Patch clamp recording from mossy fiber terminals in hippocampal slices. The hippocampal slice preparation was first brought to the united states from per andersons labinitially by tim teyler followed shortly after by phil schwartzkroin. Schematic shows brain slice, patch pipette and microscope objective. Preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology. 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. Attach the pipettes to the electrode holders of the patchclamp amplifier headstages and turn into position. Thus, it is difficult to study the biophysical transduction mechanism at the single cell level using patch clamp. 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. 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. 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. Patchclamp recording in brain slices with improved slicer. Introduction before the introduction of gigohm seal patch clamp techniques hamill et al. We discuss the patch clamp technique and how it contributes to our understanding of voltage gated ion channels.
Electrophysiological properties of ca1 pyramidal neurons. Wholecell patchclamp recordings from morphologically. Pairedpulse modulation of uipscs at individual gabaergic synapses was tested. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Although in vivo current clamp analysis is more reliable, it is technically challenging to get sufficient data from conscious animals. Chemical hypoxia in hippocampal pyramidal cells affects membrane potential differentially depending on resting potential. Wholecell patch clamp recordings of gaba a receptor currents were obtained from hippocampal neurons ca1 pyramidal neurons and dentate granule cells. 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. Highlightssimultaneous epileptiform lfps and singlecell activity can be recorded in the membrane chamber. It discusses anesthesia techniques and monitoring as well as spinal and cranial anatomy. Wholecell patchclamp recordings from morphologically and. 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. Wholecell patchclamp recording from acute brain slices of transgenic.
Whole cell patch clamp recordings from morphologically digitimer. Hippocampal slice recorded by 3brains mea biochips. Brain slices have become an integral part of synaptic and cellular physiology since the pioneering studies by henry mcilwain li and mcilwain, 1957. Since each pyramidal neuron has different cellular properties, an. 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. Epsc a was blocked by 6cyano7nitroquinoxaline2,3dione cnqx and epsc b was blocked by d2amino5. In summary, brains were rapidly removed and placed in icecold sucroseartificial cerebrospinal fluid. Wholecell patchclamp recordings in brain slices protocol jove.
Preparation of brain slices for patch clamp recording. 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. Under current clamp in the single neuron, ad is recorded as a sharp depolarization within 6 min of o2glucose deprivation ogd. Pairedpulse modulation at individual gabaergic synapses. Chemical hypoxia in hippocampal pyramidal cells affects. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. Dual electrophysiological recordings of synapticallyevoked astroglial and neuronal responses in acute hippocampal slices. Patch clamp recording in brain slices with improved slicer technology. The pipette in the photograph has been marked with a slight blue color. We further examine how the channels are affected by different gaba concentrations, other drugs and intra and extracellular factors. 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. Can anyone help with the difference of hippocampal slices. The hippocampal slice preparation was first brought to the united states from per andersons lab. Quantal analysis of inhibitory synaptic transmission in.
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. 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. Attach the pipettes to the electrode holders of the patch clamp amplifier headstages and turn into position. Traditionally, visuallyguided patch clamp in brain slices using submerged recording conditions has been required to. Wholecell patchclamp recordings of an nmda receptor. Prolonged life of human acute hippocampal slices from. Bischofberger j1, engel d, li l, geiger jr, jonas p. Patchclamp recording in brain slices with improved slicer technology. While patch clamp recordings can be obtained blind without visual control, infrared video ir video microscopy allows visual identification of. Interneuron firing can be linked to epileptiform high frequency activity. 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 major limitation of the slice patch clamp technique is the recording in slice preparation, which may not reflect what happens in vivo.
Free access to all jove education videos until june 15. Hippocampal neuronal circuits have long been the subject of intense. A patch clamp study in adult mouse hippocampal slices. 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. The method of slice preparation and the patch clamp recording technique were essentially similar to. In initial experiments, ca1 pyramidal neurons and dentate granule cells were acutely isolated from hippocampal slices.
Paired whole cell recordings in organotypic hippocampal. The technique of patch clamp recording in brain slices is applicable to a large. Hippocampal slices for patch clamp and field potential, are prepared from mice using coronal, horizontal. Simultaneous imaging and current clamp recordings show that the increased lt front and sudden depolarization are coincident. The generation and propagation of networkdriven activities can be studied. Wholecell patchclamp recordings from morphologically jove. Ca1 pyramidal neurons in adult animals 1012 weeks old were analyzed when biochemical evidence of transgene expression was most clear. 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.
Booker 1, jie song 1, imre vida 1 1 institute of integrative neuroanatomy, neurocure cluster of excellence, charite universitatmedizin. 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. Fast ripples, unique to chronic epilepsy, can be modeled in ex vivo tissue from tenttreated rats. 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. Using improved patch clamp techniques in brain slices, we recorded from mossy fiber terminals in the ca3 region of the. P21p28 hippocampal slices were obtained as described in. Hippocampal synaptic plasticity in mice overexpressing an.
