This work is supported by a National Science Foundation award (1145581) to LWG.

NOT: Chicago Illinois Üniversitesi Hayvan Bakım ve Kullanım Komitesi tarafından onaylanan tüm prosedür, Ulusal Sağlık Enstitüleri kurallarına uygun olarak gerçekleştirilmiştir. 1. Çözümleri ve Kültür Medya Hazırlıklar <ol><li> Altı aya kadar -20 ° C&#39;de tüm çözümleri tutun. 3 aya kadar 4 ° C &#39;de kültür ortamı tutun. </li><li> DMEM ile 100 ml ITSX (İnsülin-Transferrin-Selenyum takviyesi-) 1 Pen-strep çözeltisi ve 1 ml kültür ortamı karıştırılarak 100 ml hazırlayın. </li><li> DMEM, 250 mi, 100 mM CaCl2, 2.5 ml ve 50 mM EDTA, 2.5 ml enzim çözeltisi karıştırarak hazırlayın. </li><li> DMEM, 225 ml FBS, 25 ml, 625 mg albumin ve 625 mg tripsin inhibitörü karıştırılarak inaktivasyonu çözeltisi hazırlayın. </li><li> 154 mM NaCl, 5,6 mM KCl, 5,0 mM HEPES, 3.6 mM NaHCO3, 5,6 mM glikoz, bir Locke çözeltisi hazırlayın. NaOH ile 7.3 pH ayarlayın. </li><li>50 mg / ml &#39;lik bir poli-D-lisin (PDL) çözeltisi hazırlayın. Kat lamelleri, 1 mg / ml &#39;lik bir nihai konsantrasyon kullanın. </li><li> 140 mM NaCI, 5 mM KCI, 2 mM CaCI2, 1 mM MgCl2, 10 mM HEPES-NaOH, 10 mM glukoz, hücre dışı bir çözelti hazırlayın. ~ 310 nmol / kg &#39;lık bir ozmolarite NaOH ile 7.3 pH ayarlayın. </li><li> 50 mM NaCI, 100 mM TEACl, 5 mM KCI, 2 mM CaCI2, 1 mM MgCl2, 10 mM HEPES, bir pipet çözeltisi hazırlayın. ~ 290 nmol / kg &#39;lık bir ozmolarite NaOH ile 7.3 pH ayarlayın. </li></ol> 2. Adrenal İzolasyon <ol><li> Büyük ve küçük diseksiyon makas ve forsepsle iki çift bir çift otoklava. Bütün bu işlemler sırasında eldiven giyin ve bir buz kovası diseksiyon tepsiye yerleştirin. </li><li> Gün, 0-3 alındığı fareler kullanır. Dekapitasyon basınçlı CO 2 tankı ile hayvan öldürülür. </li><li> Cervi&#39;de gelen omurga aşağıdaki yavru geri kesmek için makas küçük kümesi yararlanın<html> cal bölgeyi kaudale. Deri altında yüzeysel kesilir ve vücut boşluğuna delmek değil emin olun. </li><li> Sonraki uzak omurga soyma cilt bu kaslar omurga net bir görünümü var maruz böylece. Buradan, servikal spinal kord bir transectional kesim yapmak ve daha sonra omurga kenarları boyunca iki paralel kesim yapmak. </li><li> Hayvanın ana gövdesini tutan forseps bir çift, omuriliğe kapmak ve böbrekler maruz dek omurga geri soymak için forseps diğer çifti kullanın. </li><li> Bu noktada, böbreklerin üstüne böbreküstü bezleri görselleştirmek. Daha sonra dikkatli bir Locke çözeltisi ile doldurulmuş bir konik tüp içine her hayvandan iki bezi yerleştirin. NOT: Bazen bezleri forseps sadık olacaktır. Bu durumda, yavaşça Forseps ve Locke çözelti içine bezlerinin bertaraf edilmesine yardımcı olmak için başka bir çift forseps kullanır. Bezleri 2 saat kadar bu durumda muhafaza edilebilir. </li></ol>e &quot;&gt; 3. Doku sindirimi <ol><li> Bu arada, kabarcık, 15 dakika boyunca% 5 CO2 +% 95 O2 ile enzim solüsyonu kullanımdan önce. Dengelenmiş çözüm pembemsi / turuncu renk içine bir parlak pembe renk döner emin olun. </li><li> 20-25 ünite / ml &#39;lik bir nihai konsantrasyonda taze olarak kabarcıklar halinde enzim çözeltisine papain ekleyin. , Papain ile enzim çözeltisi içerisinde 37 ° C&#39;de 40-60 dakika boyunca, her bir hayvandan bezlerinin her çift inkübe edin. </li><li> Her tüpe inaktivasyon çözeltisi% 75 ilave edildikten sonra 10 dakika boyunca 37 ° C&#39;de inkübe edin. Bu inkübasyon papain enzim aktivitesini eylemsiz hale getirir. </li><li> Inaktivasyonu çözeltisi ile 10 dakika süreyle inkübe edildikten sonra, yavaşça yeni etiketli tüpe