Name: patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices
Uploaded: 2012-01-19T13:00:00
Description: Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

Мы благодарим д-р Фред Rieke за его любезное объяснение агар встраиваемый сетчатки подготовки срез, когда мы начали использовать протокол в нашей лаборатории. Мы также благодарим Лори Vaskalis для иллюстрации схематический обзор и д-ра. Veeramuthu Балакришнан и Soyoun Чо за полезные замечания по тексту и видео. Эта работа была поддержана NEI-NIH RO1 гранта, а также был частично поддержан Корейский исследовательский грант Фонда финансируемый правительством Кореи [KRF-2008-357-E00032].

1. Внешние и внутренние решения <ol><li> Подготовка нарезки решение (низкое содержание кальция) с 10-кратным маточного раствора и добавить MgCl 2, CaCl 2 и D-глюкозы ежедневно. Окончательный 1x решение состоит из (в мм): 119 NaCl, 2,5 KCl, 3,2 MgCl 2, 0,25 CaCl 2, 12 D-глюкозы, 0,2 L-аскорбиновой кис?...

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Критический и трудный шаг в нашем протокол передачи части сетчатки в агар решение (протокол 3.4). Необходимо тщательно удалить стекловидного тела и остаточной решение ломтик от сетчатки части и передать ее без искажений или изгиба. Для того, чтобы достичь этого, мы используем не...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Низкая температура гелеобразования-агар </td><td> Сигма </td><td> A0701 </td><td> Агарозном типа VII- </td></tr><tr><td> Патч пипетки </td><td> Инструменты Всемирной Precision </td><td> 1B150F-4 </td><td> Толстостенные (1,5 мм наружный диаметр) боросиликатного стекла </td></tr><tr><td> Вертикальная съемник </td><td> Narishige </td><td> PP830 </td><td></td></tr><tr><td> Стоматологический воск </td><td> Cavex </td><td></td><td></td></tr><tr><td> Весна ножницы </td><td> Инструменты изобразительных наук </td><td> 15003-08 </td><td></td></tr><tr><td> 45 ° угловой наконечник тонкой щипцы </td><td> Инструменты изобразительных наук </td><td> 11251-35 </td><td></td></tr><tr><td> Бритва </td><td> Personna </td><td></td><td> Обоюдоострый, очищено 70% этанолом и H 2 O </td></tr><tr><td> Цилиндрические трубки </td><td> Fisherbrand </td><td> 03-338-1B </td><td> Полиэтилен образец флаконах 2,5 мл </td></tr><tr><td> Гиалуронидаза </td><td> Сигма </td><td> H6254 </td><td></td></tr><tr><td> Vibratome слайсер </td><td> Leica </td><td> VT1000S или VT1200S </td><td></td></tr><tr><td> Вертикальный микроскоп </td><td> Олимп </td><td> BX51WI </td><td></td></tr><tr><td> 60x воде погружения цель </td><td> Олимп </td><td> LUMPlanFl </td><td> Н. А. 0,90 </td></tr><tr><td> ПЗС-камера </td><td> Sony </td><td> XC-75 </td><td></td></tr><tr><td> Камера контроллер </td><td> Hamamatsu </td><td> C2400 </td><td></td></tr><tr><td> Монитор &lt;/ TD&gt; <td> Sony </td><td></td><td> 13 &quot;черно-белый монитор </td></tr><tr><td> Шприц фильтр </td><td> Nalgene </td><td></td><td> 0,2 мкм </td></tr><tr><td> Микроманипулятор </td><td> Саттер инструмент </td><td> MPC-200 </td><td></td></tr><tr><td> Блокировка усилителем </td><td> Хека </td><td></td><td> EPC-9/10 усилители имеют программную эмуляцию </td></tr><tr><td> Вращающийся диск лазерной конфокальной микроскопии </td><td> Yokogawa </td><td> CSU-X1 </td><td> Живая клетка изображений после патча зажим целом записи ячейки </td></tr><tr><td> Slidebook программного обеспечения </td><td> Интеллектуальные изображений Инструменты (3i) </td><td></td><td> Изображениями получения и анализа данных </td></tr><tr><td> Параформальдегид </td><td> Сигма </td><td> P6148 </td><td></td></tr><tr><td> Фосфатный буфер решение </td><td> Г. И.BCO </td><td> 70013 </td><td></td></tr><tr><td> SuperFrost слайдов </td><td> Fisher Scientific </td><td></td><td> Слайд стекла </td></tr><tr><td> Анти-выцветания агентов </td><td> Biomeda корп. </td><td></td><td></td></tr><tr><td> Конфокальной лазерной сканирующей микроскопии </td><td> Carl Zeiss </td><td> LSM 710 </td><td> Изображениями основных тканей </td></tr><tr><td> Шпатель </td><td> Fisherbrand </td><td> 21-401-25B </td><td></td></tr><tr><td> Мануэль вертикальной резки </td><td> Narishige </td><td> ST-20 </td><td></td></tr><tr><td> Орегон Зеленый BAPTA 488-1 </td><td> Invitrogen </td><td> O-6806 </td><td> Са 2 + чувствительны флуоресцентного красителя </td></tr><tr><td> Alexa Fluor 555 гидразида </td><td> Invitrogen </td><td> -20501MP </td><td> Флуоресцентная краска </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

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

Здесь мы опишем протокол для подготовки агара встраиваемый сетчатки ломтиками, которые подходят для электрофизиологии и Ca 2 + изображения. Этот метод позволяет исследовать ленточного типа синапсов в сетчатке микросхем с использованием прямых патч-зажим записи одного пресинаптических нервных окончаний.

Патч-зажим измерения емкости и Са 2 + Изображений на Одноместный нервных окончаний в сетчатке глаза Ломтики

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

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Neuroscience

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

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

Патч зажим Recordings от мыши нейронов сетчатки в темно-адаптированной фрагмент подготовка

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

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Парные Patch Clamp Recordings от Мотор-нейрон и целевые скелетных мышц в данио рерио

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

A Computer-assisted Multi-electrode Patch-clamp System

Компьютерный многоэлектродной патч-зажим система

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Визуализация эндосоме Dynamics в живых нервных окончаний с Четырехмерное флуоресценции изображений

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

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

Методы клеточной прилагается измерения емкости в мышь надпочечников хромаффинных Cell

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

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Электрофизиологических и морфологических характеристик нейронов микросхем при остром мозга срезов при использовании парных патч-зажим Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

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

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. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Са изображений<sup&gt; 2+</sup&gt; Динамика колбочек аксонов мыши Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole-cell Patch-clamp Recordings in Brain Slices

Цельноклеточной патч-зажим Записи в головном мозге Ломтики

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Применение автоматизированного патч-зажима для изображений для изучения нейронов в средах мозга

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...