Targeted axonattached recording with fluorescent patch clamp pipettes in brain slices. Patchclamp techniques applied to brain slices springerlink. Wholecell patch clamp recordings and pharmacological techniques have been used to obtain low noise recordings of 2 excitatory postsynaptic synaptic currents termed epsa a and epsc b evoked by stimulation of the schaffer collateralcommissural pathway in rat hippocampal slices. Whole cell patch clamp an overview sciencedirect topics.
The current, amplitude, and kinetics of the transgenic nmda receptors were evaluated using patch clamp recordings from visually identified cells in acute hippocampal slices under irdic microscopy. Wholecell patchclamp recording from acute brain slices of. Cortical processing depends on orchestrated activity across distributed neuronal assemblies, and. While patch clamp recordings can be obtained blind without visual control, infrared video ir video microscopy allows visual identification of. Patchclamp recording in brain slices with improved slicer technology. Automatic tracking of cells for video microscopy in patch clamp experiments. Sep 30, 2014 wholecell patchclamp recordings from morphologically and neurochemicallyidentified hippocampal interneurons sam a. The slice patch clamp technique is a powerful tool for investigating learning induced neural plasticity in specific brain regions. Quanta1 analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices.
Jul 10, 2016 whole cell patch clamp technique is another alternative method to obtain specific information about hippocampal cell function by studying the ion channels and associated receptors, as well as the other components of the cellular machinery that control cell function. Brain slices have become an integral part of synaptic and cellular physiology since the pioneering studies by henry mcilwain li and mcilwain, 1957. Slice patch clamp technique for analyzing learninginduced. Patch clamp of a nerve cell within a slice of brain tissue. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Brain slice electrophysiology video protocol abcam. Patch clamp wholecell recordings allow us to draw the conclusion that gdps in the intact hippocampus have the same principal features as in hippocampal slices. Epsc a was blocked by 6cyano7nitroquinoxaline2,3dione cnqx and epsc b was blocked by d2amino5. Wholecell current recording using the patch clamp technique revealed synaptic currents ranging from less than 10 to 200 pa in symmetrical cl conditions, at a. Here we have described the requirements for establishing successful paired whole cell recordings in organotypic hippocampal slice cultures. Turn on the power supply to the light source, tv monitor, and amplifier 2. Neuronal electrophysiology this application note includes a complete protocol for the dissection of rat brain, the preparation of brain hippocampal slices for acute and long term experiments, suggestions for longterm cultures, for mea system configurations, and for data analysis. New illumination technique for irvideo guided patchclamp. By carefully heating and pulling a small glass or quartz capillary tube, a very fine pipet can be formed.
Hippocampal slices were pretreated with dmso control or phenytoin for 20 min before electrical recordings and were continuously superfused in the same acsf during the recordings. Pairedpulse modulation of uipscs at individual gabaergic synapses was tested. Synaptically connected neurones were identified in the granule cell layer of slices of 17 to 21dayold rat hippocampus. Dual electrophysiological recordings of synapticallyevoked astroglial and neuronal responses in acute hippocampal slices. For field recordings nn refers to numbers of slices mice. With this technique, we demonstrate visually guided wholecell patchclamp recording of na and k currents as well as combination of wholecell recording with fluorescence imaging of hippocampal and entorhinal cortex neurons in biomembrane slice. Conventional wholecell patch pipette recordings were obtained from visually identified cells within 75. Interneuron firing can be linked to epileptiform high frequency activity. Aug 15, 2000 wholecell patchclamp recordings were made from visualized pyramidal cells or interneurons in the ca1 area of rat hippocampal slices as described previously dodt and zieglgansberger, 1990. Patch clamp methods and protocols of the methods in molecular biology series. Wholecell patchclamp recordings from morphologically jove. To measure whats happening in or on a single, living cell, scientists use a technique called the patch clamp which requires an extremely fine pipet held tightly against the cell membrane. Aug 01, 2002 extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisensetreated rats. Dodt hu 1992 infrared video microscopy of living brain slices.
Attach the pipettes to the electrode holders of the patchclamp amplifier headstages and turn into position. Brain slice electrophysiology video protocol youtube. For patch clamp recordings nnn1 refers to numbers of cells slices mice. Bischofberger j1, engel d, li l, geiger jr, jonas p. Wholecell patch clamp recordings from morphologically and neurochemicallyidentified hippocampal interneurons. Hippocampal slice recorded by 3brains mea biochips. The technique of patchclamp recording in brain slices is applicable to a large. Patch clamp recordings of thermal effects of magnetic stimulation on the physiological characteristic of rat hippocampal neurons yu zheng1, lei dong 1, ying kong 1, hui hong1, yang gao, zhe zhao and huiquan wang 1 school of electronics and information engineering, tianjin polytechnic university, tianjin 300387, china.