aktarın ve bezleri bezleri kültür ortamı ile 3 kez yıkayın. </li></ol> 4. Hücre Ayrılma ve Kaplama <ol><li> Yıkama işleminden sonra, kültür ortamı içine 200 ul iki bezi yerleştirin ve sonra yavaşça bezleri ve titreaşağı 200 ul pipet pipet ucu ile artık çözelti içinde dokusunun büyük bir parçası kalmayıncaya kadar. Not: titre zaman, 200 ul pipet ucu aşağıya doğru bir kuvvet ile tüpün alt bezleri ve muhafaza ve yavaşça doku girerler. , Yavaş, sürekli darbeleri kullanmak için belirli olun asla hızlı veya ani pipetlerken. </li><li> 450 ul kadar kültür ortamı toplam hacmi getirmek için ek bir 250 ul kültür ortamı ekleyin. </li><li> Plaka, bir 60 x 15 mm kültür tabağına yerleştirilir yedi ila 12 mm PDL önceden kaplanmış lamelleri, her biri üzerine, bu, hücre-ihtiva eden çözelti, 60 ul. </li><li> Bir kez kaplama, hücre canlılığı için bir ortam sağlamak için 30 dakika boyunca% 5 CO 2 sürekli bir akış / arz ile 37 ° C ayarlanır ve muhafaza kuvöz içine koyunuz. İnkübasyondan sonra, yavaşça kültür kabı içine önceden ısıtılmış kültür ortamı 5 ml ilave edilir ve 24 saat süreyle tekrar inkübatör içine yemeğin geri hücre ataşe öncesinded kayıtları. Not: Hücreler tipik olarak, en fazla 4 gün boyunca elektrofizyolojik kayıtları için kullanılabilir. </li></ol> 5. Hücre Tanımlama ve gigaohm Seal Oluşumu <ol><li> Hücre dışı çözelti içine 12 mm&#39;lik bir örtücü cam yerleştirin. Bütün böbreküstü bezleri hücre hazırlanması için kullanılan bu yana, kortikal hücreleri (küçük ve) hücreleri kromafin daha sönük kültür içinde kromaffin hücreleri (kahverengimsi / bej rengi ve mükemmele yakın bir dairesel formlu, genellikle çok parlak (Şekil 1) karıştırın. </li><li> Programlanabilir P-97 çektirmenin kullanarak dört aşamada yama pipetler çekin. Daha sonra cam kapasitenin azaltılması ve yardımcı olacak bir erimiş mum içine pipet ucu batırın pipet içerisinden ucu &quot;darbe-away&quot; lehçe bu balmumu ateş. Not: Yama pipet çözeltisi ile doldurulan, yama pipetler genellikle yaklaşık 2 M? Bir dirence sahiptir. </li><li> Birkaç metre içinde hücreye yakın yama pipet Yaklaşımicrons. Hücreye bağlı bir konfigürasyon elde etmek için, hücreye yakın yama pipet yaklaşması sırasında herhangi bir hafif hücre deformasyon için bak. Bir GΩ mühür elde edilinceye kadar nazik vakum uygulayın. </li></ol> 6. Hücre-Ekli Kapasite Kayıtlar <ol><li> Bir mühür ortaya GΩ sonra, EPC-7 artı yama-kenet büyütücü kullanımı ile hücre bağlı kayıt yapmak ve iki fazlı bir analog kilidi amplifikatör (ekipman ekli tabloya bakınız). Kilit-amplifikatör 50 mV (rms) genlik ve 20 kHz frekans ile bir sinüs dalgası uygulayın. </li><li> 1 msn zaman sabiti ve 24 db de kilit-amplifikatör çıkış filtre ayarlayın. Kilit-amplifikatör gibi nazik emme bakliyat tarafından üretilen geçici kapasite değişiklikleri sadece yama iletkenlik (Re) iz içine hiçbir projeksiyon ile yama kapasitans (Im) iz görünür ki (Şekil 2) fazını ayarlayın. NOT: bir yeniden sistemin kalibrasyonu için ayrıntıları bakın19 Ference. </li><li> &quot;Aşağıya doğru&quot; adımlar (Şekil 3) ile tipik kapasite değişiklikleri belirlemek. NOT: Bir katı kayıt Tek veziküllerini içselleştirerek gösterir birçok aşağıya doğru adımlarla bir &quot;merdiven&quot; tipi benzerliği olarak görülecektir. Yama işlemi eylem akım tipik olarak en hücrelerinden kaydedilmiş olacağından mekanik uyarım olarak hizmet vermektedir. </li><li> Bu dosya içinde aa / gg / yıl ve kendi bireysel numaralandırma (xx) gibi her hücre bağlı kayıt: Bireysel tarih dahil klasörler gibi dosyaları isim. </li></ol>

