In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine dabbased processing of brain slices. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. Drexel university college of medicine goal of physiological recording to detect the communication signals between neurons in real time. Automated wholecell patch clamp electrophysiology of. Voltageclamp experiments to record membrane currents from neurons utilize wholecell patchclamp recordings with relatively low resistance microelectrodes 35 m. Whats the ideal time to have a better biocytin filling success rate.
The enzyme biotinidase cleaves biocytin and makes biotin available to be reused by other enzymes. The patchclamp recording technique was developed by erwin. This protocol describes the use of biocytin to visualize and distinguish the morphology of glia and neurons in rat brain slices. Immediately before beginning surgery, use the electrode puller to prepare between 10 and 50 patch pipet electrodes e. Neurobiotin 488 tracer is readily fixed with standard aldehyde based fixatives.
Microchip amplifier for in vitro, in vivo, and automated. Several patch clamp configurations can be used depending on the research interests, but in all cases, electrophysiological. The patch clamp technique is a significant tool in electrophysiology for high resolution investigations of the ionic current activities through the membrane of living biological cells. The cellimpermeant, fixable, polar tracer alexa fluor 488 biocytin combines the greenfluorescent alexa fluor 488 fluorophore with biotin and an aldehydefixable primary amine. If the pipette solution contains biocytin a biotin. The electrode is sealed to the patch of membrane, and the cell remains intact. The technique provides critical insight into wide ranging applications in the fields of neuroscience, biology, pharmacology, and many other related biomedical research disciplines. Wholecell patch clamp electrophysiology of neurons is a gold standard technique for highfidelity analysis of the biophysical mechanisms of neural computation and pathology but it requires great skill to perform. If the connection probability is low as can be expected for most excitatory connections, proceed as follows. To understand how a neuron can be excited, or to study the behavior of membrane potential, a current clamp is used. Organization of the intermediate gray layer of the. Is there a technique for staining living cell for patching clamping.
Electrophysiological recording techniques wenjun gao, ph. To measure whats happening in or on a single living cell, scientists use a technique called the patch clamp which requires an extremely fine pipette held tightly against the cell membrane. I am trying to figure out the optic time for biocytin filling with whole cell patch pipette. Synaptic properties of som and cckexpressing cells in. Here, we summarized current applications and recent research progress using the in vivo patchclamp recording method and focused on its role in the functional dissection of different synaptic inputs. The black circle in the figure indicates the location of. The key factors of a successful in vivo patchclamp experiment and. The currentclamp lab and voltageclamp lab are organized in the same way. Acc neurons were electrophysiologically characterized and stained with biocytin at the end of the experiments. Currentclamp is a method of intracellular recording involving measurement of the. In vivo twophoton targeted multiple wholecell patchclamp setup. Sep 29, 20 this screencast describes iv and vi relations derived from patch or current clamp data, and how to calculate conductance or resistance from the data. Whats the ideal time to have a better biocytin filling success rate with whole cell patch clamp recordings.
Currentand voltageclamp experiments in virtual laboratories. During the experiment, keep brain slices in an experimental chamber with a glass coverslip at the bottom. The superior colliculus sc is composed of welldifferentiated, alternating gray and white layers that can be easily identified in the living slice. Biocytin is a chemical compound that is an amide formed from the vitamin biotin and the amino acid llysine. Current clamp traces of dopamine cell spontaneous activity in red overlaid and, on the right, dic image of a putative dopamine neuron. Here, by combining wholecell patchclamp recordings and biocytin labeling with full anatomical reconstruction of the filled neurons and ultrastructural analysis of their synaptic contacts, we have elucidated the cellular organization and efferent connections of one of the main itc clusters in mice. The patch of membrane contained within the electrode tip is then ruptured to allow. A simplified circuit schematic of a traditional patchclamp amplifier for whole cell voltageclamp experiments is shown in fig. Dopamine th, green neurons backfilled by including biocytin red in the recording pipette during whole cell patch clamp experiment. This allows for the recording of currents through single ion channels in that patch of membrane, without disrupting the interior of the cell. May 22, 2015 here, we summarized current applications and recent research progress using the in vivo patchclamp recording method and focused on its role in the functional dissection of different synaptic inputs. Patch pipettes are used to load biocytin into different cell types. Whats the ideal time to have a better biocytin filling.
