Many patch clamp amplifiers do not use true voltage clamp circuitry, but instead are differential amplifiers that use the bath electrode to set the zero current ground level. By providing access to a cells intracellular environment, the wholecell patch clamp technique allows one to record the. In this chapter we describe a robust and practical protocol for preparing brain slices from mature adult mice that are suitable for patch clamp analysis. Cellular mechanisms of brain function 10,787 views 9. Wholecell patchclamp recordings provide exceptional access to spiking and synaptic neural activity.
Since the standard brain slice setup employs a water immersion objective it was not possible to place the magnetic coil above the brain slice. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Attach the pipettes to the electrode holders of the patchclamp amplifier headstages and turn into position. Patchclamp technique in brain slices springer nature experiments. A detailed stepbystep description of the standard patch clamp protocol and labome survey results for vibratomes and patchclamp amplifiers. A robust ex vivo experimental platform for molecular. Using fine control micromanipulators, descend the recording pipettes to the region of interest within the brain slice. This book is the most current single how to manual and text on patch clamp techniques. This protocol is a practical guide to the nmethyldglucamine nmdg protective recovery method of brain slice preparation.
One way to investigate ion channels is to use patch clamping. Introduction the patch clamp is a laboratory technique in electrophysiology that allows investigation of the electrical excitability of neurons and the functional properties and densities of ion channels. The slice patch clamp technique is a powerful tool for investigating learning induced neural plasticity in specific brain regions. For our performance analysis, we focused on experiments in the mouse subiculum, rat presubiculum and rat motor cortex to present an applicable use case for labs working with rodent slices. Electrophysiology from greek, elektron, amber see the etymology of electron. This image shows a neuron patched with the automated imageguided patch clamp system in the brain slice.
Steps are outlined for carrying out the protective recovery brain slice technique using an optimized nmdg artificial cerebrospinal fluid acsf media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. Among the different patch configurations that can be achieved, wholecell patchclamp recordings. These ion channels play a major role in cell physiology. Wholecell patch clamping of mammalian neurons is now routinely performed by many laboratories working on preparations ranging from cultured neurons to in vitro neurons in the slice. Patch clamp recordings from a pyramidal neuron and fast spiking interneuron in neocortical slices derived from a 2. Additionally, metadata for the brain slicing, the whole cell patch clamp and the stimulus including protocols and agents involved in the generation of the recording traces are captured.
Ye jh, zhang j, xiao c, kong jq 2006 patchclamp studies in the cns illustrate a simple new method for obtaining viable neurons in rat brain slices. Metadata is collected on the subject used in the experiment, the slice, the patched cell which was recorded as well as the recording traces and protocols. This technique enables scientists to perform pharmacological studies in defined brain regions by directly applying known concentrations of drugs, which can. Patchclamp recordings of rat neurons from acute brain. The patchclamp method requires cells that have clean, smooth membranes, so it has been primarily applied to neurones in culture or to cells acutely dissociated enzymatically. Success rate of patchclamp recordings can vary greatly depending on tissue health, species, brain region and cell type. New robotic technology may provide more insight into neurological diseases, which affect about onethird of americans. Preparation of acute brain slices using an optimized nmethyldglucamine protective recovery method. Introduction for several decades the vast majority of brain slice physiologists have relied upon a protective cutting method for preparing healthy brain slices from juvenile and adolescent animals. For further theoretical learning, many books and articles have been published on both wholecell patch clamp recording in brain slices 3,2124 and on methods that can refine the solutions used 2527 in order to enhance cell viability.
Interface brain slice chamber from automate scientific. The visual selection of specific cells within an ex vivo brain slice, combined with wholecell patch clamp recording and capillary electrophoresis cemass spectrometry msbased metabolomics, yields high chemical information on the selected cells. The function of nerve and muscle cells relies on ionic currents flowing through ion channels. Combined with intracellular dye labeling, this approach can be. Wholecell patchclamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the. Brain slice electrophysiology involves the ex vivo measurement of neuronal activity in acutely prepared brain slices using either extracellular or intracellular patchclamp recordings.
Flow rate 1 mlmin this is what we do for mice but i see most rat is a higher flow rate slices maintained at 30 degrees c with oxygenated. The patchserver does not work with intact brain slice tissues, but it can patch cells from brain slices after they have been freshly dissociated. An important consideration for patch clamp analysis in adult brain slices is the fluorescent labeling of cellular populations under investigation. Functional electron microscopy, flash and freeze, of. This method involves the preparation of relatively thin slices takahashi, 1978 of brain tissue 120300,am in thickness, followed by localized, visually controlled removal of neuropile covering the neuronal somata. Experiments on neurones within slices of brain tissue were restricted to a few special cases 47. What are the basics of brain slice electrophysiology. The technique of patchclamp recording in brain slices is applicable to a large variety of cell types in slices from nearly all areas of the central nervous system. Wholecell patchclamp recording from acute brain slices of transgenic. Patchclamp techniques applied to brain slices springerlink.
