Name: culturing and electrophysiology of cells on nrcc patch-clamp chips
Uploaded: 2012-02-07T12:00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips at JoVE.com

Авторы признают, Алексей Богданов для изготовления патч-зажим фишки в CPFC и Hue Чан, Чжао Пин и Мэтью Шиу за помощь в сборке. Naweed Саид был поддержан Канадским институтом исследований в области здравоохранения (CIHR) гранта. Коллин Лук является получателем NSERC и Альберта Heritage Foundation по медицинским исследованиям (AHFMR) studentships.

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Нет конфликта интересов объявлены.

Патч зажим NRCC чип допрос платформа является потенциально мощным инструментом для высокой информативности фармацевтических анализов и исследовать в лабораторных моделей болезни. Его преимущества по сравнению со стеклом пипетки низким сопротивлением доступа, что является преимуществом для исследования крупных клеток, и, несмотря на несколько большую емкость приведет к сопоставимой динамики для небольших клетках. Спонтанное клетки диафрагмы тюленей обычно получается, и все вступление клетки было обнаружено, что спонтанное 14. Четкое различие между чипами и метод стеклянной пипетки является тот факт, что зонд входит в состав блюда культуре клеток и не вручную в контакте с клеточной мембраной. Культивирование клеток, возможно, часть функциональных сетей, приводит к более биологически соответствующих моделей, как болезнь моделей, и другой механизм для обеспечения высокой клетки для исследования уплотнения 16. Однако, в отличие от клеточных суспензий, стремление cannoт быть использованы для размещения клеток на зонд. Улитка нейронов, как и другие крупные клетки, которые поддаются лечению с ручным позиционированием на вершине зонда. Для небольших клеток требуется более длительное время культуре, мы устраняет необходимость для любой манипуляции и сохранить высокую вероятность получения печатью полипептидов структурирование сцепление на вершине зонда поместить клетки в верхней части датчика и продемонстрировал размещения ячеек на Зонд 17,18. NRCC также разрабатывает полиимида микрофлюидных патч зажим чип 19 с емкостью сравнимой с стеклянной пипетки. Конечной целью этого проекта является несколькими датчиками патч зажим чип, который позволяет одновременное наблюдение за электрофизиологических активности нескольких нейронов участвует в сеть поведения при разрешении отдельных ионных каналов 14. Этот метод является высокое разрешение дополнительный метод к многоэлектродной массивы 20.

culturing and electrophysiology of cells on nrcc patch-clamp chips

Благодаря своей изысканной чувствительностью и возможностью контроля и управления отдельными ячейками на уровне ионных каналов, исправления зажима является золотым стандартом электрофизиологии применяется к болезням моделей и фармацевтической экранов, так 1. Метод включает в себя традиционно мягко контакт клетка со стеклянной пипетки заполнен физиологическим раствором, чтобы выделить участок мембраны в соответствии с его вершины 2. Электрод вставляется в пипетку захватывает ион-канал деятельности в участок мембраны или, в случае разрыва, по всей клетке. В последнее десятилетие, патч-зажим чипы были предложены в качестве альтернативы 3, 4: приостановлено фильма отделяет физиологическую среду из питательной среды, а также отверстие микроизготовленном в фильме заменяет вершине пипетки. Патч зажим чипы были интегрированы в автоматизированные системы коммерческого и для высокопроизводительного скрининга 5. Для инкроблегчить пропускной способности, они включают в себя жидкостного доставки клеток от подвески, их позиционирование на диафрагму на всасывании и автоматизированные процедуры для выявления клетки к зонд печатей и вступить в целом режим клетки. Мы сообщили об изготовлении кремниевых патч зажим чип с оптимизированным сопротивлением и формы отверстия, что позволяет высокое качество записи потенциалов действия в культуре нейронов улитки 6, в последнее время, мы также сообщили о прогрессе на допрос млекопитающих нейроны 7. Наш патч зажим чипы изготавливаются на канадских Photonics Изготовление центр 8 коммерческих литейного производства, и доступны в больших серий. Мы готовы участвовать в совместных с электрофизиологов для проверки использования NRCC технологий в различных моделях. Чипы используются в соответствии с общей схемой представлена ​​на рисунке 1: кремниевый чип находится на дне флакона оргстекла культуры и задней части диафрагмы связан с подземными ChannЭль оснащены труб на обоих концах пакета. Клетки культивируют во флаконе и клетки в верхней части датчика контролируется измерительного электрода вставляется в channel.The двумя внешними портами жидкостный обмен облегчить решение с минимальным вмешательством в клетке, это преимущество по сравнению со стеклом пипетки для внутриклеточных перфузии. <img alt="Рисунок 1" src="/files/ftp_upload/3288/3288fig1.jpg" /> Рисунок 1. Принцип измерения с использованием NRCC патч зажим чип Мы подробно протоколы для стерилизации и премьер-чипов, загрузить их со средой, пластинка их с клетками, и, наконец, использовать их для электрофизиологических записей.

Watch this Scientific Journal Video about Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips at JoVE.com

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Мы покажем, как плоская патч-зажим чипов, изготовленных в Национальный исследовательский совет Канады стерилизуют, грунтовка, загружается со средой, покрыты клетками, и используется для электрофизиологических записей.

Культивирование и электрофизиологии ячеек на NRCC Патч зажим фишки

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

culturing-and-electrophysiology-of-cells-on-nrcc-patch-clamp-chips

Research

JoVE Journal

Neuroscience

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch Clamp Записи в внутренних волосковых клеток уха, изолированных от данио рерио

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

Всего зажим патч для сотовых изучения механизмов Инфракрасный нервного возбуждения

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Выделение сенсорных нейронах<em&gt; Aplysia саЩогтса</em&gt; Для патч зажим Recordings глутаматергической Токи

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Патч зажим Recordings от эмбриональных данио рерио Mauthner клеток

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Цельноклеточная Записи патч-зажим от Morphologically- и нейрохимически выявленных гиппокампа Интернейроны

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Субклеточные патч-зажим Recordings от Somatodendritic домена нигральных дофамина нейронами

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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.

Патч зажим Запись Starburst амакриновые клетки в плоскопанельных горе Подготовка деафферентированной 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 ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.