这项工作是支持由美国国立卫生研究院资助EY17606（APS）。

1。准备电极<ol><li>准备记录和参比电极浸泡在1M氯化钠，并推动它们之间的15V〜20秒1 Hz的正弦波</li><li>请使用微量拉马（萨特仪器的P - 97）的记录电极。对于补丁移液器使用硼硅玻璃1.2毫米外径和内径0.69毫米（萨特仪器，CAT＃B120 - 69 - 10）的一个。通过电极尖端测量串联电阻。根据靶细胞的大小选择枪头直径;胞体〜使用直径在20微米〜5MΩ胞体〜在使用直径5微米〜12MΩ。 </li><li>准备地面/参比电极。用注射器填充1毫米氯化钠一小部分聚乙烯管（Intramedic PE205），其中包括3.5％的琼脂（Sigma公司; CAT＃A7002）在1M氯化钠。这超过了Ag / AgCl参比电极放置琼脂桥。 </li></ol> 2。准备解决方案<ol><li>外部液，含有碳酸氢盐（1升）的艾姆斯媒体:1艾姆斯瓶"中等（Sigma公司; CAT＃A1420），1.9克碳酸氢钠 3，7毫升的青霉素，链霉素（Sigma公司; CAT＃P0781），以防止细菌增长。调整渗透压〜280 mOsm。 </li><li>切片的解决方案，艾姆斯介质pH缓冲HEPES（1升）:1瓶Ames的培养基，0.887克的氯化钠，2.38克HEPES，7毫升的青霉素，链霉素。使用1M NaOH和/或1M盐酸调节pH值至7.4〜。调整渗透压〜280 mOsm。 </li><li> 10 -天门冬氨酸钾（K - ASP）移液器内部的解决方案（毫米）:125的K -天门冬氨酸，氯化钾，10 HEPES，5 NMG - HEDTA氯化钙2，1的ATP GTP镁毫克，0.2，0.5， pH值调整到〜7.3 NMG - OH和渗透压调整〜280 mOsm。 </li><li>稳定低胶凝温度琼脂糖包含视网膜（按重量的5％），琼脂7.5克（Sigma公司;猫＃A7002）逆止琼脂融化在150毫升DH 2 O </li></ol> 3。实验日<ol><li>解冻〜1.5毫升的K - ASP内部解决方案，您所选择的荧光团从冰箱。稀释至适当浓度，应凭经验确定的荧光。 </li><li>倒艾姆斯含有碳酸氢钠3（约200毫升）的避光贮存容器，然后将水浴（30-32℃）的地方"媒体和平衡5％的CO 2 / 95％O 2气体</li><li>填充〜110毫升含有HEPES，放置在冰艾姆斯媒体的瓶子和100％O 2的平衡。 </li><li>广场余下的Ames的媒体，包含一个法拉第笼顶部的加热水库碳酸氢钠3，和5％的CO 2 / 95％O 2气体的平衡。封口膜可用于捕获的气体在上述解决方案的空间。 </li><li>准备加入0.75克的低胶凝温度琼脂糖（3％）; HEPES和热量的解决方案〜115 ° C至融化琼脂糖（Sigma公司猫＃A0701）25毫升艾姆斯媒体。搅拌，直至溶液清晰​​，无气泡，并在存储水浴（42℃）。 </li><li>填充的K - ASP的内部解决方案的一个记录的吸液管和检查尖阻力。 </li></ol> 4。开始实验<ol><li>安乐死鼠标，迅速取出眼用弯剪刀。应被用于所有的程序，以防止视觉色素漂白红外护目镜。 </li><li>放在滤纸上，以防止他们从滚动，使用超薄双面刀片眼睛，使角膜缝钳。 </li><li>尽快刺破眼球，转移到与Ames的媒体充满了菜，他们的眼睛。 </li><li>使用角膜为切入点的缝隙，小剪刀剪去其余部分角膜，晶状体和玻璃体后可以去掉。小心视网膜仍然附着眼罩，并存储在黑暗眼罩在32 °艾姆斯"媒体与5％的CO 2 / 95％O 2的平衡彗星。 </li></ol> 5。切片<ol><li> Hemisect一个眼罩，和视网膜脱离的视网膜色素上皮。取出的视网膜边缘，让组织平躺。 </li><li>使用乳胶泡一个小玻璃巴斯德吸管，以填补35毫米的Petri盘与融化的琼脂，并通过琼脂拖过的镊子，以检验其一致性。 </li><li>当你可以开始看到琼脂凝固: <ol class="lalpha"><li>视网膜传送至琼脂顶部。 </li><li>使用一个Kimwipe扭曲，以吸收周围的视网膜多余的溶液。 </li><li>广场2-3滴琼脂直接在视网膜上，并迅速放在一个塑料圆筒视网膜形成了它周围的墙壁。然后往下淌的塑料圆筒两侧额外融化的琼脂，而围绕琼脂内的视网膜。 </li><li>转让的Petri菜吨Ø冰水浴冷却。 </li></ol></li><li> 〜45秒后从冰水浴中删除的组织和使用柱塞挤出琼脂块的塑料圆筒，从视网膜最远的一侧推。 </li><li>使用一个双面刀片切出一个小数据块包含视网膜琼脂和到位，对试样的光盘上的琼脂逆止强力胶块。 </li><li>标本光盘传输振动切片机托盘，倒入托盘允许与视网膜块完全淹没冰冷艾姆斯HEPES。 </li><li>视网膜片〜200微米的厚度，可以切割​​最大的刀片振动速度和向前的刀片速度设定等，它需要4至5秒，穿过视网膜上的每个切片。 </li><li>使用11号手术刀刀片一次删除每个可用的片之一</li><li>含有视网膜的好片放入录制商会和加权对着干的尼龙线片锚。尼龙线按住琼脂周围的视网膜，使视网膜录音通畅。 </li><li>录音室转移到直立显微镜阶段，关闭窗帘，打开红外光源，红外感光摄像头，以查看片。 </li></ol> 6。记录<ol><li>一旦一个良好的视网膜片（完整的感光细胞和适当的切削角度）确定开始灌注视网膜率大于5 mL / min的加热艾姆斯媒体被加热到35 37 ° C和5％的CO 2平衡/ 95％O 2。 </li><li>一些表面的损害可能是由于切割过程明显。这通常是一层薄薄的细胞，可以使用一个破碎的一角吸管去除，并轻轻的吸吮了死细胞机构。这通常会发现下面的活细胞。确定一个健康的细胞体，其位置位于利益阶层。 </li><li>填写与KAsp内部解决方案的一个微量，并申请通过吸管尖正面（向外）的压力，降低进录音室吸管尖，使用显微细胞水平。 </li><li>移动枪头，这样它的酒窝细胞膜，并适用于温柔负（向内）的压力，使高抗密封。全细胞模式，可以实现应用负压电极进入细胞打破。 </li><li>刺激视网膜组织使用的LED就可以唤起反应轻。 </li><li>照片，录音唤起从吸管内部解决的荧光团的荧光的细胞。 </li></ol> 7。代表性的成果<img src="/files/ftp_upload/2107/2107fig1.jpg" alt="图1" /> 图1在这里，一个暗适应视网膜神经元增加强度的光闪烁光诱发的潜力。 <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="图2" /> 图2:从视网膜切片的细胞已被填充的荧光，路西法黄河荧光图像。诱发荧光显示的细胞补丁录音形态。