<ol>
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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Hücre bağlı kapasite ölçümleri başarılı bir şekilde yüksek kalitede ses kayıtları elde etmek için bazı kritik adımları gerektirir: böbreküstü bezlerinden hazırlanmış 1) canlı ve sağlıklı hücreleri; Lamelleri 2) PDL kaplama; 3) gigaohm yalıtım oluşturma; 4) sistem gürültü seviyesi; ve 5) faz düzeltme. Hayvan ameliyatı için bir potansiyel yapabilirsiniz değişiklikler en uygun dirayeti cerrahi yaklaşımı ayarlamak ve diseksiyon sırasında zarar görmesi...

Sinaptik nörotransmiter içeren sinaptik veziküllerin ekzositozu aracılık eder ve bu veziküller uzun vadede nöronal iletişim sağlamak için sinir terminal içinde yerel endositik geri dönüşüm geçmesi gerekmektedir. Sinaptik vezikül döngüsü, bir bütün olarak, hücresel haberleşme daha iyi bir kavramaya yönelik gerekli bir temel teşkil eden moleküler makine anlaşılması, sinaptik iletim temel rolü göz önüne alındığında. Hücre modeli sistemleri arasında, adrenal kromafin hücre sinaptik vezikül geri dönüşüm altında yatan moleküler makineler içine en kesin içgörü bazı sağlamıştır. Ekzositoz, nörotransmitter açıklamasında son adım, son derece okudu ve adrenal chromaffin hücrenin 1,2 kullanımı yoluyla incelenmiştir. Aslında, salgı granüllerin oluşumuna, hedefleme, yerleştirme ve füzyon orkestra moleküler oyuncu büyük çapta div uygulanmasından tespit edilmiştirkromafin hücreleri 1 erse teknikleri. Ayrıca, Ekzositoz katılan protein makine tek-vezikül çözümü için izin için bir fırsat sağlayarak, kromaffin hücre vezikül füzyonu 3 soruları yanıtlamak için güçlü bir model kalır. Hücre bağlı kapasitans ölçümleri ilk Ekzositoz 3 sırasında tek vezikül füzyon çözümünde kullanılmıştır. ~ Çapı 60 mil gösterilmiştir kadar küçük veziküller Ekzositoz hücre konfigürasyonda bağlı olarak 4-7 yama kelepçe tekniğiyle birlikte hücre zarı admitans ölçümleri ile tespit edilebilir. Admittans bir devre ya da cihaz, bir akımın akmasına izin verecek kadar kolay bir ölçüsü olarak tanımlanır; Bu empedansının tersidir. Böylece, giriş ölçümler membran kapasitans anlaşılmasını sağlamak. Bu, plazma membranı içine veziküler membranın eklenmesi ile gerçekleştirilir; Bu birleşme yüzeyinde değişiklikler ortayaalan 8. Her bir füzyon vezikül membran kapasitans 9,10 kademeli bir artışa neden olur. Ayrıca, bu geçiri ölçüm membran iletkenliğini ve eksositotik olay 3 sırasında füzyon gözenek iletkenlik sağlar. Bu tekniğin Ekzositoz sırasında tek vezikül kinetiği belirlenmesi eşsiz bir araç sağladı gibi, bizim laboratuvar son zamanlarda tek kesecikler 11,12 endositozu algılamak için bu kavramı başvurdu. Bizim özel ilgi birçok hücreleri 13 ve nöronal terminalleri 14,15 sinaptik vezikül endositoz için bir ana yol olarak temel bir temizlik bileşeni olarak kabul edilmiştir klatrin aracılı endositoz (CME), olduğunu. CME Ancak kinetiği nedeniyle tekil endositik olayların izlenmesinde teknik sınırlamalara tam olarak anlaşılamamıştır, biyolojik olarak önemli olduğu bilinmektedir. Kromaffin hücreleri ve nöronlar arasındaki 1 ekzositik mekanizmalarında benzerlikler dikkate