Name: recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques
Uploaded: 2015-02-11T14:00:00
Description: Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.

О себе Этика: Процедуры, связанные с животными предметы были одобрены уходу и использованию комитета за животными (IACUC) в Wayne State University в. 1. Подготовка экспериментального решения <ol><li> Подготовка рассекает раствор 1 день 1 неделю до фактического эксперимента. Используйте ?...

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Intracellular recording.</a> J Neurophysiol. 32 (3), 339-355 (1969).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annu Rev Physiol. 46, 455-472 (1984).</li><li>Lukasiewicz, P., Werblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+slowly+inactivating+potassium+current+truncates+spike+activity+in+ganglion+cells+of+the+tiger+salamander+retina.">A slowly inactivating potassium current truncates spike activity in ganglion cells of the tiger salamander retina.</a> J Neurosci. 8 (12), 4470-4481 (1988).</li><li>Kaneko, A., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+L-glutamate+on+the+anomalous+rectifier+potassium+current+in+horizontal+cells+of+Carassius+auratus+retina.">Effects of L-glutamate on the anomalous rectifier potassium current in horizontal cells of Carassius auratus retina.</a> J Physiol. 358, 169-182 (1985).</li><li>Kamermans, M., Werblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA-mediated+positive+autofeedback+loop+controls+horizontal+cell+kinetics+in+tiger+salamander+retina.">GABA-mediated positive autofeedback loop controls horizontal cell kinetics in tiger salamander retina.</a> J Neurosci. 12 (7), 2451-2463 (1992).</li><li>Cook, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+retinal+dystrophy+in+the+rat.">Inherited retinal dystrophy in the rat.</a> J Cell Biol. 14, 73-109 (1962).</li><li>Eggers, E. D., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(A),+GABA(C)+and+glycine+receptor-mediated+inhibition+differentially+affects+light-evoked+signalling+from+mouse+retinal+rod+bipolar+cells.">GABA(A), GABA(C) and glycine receptor-mediated inhibition differentially affects light-evoked signalling from mouse retinal rod bipolar cells.</a> J Physiol. 572 (Pt 1), 215-225 (2006).</li><li>Ichinose, T., Lukasiewicz, P. 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Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings. 
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the ...

The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms. 
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6. 
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mice (28-60 days old, male)</td>
			<td style="width:145px;">Jackson laboratory</td>
			<td style="width:203px;">C57BL/6J strain</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; medium powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:203px;">A1420</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:145px;">Nikon</td>
			<td style="width:203px;">SMZ745</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:203px;">#4, #5, #55</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_scissors</td>
			<td style="width:145px;">Roboz</td>
			<td style="width:203px;">RS-5605</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_surgery knife</td>
			<td style="width:145px;">Surgistar</td>
			<td style="width:203px;">7514</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">razor blade (for chopper)</td>
			<td style="width:145px;">EMS</td>
			<td style="width:203px;">71970</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chopper</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">infrared viewer</td>
			<td style="width:145px;">Night Owl Optics</td>
			<td style="width:203px;">NOBG1</td>
			<td>It shows bright view.&#160; Focusing small objects is an issue.</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">infrared pocket scopes</td>
			<td style="width:145px;">B.E. Meyers</td>
			<td style="width:203px;">OWL Gen 3 NV pocketscope</td>
			<td>excellent view</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puller</td>
			<td style="width:145px;">Sutter</td>
			<td style="width:203px;">P-1000</td>
			<td>excellent.&#160; Make consistent size pipettes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dark box</td>
			<td style="width:145px;">Pelican</td>
			<td style="width:203px;">dark box</td>
			<td>excellent</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">patch clamp system</td>
			<td style="width:145px;">Scientifica</td>
			<td style="width:203px;">slice scope 2000</td>
			<td style="width:323px;">Excellent setup.&#160; Most key components are included in one package.&#160; Micromanipulators are excellent.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amplifier</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">multiclamp 700B</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acquiring software</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">pClamp software</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:216px;">light source (LED)</td>
			<td style="width:145px;">Cool LED</td>
			<td style="width:203px;">pE-2 4 channel system</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD camera</td>
			<td style="width:145px;">Q-imaging</td>
			<td style="width:203px;">Retiga 2000</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques

Сетчатка является воротами в зрительной системе. Чтобы понять, визуальные механизмы обработки сигнала, мы исследуем функции сетчатки нейронных сетей. Нейронов сетчатки в сети включают в себя многочисленные подтипы. Более 10 подтипов биполярных клеток, ганглиозных клеток и амакринных клеток были определены морфологических исследований. Несколько подтипов нейронов сетчатки, как полагают, кодируют различные особенности визуального сигналов, таких, как движение и цвет, и форма несколько нервных путей. Тем не менее, функциональные роли каждого нейрона в визуальной обработки сигнала, полностью не поняты. Метод локальной фиксации полезно для решения этой фундаментальный вопрос. Здесь протокол к записи легкие, вызвали синаптические реакции в мышиных нейронов сетчатки с использованием локальной фиксации записи в темно-адаптированы условиях предоставлена. Глаза мыши темно-адаптированы O / N, и сетчатки препараты ломтик расчленены в темной комнате с помощью инфракрасного освещения и зрителей. Инфракрасный свет неактивировать фоторецепторы мыши и, таким образом, сохраняет свой свет реагировать. Патч зажим используется для записи световых вызвали ответы в нейроны сетчатки. Флуоресцентный краситель вводится и в процессе записи, чтобы характеризующих нейронов морфологические подтипы. Эта процедура позволяет определить физиологические функции каждого нейрона в сетчатке мыши.

Представитель препарат срез не показан на рисунке 1. Препарат срез в прямом положении, показывающий фоторецепторов ганглиозных клеток в плоскую поверхность, а не отделение от фильтровальной бумаги. Если срез наклонены, только частью подготовки находится в фокусе, что делает е...

Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Запись Light-вызывала постсинаптические реакции нейронов темно-адаптированных, мышь сетчатки ломтик препаратов с использованием методов локальной фиксации

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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