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, 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=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

片脊椎动物视网膜的录音已经几十年 1,2，已经相当成功，提供了一个如何视网膜电路编码入射光更深入的了解。振动切片，而不是直接用刀片切割的优点是视网膜切片，在表面产生较少的损害。具有吸吸管，这是容易消除这种损害健康细胞和目标，只是在表面之下，他们的连接保留在视网膜的电路，其中大部分的细胞类型已经确定了5,6，轻，显示强劲的反应。因此，从暗适应视?...

<table><tbody><tr><td>Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Borosilicate glass</td><td>Sutter Instrument Co.</td><td>B120-69-10</td><td></td></tr><tr><td>Polyethylene tubing</td><td>Intramedic</td><td>PE205</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A7002</td><td></td></tr><tr><td>Low gelling temp agarose</td><td>Sigma-Aldrich</td><td>A0701</td><td></td></tr><tr><td>Ames&amp;rsquo; Medium</td><td>Sigma-Aldrich</td><td>A1420</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr></tbody></table>

patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

启动时，在我们的视网膜感光细胞的视色素吸收光能量的光子，并启动一个封闭的环核苷酸门控通道在细胞膜，导致细胞内事件级联我们的视觉体验。膜电位的变化，导致神经递质的释放杆和锥突触终端数量的减少反过来。为了测量在感光膜电位光诱发的变化如何导致在视网膜上下游活动，科学家们从几十年 1,2的视网膜切片准备的电生理记录。在过去，这些片被切断与视网膜组织，是连接到滤纸上的刀片手动，近年来已开发的另一种方法的切片，使视网膜组织中嵌入胶凝温度琼脂低和在阴凉的解决方案与切片振动切片3,4。这种准备产生表面损伤少和非常强大的光诱发反应的视网膜切片。在这里，我们的文件，此过程中如何可以根据红外光，以避免视觉色素漂白。

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

在这里，我们描述了产生暗适应对小鼠视网膜电生理记录片的过程。

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

Research

JoVE Journal

Neuroscience

14.4K 次观看.  University of Southern California. 在这里，我们描述了产生暗适应对小鼠视网膜电生理记录片的过程。

视频: Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

科研

神经科学

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

生物学

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.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

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

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.

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

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

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

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

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

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.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

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

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

研究小鼠分裂视网膜中的双极和水平神经元细胞

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

作者聚焦：使用 Split Retina 技术增强访问和加速实验 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

摘要

探索更多视频

此视频中的章节