By carefully heating and pulling a small glass or quartz capillary tube, a very fine pipet can be formed. Automatic tracking of cells for video microscopy in patch. Whole cell patch clamp an overview sciencedirect topics. The patch clamp technique allows the investigation of a small set or even single ion channels. Im a fairly experienced slice physiologist but a current short project requires me to patch neurons in cortical hippocampal cultures i need. Turn on the power supply to the light source, tv monitor, and amplifier 2. However, this section contains fairly recent material and is not as in depth as slice patchclamp recordings. 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. Paired recordings can also be performed in multiple preparations, including acute slices and dissociated culture systems 26,27. 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. Booker, 1 jie song, 1 and imre vida 1 1 institute of integrative neuroanatomy, neurocure cluster of excellence, charite universitatmedizin. Patch clamp methods and protocols of the methods in molecular biology series.
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. 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. Apical and basal dendrites, as well as the dyecontaining patchpipette can be clearly seen in this plane of focus. Patch clamp of a nerve cell within a slice of brain tissue. 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. The technique of patchclamp recording in brain slices is applicable to a large. Dual electrophysiological recordings of synaptically.
Place your chamber onto the microscope stage, and tack the chamber down with tacky wax. Brain slice electrophysiology video protocol duration. A single ion channel conducts around 10 million ions per second. Frontiers a new approach of modified submerged patch clamp. Wholecell patch clamp recordings from morphologically and neurochemicallyidentified hippocampal interneurons. Conventional wholecell patch pipette recordings were obtained from visually identified cells within 75. Using fine control micromanipulators, descend the recording pipettes to the region of interest within the brain slice. 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. 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. 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.
Organotypic cultures of hippocampal slice preparation otc. 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. New illumination technique for irvideo guided patchclamp. Aug 01, 2002 extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisensetreated rats.
Preparation of acute hippocampal slices from rats and. Wholecell patch clamp recordings from morphologically and neurochemicallyidentified hippocampal interneurons sam a. Slices were constantly perfused with room temperature 2124c acsf at a rate of 22. Hippocampal circuits are comprised of excitatory principal cells 80% and a smaller 1020%, but highly. Targeted axonattached recording with fluorescent patch. 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.
Thin slice cleaning and patch clamp techniques were shown. Patchclamp recordings from the soma and dendrites of neurons in. Quanta1 analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices. Discover how to successfully perform brain slice electrophysiology. For field recordings nn refers to numbers of slices mice. 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. Invivo patchclamp technique this chapter covers invivo patchclamp recordings in the mouse spinal cord and somatosensory cortex. Voltage imaging and optogenetics reveal behaviourdependent. 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. For patch clamp recordings nnn1 refers to numbers of cells slices mice. Wholecell patchclamp recordings from morphologically ncbi. Effects of halothane on glutamate receptormediated. Wholecell patchclamp recording from acute brain slices of. Electrophysiology and morris water maze to assess hippocampal.
Patchclamp recording from mossy fiber terminals in. Patchclamp technique in brain slices springer nature experiments. Rat hippocampal slice recorded with high resolution mea. Brain slice electrophysiology video protocol abcam. 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. Targeted axonattached recording with fluorescent patch clamp pipettes in brain slices. Paired whole cell recordings in organotypic hippocampal slices.
Issue 69, physiology, anatomy, medicine, hippocampus preparation, acute brain slice, electrophysiology, patch clamp, neurons, astrocytes, astroglial, neuroglial. Patch clamp recording from cells in sliced tissues alasdair j. Automatic tracking of cells for video microscopy in patch clamp experiments. 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. A new approach of modified submerged patch clamp recording. Ff and snr of optically recorded postsynaptic potentials psps. Wholecell patchclamp recording from acute brain slices of transgenic animals, expressing. Sep 30, 2014 wholecell patchclamp recordings from morphologically and neurochemicallyidentified hippocampal interneurons sam a. Mechanical injury of the cns frequently results from accidents but also occurs in the course of neurosurgical interventions. 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. View all our video protocols with written procedures at. Patchclamp techniques applied to brain slices springerlink.
Pdf patchclamp recording from mossy fiber terminals in. Patch clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. To answer this question, wholecell patch clamp recordings from pvbcs were performed in current clamp mode. Dodt hu 1992 infrared video microscopy of living brain slices. Visually guided patchclamp recordings in brain slices. Cortical processing depends on orchestrated activity across distributed neuronal assemblies, and. A great variety of anatomical and physiological changes have been descri. Slice patch clamp technique for analyzing learninginduced. Here we have described the requirements for establishing successful paired whole cell recordings in organotypic hippocampal slice cultures. The slice patch clamp technique is a powerful tool for investigating learninginduced neural plasticity in specific brain regions. 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. Rhythmic synaptic activity induced by mechanical injury of. Can anyone help with the difference of hippocampal slices oientation for electrophysiology. 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.
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