With the latest developments in the traditional patch techniques such as wholecell and single channel as well as perforated patch, fast drug application, loose patch. In vitro whole cell patch clamp recording neuroshapes. The coil was thus positioned between the condenser and the specimen table figure 1a. References jonathan improved adult brain slice methods. We use the patchclamp technique 9,10 to study the functional properties of the gabaa channels in alive neurons in hippocampal brain slices and record the singlechannel and wholecell currents. Patch clamp recording from cells in sliced tissues the university of. In this paper, recent researches on how acupuncture might modulate. Slice patch clamp technique for analyzing learninginduced. Introduction the patch clamp method in cellular electrophysiology has been a motor for the progress in experimental cell. Purdue university researchers developed a robotic system that provides an advanced way to study the brain and how the. To do this, the neuron or slice in your case would be put into a bath solution containing the appr. Brain slice electrophysiology video protocol youtube. Exploring the method of preparing brain slices for patch. Patch clamp of a nerve cell within a slice of brain tissue.
In addition, the patchclamp technique has become a powerful method for investigating the mechanisms underlying the effects of acupuncture. Continuing the research of the bestselling first edition, patchclamp analysis. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation. Wholecell patchclamp recordings in brain slices protocol jove. A lamprey striatal brain slice preparation for patchclamp recordings a lamprey striatal brain slice preparation for patchclamp recordings ericsson, jesper. This protocol describes basic procedural steps for performing wholecell patch clamp recordings. Highthroughput microcircuit analysis of individual human. This method reduces swelling and damage in superficial layers of the slices and improves the success rate for targeted patch clamp recordings, including recordings from fluorescently labeled. New standard in electrophysiology and deep tissue imaging. To overcome the inaccessibility of intact axonal fibers, we developed a novel protocol for direct patchclamp recordings from intact unmyelinated axons in cortical brain slices with an. New robotic technology may provide more insight into. Brain slices have become an integral part of synaptic and cellular physiology since the pioneering studies by henry mcilwain li and mcilwain, 1957. Cellular and molecular events can be investigated using electrophysiological techniques. The patchclamp technique, an electrophysiological technique that has been developed in the late 1970s 1,2, is a primary tool for studying single or multiple ion channel functions in live tissue.
There are all different types of electrophysiology done in the brain. Patch clamp methods and protocols of the methods in molecular biology series. The lamprey brain slice preparation presented has been developed to study the striatal microcircuits and inputoutput systems with patchclamp recordings. The protective recovery method has even been applied successfully in previously unforeseen experimental contexts, such as, improved preservation of vasculature and pericytes in adult cortical brain slices 50, patch clamp recording from transplanted interneuron populations in 11. A patchclamp setup was modified to allow magnetic stimulation of cortical brain slices. However, such a development is critically important to use the technique for rigorously identified central synapses and to correlate structural and functional analysis of. The pipette in the photograph has been marked with a slight blue color. There are a number of configurations of wholecell or cellattached patch clamp recordings we often use, these includes. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The whole cell, inside out patches, and membrane techniques are well illustrated.
Whole cell patch clamp recordings from morphologically digitimer. At first glance this technique has significantly advanced electrophysiological studies of central neuronal excitabilty. The most basic would be simply measuring the resting potential of a neuron. A lamprey striatal brain slice preparation for patchclamp. Brain slice electrophysiology video protocol duration. The patch techniques for brain and spinal slices in culture has several chapters in this book.
This method allows investigation of ion channels in detail and recording of the electric activity of different types of cells, mainly excitable cells like neurons. Conclusion the activity of neurons in the brain slice is associated with age,solutions and processes of operation, thus in experiments we should strictly control these factors. Thus, only the recorded neurone will be filled with a fluorescent dye that will subsequently be visualized on the confocal microscope following the treatment of the slice with this protocol. The method involves dissection of the central nervous system, brain slice preparation, identification of the striatum, visual identification of striatal neurons and patchclamp recordings. Advanced techniques, second edition collects three more years of research in the everexpanding study of the cell membrane. Whole cell patch clamp an overview sciencedirect topics. The patchclamp technique allows the investigation of a small set or even single ion channels. A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Brain slice patchclamp recordings and laserspritzers for optogenetic interogations. The activity of brain slices depended on various experiment conditions. The implementation of this technique by early adopters has facilitated detailed investigations into brain function.
Standard patch clamp techniques hamill, marty, neher. This is especially true for whole cell patch clamp investigations. The technique of patchclamp recording in brain slices is applicable to a large variety of cell types in slices from nearly all areas of the central nervous system cns in animals at many different stages of development blanton et al. Experienced brain slice electrophysiologists are well aware of the difficulties for preparing healthy brain slices from adult animals, and as such the vast majority of acute brain slice studies have been limited to juvenile or adolescent animals. Recipes jonathan improved adult brain slice methods. Effects of body temperature on neural activity in the. A single ion channel conducts around 10 million ions per second. The adult human ex vivo brain slice culture method was modified based on existing procedures previously optimized for juvenile rodent hippocampal slice culture and patch clamp electrophysiology 26. We combined wholecell patchclamp recordings in brain slices and behavioral assays to test the hypothesis that nps activates synaptic inhibition of amygdala output. In particular, the patchclamp method provides detailed information. Targeted axonattached recording with fluorescent patch.
Specific components of the patchserver the patchserver is an addon tool for automating a manual patchclamp setup. We further examine how the channels are affected by different gaba concentrations, other drugs and intra and extracellular factors. Wholecell patchclamp recordings in brain slices protocol. This method has been applied to neurons in the central nervous system of drosophila and allows researchers the opportunity to study the function of their neurons of interest within the context of native circuits in a genetically tractable model system. Preparation of acute brain slices using an optimized n. Acute brain slice methods for adult and aging animals.