To the best of my knowledge, biocytin can be used to identify the patched cell but only after the. Patch clamp is the gold standard technique for highfidelity analysis of the electrical properties and functional connectivity of neurons. Identity of a pathway for saccadic suppression pnas. The patch clamp is a laboratory technique in electrophysiology that allows. We recently developed patchseq, which combines wholecell patch clamp recording with singlecell rna. Pcth 404 principles and applications of patch clamp recording. Alexa fluor 594 biocytin, sodium salt biocytin alexa fluor. An introductory guide to patch clamp electrophysiology is a concise introduction to the basic principles and practical applications of this important technique. This tracer can be viewed directly under fluorescence microscopy. Extension arms also allow placement of apparatus at various distances from labframes without compromising the integrity of your experiment. After the experiment, the integrity of the seal was veri.
The inset is a drawing of the patchclamped cell, which was filled with biocytin during the recording to reveal its dendritic morphology. As an intermediate in the metabolism of biotin, biocytin occurs naturally in blood serum and urine. Polar tracers are commonly used to investigate cellcell and cellliposome fusion as well as membrane permeability and transport through gap junctions or cell uptake during pinocytosis. Wholecell recording of neuronal membrane potential. The key factors of a successful in vivo patch clamp experiment and possible solutions based on references and our experiences were also discussed.
Patchclamp is the gold standard technique for highfidelity analysis of the electrical properties and functional connectivity of neurons. Rather than penetrating the cell with sharp electrodes as is traditionally performed in voltage clamp experiments, in the patch clamp technique, blunttipped glass pipettes are used in such a way that, when pressed gently against the membrane of a cell, they isolate a small area of membrane. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Biocytin staining of glia and neurons in brain slices.
Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on. Immunostaining of biocytinfilled and processed sections for. Electrophysiological recording techniques drexel university. The cellimpermeant, fixable, polar tracer alexa fluor 594 biocytin combines the redfluorescent alexa fluor 594 fluorophore with biotin and an aldehydefixable primary amine. Control of cerebellar granule cell output by sensoryevoked. Patch clamp electrophysiology, voltage clamp, action.
Oct 20, 2015 patchclamp recordings from granule cells in vitro were made at 3335 c in cerebellar slices 250 m thick prepared with standard techniques. Us20225963a1 automated cell patch clamping method and. The robot in this work demonstrates the most automated in vivo patchclamp experiment to date, by enabling production of multiple, serial intracellular recordings without human intervention. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Intracellular staining with biocytin is then revealed using a standard abc kit. Neurobiotin 488 tracer is a trifunctional molecule designed for neuronal tracing and cell filling. It includes current clamp and voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. Mar 30, 2011 here, by combining wholecell patch clamp recordings and biocytin labeling with full anatomical reconstruction of the filled neurons and ultrastructural analysis of their synaptic contacts, we have elucidated the cellular organization and efferent connections of one of the main itc clusters in mice. A patch clamp recording of current reveals transitions between two conductance states of a single ion channel. Aug, 2016 variations in patch clamp 1cellattached or oncell patch. By carefully heating and pulling a small glass or quartz capillary tube, a very fine pipette can be formed. Depending on the type of synaptic connection three different approaches are used to find synaptically coupled neurons. Figure 1 is a photograph of an unstained in vitro slice and illustrates these layers as they appear in the living slices during a patchclamp experiment. We recently developed patch seq, which combines wholecell patch clamp recording with singlecell rnasequencing.
The difference between successive resistance levels is computed and compared to a. The interpretation of currentclamp recordings in the cell. Jun 11, 2014 during paired wholecell patchclamp recordings of synaptically connected interneurons, singleaction potentials were evoked by brief depolarizing current injection in the presynaptic interneuron 12 ms, 400 pa, and uipscs were recorded. To characterize acc neurons of adult mice in vivo, we carried out wholecell patchclamp recordings from neurons in the superficial layers of adult mouse acc under anesthesia. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. About miriam melis dipartimento di scienze biomediche. The voltage clamp technique allows an experimenter to. A single ion channel conducts around 10 million ions per second. Inputspecific synaptic location and function of the. Keep the slices in the incubation chamber for at least 30 min to 1 hr either at rt or at 30 c, depending on the type of experiment. Electrophysiological and morphological characterization of neuronal microcircuits in acute brain slices using paired patchclamp recordings. Calculating conductanceresistance from patchcurrent clamp. For both in vivo and in vitro experiments, patch pipettes 57 m.