alındığında, plchromaffin hücreleri fisyon mekanizmalar büyük olasılıkla nöronlarda sinaptik vezikül endositoz için geçerli olabilir ausible. Hücre bağlı kapasite ölçümleri bireysel endositik olayları izlemek ve en yöntemleri çözemiyorsanız fisyon kinetik analiz için kullanılmıştır. Bizim hücre bağlı kayıtlarında, 20 kHz&#39;de bir sinüs dalgası tutma potansiyelinden üzerine biner, ve çıkış akımı yükseltici kilitli iki fazdan, başka bir kanalda bir kanal ve membran kapasitans membran iletkenliği ayrılır 16- 18. Membran iletimini ve kapasitanstaki değişimlere kaynaktan, tek bir olasılıkla kabarcık kıstırma önce plazma zarına içe vezikül bağlayan boru şeklindeki zar boynuna karşılık fisyon-gözenekli, kinetiğini hesaplayabilir. Topluca, bu teknik bize CME sırasında vezikül fisyon düzenleyici mekanizmaları incelemek için fırsat verir.

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			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
			<td rowspan="2" style="width:88px;">HEKA</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

Hücre canlılığı ve gigaohm sızdırmazlık kalitesinin hücre bağlı kapasitans kayıtlarının kalitesini belirlemek için önemlidir. Bu nedenle, önceki elektrofizyolojik kayıtları ve tipik yaşayabilir hücrelerin, Şekil 1 &#39;de gösterilmiştir etkili ve verimli bir hücre kültürünü temin etmek önemlidir. Teori ve zaman yüksek kaliteli bir yalıtım elde edilir gigaohm yararlı olacaktır. Bir açık protokol Aşama 5.3 &#39;te tarif edildiği gibi Patch-Pipeti hücre yaklaşmaktad...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Fare Adrenal Kromafin Cell Hücre bağlı Kapasitans Ölçümler Yöntemleri

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.7K Görüntüleme.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Video: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Fare önbeyin içine Ultrason Rehberliğinde Mikroenjeksiyon In Utero E9.5 az

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Nörobilim

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Çoklu-fare nöroanatomik Manyetik Rezonans Görüntüleme

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Gelişmekte olan Fare Retina in vivo Elektroporasyon olarak

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-kelepçe Kapasitans Ölçümler ve Ca 2 + Retina Dilimleri Tek Sinir Terminalleri Görüntüleme

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Bir Fare Modelinde İntravenöz Öz Yönetim Yöntemleri

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

NF-KB-bağımlı yetişkin nöron Modülasyon ve Analiz Yöntemleri

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Fare Siyatik Sinir Vivo elektrofizyolojik ölçümlerde

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Gelişmekte Fare Embriyonik Cortex mitoz Canlı Görüntüleme

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Protein Analizi için Fare Retinasındaki Fotoreseptör Hücre Bölmelerinin İzolasyonu İçin İki Peeling Yöntemi

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Drosophila Fotoreseptörlerinde Fotopigment Düzeylerini Ölçmek için Elektrofizyolojik Yöntemler

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...