim Kültürler

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotipik Dilim Kültürleri Purkinje Hücre Dendritik Morfoloji Analizi

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Bir Amfibi Retina Dilim hazırlanması Fotoreseptörler ve İkinci dereceden nöronlar eş zamanlı tam hücreli Kayıtlar

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotipik Dilim Kültürler Oligodendrosit Dinamikler ve miyelinasyonun incelemek için

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Organotipik Hipokampal Dilimleri eşleştirilmiş Tüm Hücre Kayıtlar

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

İşitsel Beyin Sapı Davranım Odyometrisi ve dış saç hücre bütün hücreli yama kelepçe kayıt Doğum sonrası sıçanlarda

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...