Reads values for gains, filter settings, membrane capacitance, series resistance and external command sensitivity. Rather than penetrating the cell with sharp electrodes as is traditionally performed in voltageclamp experiments, in the patchclamp technique, blunttipped glass pipettes are used in such a way that, when pressed gently against the membrane of a. The interpretation of currentclamp recordings in the cellattached patchclamp configuration. Patch clamp recording from enteric neurons in situ nature. Immunostaining of biocytinfilled and processed sections. To understand the classical current clamp experiment and to study the nature of spiking using current injection. Telegraph communication to set channel scales based on voltages supplied from a wide range of patch clamp amplifiers. First, we added biocytin into the internal solution, which is the standard dye used. Improved biocytin labeling and neuronal 3d reconstruction.
The key factors of a successful in vivo patchclamp experiment and possible solutions based on references and our experiences were also discussed. In voltageclamp mode, an operational amplifier op amp is used in a feedback loop to hold or clamp the cell membrane potential to a userspecified value v clamp. Start studying pcth 404 principles and applications of patch clamp recording. Patch clamp recording is an extremely useful technique for investigating the biophysical properties of the ion channels that control neuronal activation. May 16, 2019 the aim of this article is to provide a description of multiple, twophoton targeted wholecell patchclamp recordings to monitor monosynaptic connectivity in vivo. The patch clamp technique allows the investigation of a small set or even single ion channels.
The following is a brief description of the specific methods we have employed to study the effects of propofol on nachbac and navms. Patch clamp recording from enteric neurons in situ. Alexa fluor 594 biocytin, sodium salt biocytin alexa. Polar tracers are commonly used to investigate cellcell and cellliposome fusion as well as membrane permeability and tra. The advantage of recording in current clamp is that the membrane potential remains unaffected by the recording technique and requires fewer adjustments during the experiment. Ercc spikein rna was used as a positive control in this experiment and gives rise to. Current clamp technique an overview sciencedirect topics.
Clamps are constructed with round extension arms, which allow the clamps to be rotated 360. The readers can refer to alternative sources for basic patch clamp recording techniques and instrumentation 1922, which are not elaborated upon here. In an automated methodology for carrying out in vivo cell patch clamping, a cell patch clamping device is automatically moved into position and targeted to a neuron. Hippocampusdependent learning processes are coordinated via a large diversity of gabaergic inhibitory mechanisms. For ligandgated ion channels or channels that are modulated by. Patchclamp recordings from neurons in vivo were obtained as previously described pern.
Individual neurons vary widely in terms of their gene expression, morphology, and electrophysiological properties. The procedure involves pressing a glass micropipette against a cell in order to isolate a small patch of membrane that contains one or more ion channels. The procedure is described for an acute 1day experiment in anesthetized mice. Available dyes include lucifer yellow, cell tracker, biocytin, alexa biocytin. Biocytin is a chemical compound that is an amide formed from the vitamin biotin and the amino acid l lysine. Several patch clamp configurations can be used depending on the research interests, but in all cases, electrophysiological recordings are produced using a glass micropipette in contact with a patch of the neurons membrane. Electrophysiological and morphological characterization of.
Currentand voltageclamp experiments in virtual laboratories tutorial contents. Direct software communication with molecular devices multiclamp 700 and axoclamp 900 amplifiers. Polar tracers are commonly used to investigate cellcell and cellliposome fusion as well as membrane permeability and trans. Patch clamp electrophysiology is the preferred method that we use to study the effects of general anesthetics on nav function. Patchclamp is the gold standard technique for highfidelity analysis of the. The steps detailed here assume that the equipment and procedures for patch clamp recordings are already established, and the description will be restricted to details related to biocytin filling. The wholecell recording technique has also been adapted for use in vivo. In contrast, using voltageclamp mode, the command potential would have to be adjusted frequently due. Control of cerebellar granule cell output by sensory. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. Neuron contact is determined by analyzing the temporal series of measured resistance levels at the cell patch clamping device as it is moved. The slices are subsequently fixed, stained, and mounted in preparation for imaging.
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