Chemical hypoxia in hippocampal pyramidal cells affects membrane potential differentially depending on resting potential. Schematic shows brain slice, patch pipette and microscope objective. Ff and snr of optically recorded postsynaptic potentials psps. P21p28 hippocampal slices were obtained as described in. Im a fairly experienced slice physiologist but a current short project requires me to patch neurons in cortical hippocampal cultures i need. The method of slice preparation and the patch clamp recording technique were essentially similar to. We further examine how the channels are affected by different gaba concentrations, other drugs and intra and extracellular factors.
Voltage imaging and optogenetics reveal behaviourdependent. This new illumination method is extremely affordable and has been proven to allow efficient irvideo guided patchclamp recording from neurons in membrane slice culture from 7 to 9dayold rat, i. We discuss the patch clamp technique and how it contributes to our understanding of voltage gated ion channels. Introduction before the introduction of gigohm seal patch clamp techniques hamill et al. The hippocampal slice preparation was first brought to the united states from per andersons labinitially by tim teyler followed shortly after by phil schwartzkroin. A single ion channel conducts around 10 million ions per second. Using improved patch clamp techniques in brain slices, we recorded from mossy fiber terminals in the ca3 region of the. In initial experiments, ca1 pyramidal neurons and dentate granule cells were acutely isolated from hippocampal slices. Slices of living brain tissue are maintained in a special chamber, and individual neurons are visualised using an upright microscope equipped with specialised optics and a video camera. Patchclamp recordings from the soma and dendrites of neurons in. To answer this question, wholecell patch clamp recordings from pvbcs were performed in current clamp mode. Whole cell patch clamp recordings from morphologically digitimer. Free access to all jove education videos until june 15.
Aokidifferential responses between ca1 pyramidal cells and granule cells to ischemic insult in rat hippocampal slices. Effects of halothane on glutamate receptormediated. The slice patch clamp technique is a powerful tool for investigating learninginduced neural plasticity in specific brain regions. These irdic videos show the morphology of neurons in living adult brain slices prepared from 5 month old mice, as imaged from the surface and proceeding down through the tissue in 10 micron increments. Wholecell patchclamp recordings of an nmda receptor. Wholecell patchclamp recordings from morphologically and.
A new approach of modified submerged patch clamp recording. Electrophysiology and morris water maze to assess hippocampal. Visually guided patchclamp recordings in brain slices. It discusses anesthesia techniques and monitoring as well as spinal and cranial anatomy. Frontiers a new approach of modified submerged patch clamp. Fast ripples, unique to chronic epilepsy, can be modeled in ex vivo tissue from tenttreated rats. This new illumination method is extremely affordable and has been proven to allow efficient ir video guided patchclamp recording from neurons in membrane slice culture from 7 to 9dayold rat, i. Hippocampal synaptic plasticity in mice overexpressing an. Wholecell patchclamp recordings from morphologically ncbi. Patchclamp technique in brain slices springer nature experiments. Rat hippocampal slice recorded with high resolution mea.
It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. Rhythmic synaptic activity induced by mechanical injury of. Patchclamp recording in brain slices with improved slicer. Paired whole cell recordings in organotypic hippocampal.
Sep 30, 2003 read new illumination technique for irvideo guided patchclamp recording from neurons in slice cultures on biomembrane, journal of neuroscience methods on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. An infrared differential interference contrast irdic video microscopy optical setup was used for all patchclamp studies stuart et al. Received from the department of anesthesiology and critical care, hadassah university hospitalhebrew university, and the department of physiology, hebrew university school of medicine, jerusalem, israel. Although in vivo current clamp analysis is more reliable, it is technically challenging to get sufficient data from conscious animals. A patch clamp study in adult mouse hippocampal slices. Pairedpulse modulation at individual gabaergic synapses. Preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology. Patch clamp recording in brain slices with improved slicer technology. Invivo patchclamp technique this chapter covers invivo patchclamp recordings in the mouse spinal cord and somatosensory cortex. Wholecell patch clamp recordings from morphologically and neurochemicallyidentified hippocampal interneurons sam a. Wholecell patchclamp recordings from morphologically. The technique of patch clamp recording in brain slices is applicable to a large. Booker 1, jie song 1, imre vida 1 1 institute of integrative neuroanatomy, neurocure cluster of excellence, charite universitatmedizin. Patch clamp recording from mossy fiber terminals in hippocampal slices.
Using improved patch clamp techniques in brain slices, we recorded from mossy fiber terminals in the ca3 region of the hippocampus, which have a diameter of 25. Patch clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Prolonged life of human acute hippocampal slices from. The hippocampal slice preparation was first brought to the united states from per andersons lab, initially by tim teyler and followed shortly after by phil schwartzkroin. Under current clamp in the single neuron, ad is recorded as a sharp depolarization within 6 min of o2glucose deprivation ogd. The technique of patch clamp 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. Discover how to successfully perform brain slice electrophysiology.
Slices were constantly perfused with room temperature 2124c acsf at a rate of 22. Patch clamp recording from cells in sliced tissues 257. Since each pyramidal neuron has different cellular properties, an. Read new illumination technique for ir video guided patch clamp recording from neurons in slice cultures on biomembrane, journal of neuroscience methods on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Simultaneous imaging and current clamp recordings show that the increased lt front and sudden depolarization are coincident. Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. However, cortical terminals have been largely inaccessible to presynaptic patch clamp recording, due to their small size. Hippocampal circuits are comprised of excitatory principal cells 80% and a smaller 1020%, but highly. Paired whole cell recordings in organotypic hippocampal slices. Can anyone help with the difference of hippocampal slices. Hippocampal slices for patch clamp and field potential, are prepared from mice using coronal, horizontal. In this study, a miniaturized ultrasound neurostimulation chip is developed to investigate the ultrasonic effects on the level of ion channels in the pyramidal neurons using whole. Mechanical injury of the cns frequently results from accidents but also occurs in the course of neurosurgical interventions.
The generation and propagation of networkdriven activities can be studied. Patch clamp recording from cells in sliced tissues alasdair j. Membrane and synaptic actions of halothane on rat hippocampal. Place your chamber onto the microscope stage, and tack the chamber down with tacky wax. Booker, 1 jie song, 1 and imre vida 1 1 institute of integrative neuroanatomy, neurocure cluster of excellence, charite universitatmedizin. Ca1 pyramidal neurons in adult animals 1012 weeks old were analyzed when biochemical evidence of transgene expression was most clear.
Apical and basal dendrites, as well as the dyecontaining patchpipette can be clearly seen in this plane of focus. Can anyone help with the difference of hippocampal slices oientation for electrophysiology. Targeted axonattached recording with fluorescent patch. Organotypic cultures of hippocampal slice preparation otc. Preparation of brain slices for patch clamp recording. However, this section contains fairly recent material and is not as in depth as slice patchclamp recordings. This system has been reported to enable recordings of sustained epileptiform activity in acute rodent hippocampal slices, with the advantage of allowing visually guided wholecell patch clamp. Traditionally, visuallyguided patch clamp in brain slices using submerged recording conditions has been required to. 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. Using improved patch clamp techniques in brain slices, we recorded from mossy fiber terminals in the ca3 region of the hippocampus, which have a diameter of 25 microm. The pipette in the photograph has been marked with a slight blue color. In summary, brains were rapidly removed and placed in icecold sucroseartificial cerebrospinal fluid. The intact hippocampal formation ihf of neonatal or young rats can be kept alive for an extended period in a fully submerged chamber with excellent morphological preservation.
Attach the pipettes to the electrode holders of the patch clamp amplifier headstages and turn into position. The hippocampal slice preparation was first brought to the united states from per andersons lab. Chemical hypoxia in hippocampal pyramidal cells affects. Wholecell patchclamp recordings in brain slices protocol jove. We use the patch clamp 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. The spatial restriction of these two neurogenic niches svz and sgz is of considerable relevance to their potential role in repair of the brain. The patch clamp technique allows the investigation of a small set or even single ion channels. Patchclamp recording from mossy fiber terminals in. Pdf patchclamp recording from mossy fiber terminals in. Wholecell patchclamp recording from acute brain slices of transgenic. Automatic tracking of cells for video microscopy in patch.
Patchclamp recordings of miniature ipscs mipscs conducted in ca1 pyramidal neurons in slices from eaac1 antisensetreated animals demonstrated a significant decrease in mipsc amplitude, indicating decreased tonic inhibition. Quantal analysis of inhibitory synaptic transmission in. Paired recordings can also be performed in multiple preparations, including acute slices and dissociated culture systems 26,27. Hippocampal neuronal circuits have long been the subject of intense. Using improved patchclamp techniques in brain slices, we recorded from mossy fiber terminals in the ca3 region of the hippocampus, which have a diameter of 25. Preparation of acute hippocampal slices from rats and. Highlightssimultaneous epileptiform lfps and singlecell activity can be recorded in the membrane chamber. Dual electrophysiological recordings of synaptically. Wholecell patch clamp recordings of gaba a receptor currents were obtained from hippocampal neurons ca1 pyramidal neurons and dentate granule cells. Brain slice electrophysiology video protocol duration. Thin slice cleaning and patch clamp techniques were shown. Irdic adult brain slice videos hippocampus ca1 pyramidal layer these irdic videos show the morphology of neurons in living adult brain slices prepared from 5 month old mice, as imaged from the surface and proceeding down through the tissue in 10 micron increments. Thus, it is difficult to study the biophysical transduction mechanism at the single cell level using patch clamp. Unitary inhibitory postsynaptic currents uipscs were recorded in synaptically coupled pairs of ca1 hippocampal interneurons and pyramidal neurons in rat brain slices by using dual patch clamp techniques.
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