2011.10.07.. TÁMOP–4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY sote_logo.jpg dk_fejlec.gif INFOBLOKK 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Peter PazmanyCatholicUniversity Facultyof InformationTechnology ELECTROPHYSIOLOGICAL METHODS OF THE STUDY OF THE NERVOUS-AND MUSCULAR SYSTEM LECTURE6 METHODSOF INTRA-AND EXTRACELLULAR MICRORECORDINGS www.itk.ppke.hu Az ideg-és izomrendszer elektrofiziológiai vizsgálómódszerei (Intra-és extracelluláris mikroelvezetések módszerei ) DOMONKOS HORVÁTH, GYÖRGY KARMOS ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 DEFINITIONS • Intracellular microrecording: a technique used to measure with precision the voltage across, or electrical currents passing through, neuronal or other cellular membranes by inserting an electrode inside the neuron • Extracellular recording: a technique used to measure a single neuron’s spike discharge or a small neuron population’s electric activity with an electrode placed in close proximity to a single neuron or small neuron population www.itk.ppke.hu ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings COMPARISON OF INTRACELLULAR AND EXTRACELLULAR RECORDINGTECHNIQUES 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 Extracellular • Recording from the extracellular medium • Recording the activity of a neuron population • Local field potentials • Multiunit activity • Single unit action potential Intracellular • Recording from the intracellular space • Recording the activity of a single neuron • Synaptic, action and membrane potentials • Ion channel and membrane current recordings • Recording from a single ion channel • Chemical substance introduction during recording ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings COMPARISON OF INTRACELLULAR AND EXTRACELLULAR RECORDINGELECTRODES –ADVANTAGES AND DISADVANTAGES 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 Extracellular • Technically easy • Low signal amplitude (10-500µV) • Low electrical noise amplifiers needed • Multiple channel (10-200) recordings possible • Available in freelymoving animals • Unable to record intracellular processes directly Intracellular • Technically complicated • High signal amplitude (1-100mV) • Low electrical noise amplifiers needed • Only few (1-4) channel recordings possible (with separate electrodes) • Unavailable in freelymoving animals • Records intracellular processes directly ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings TYPES OF INTRACELLULAR AND EXTRACELLULAR RECORDINGELECTRODES 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 Extracellular • Micropipette • Single microwire • Tetrodeand microwiremultielectrode • Silicon-based multielectrodes Intracellular • Micropipette • Sharp microelectrode • Patch-clamp electrode ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings TYPES OF INTRACELLULAR RECORDING ELECTRODES 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 Sharp microelectrode • Sharp glass micropipette • Sharpness: tip diameter << 1µm • High electrode impedance • Penetrates the cell by external pressure • Low insulation resistance • High leakage current around the electrode • Suitable for in-vivo experiments • Accesses deep-layer cells • ‘Blind’ recordings • Planning of cell targeting unavailable • Membrane potential measurements • Constant current injection • Current clamp • Low suitability for membrane channel current recordings • Unsuitable for single membrane channel recordings ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings • Diameter of tip: 0.03-0.06µm • Length of neck: 6-14mm • Electrode impedance: 30-200MOhm • Filled with: 2M potassium acetate (and Neurobiotintracer) 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 TYPES OF INTRACELLULAR RECORDING ELECTRODES Sharp microelectrode ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings • Connected to preamplifier through a non-polarizable Ag/AgClelectrode because of DC recording 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 TYPES OF INTRACELLULAR RECORDING ELECTRODES Sharp microelectrode MW_intracellular_electrode3 http://www.scholarpedia.org/article/File:MW_intracellular_electrode3.jpg Sharp microelectrode with connecting electrode inside ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings TYPES OF INTRACELLULAR RECORDING ELECTRODES 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 Patch-clamp electrode • Glass micropipette less sharp than sharp microelectrode • Tip diameter < 1µm • Lower electrode impedance • Cell membrane sealed to the electrode by suction • High insulation resistance • Low leakage current around the electrode • Not idealfor in-vivo experiments • Deep-layer cells inaccessible • Used in brain slices or cell cultures • Cell targeting well planned • Membrane potential and membrane current measurements • Constant current injection, constant membrane voltage • Current clamp, voltage clamp • Excellently suitable for membrane channel current recordings • Suitable for single membrane channel recordings ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings • Diameter of tip: 1-3µm • Length of neck: 3-4mm • Electrode impedance: 1-10MOhm • Filled with: solution with similar ion composition to the intracellular medium (and Neurobiotintracer) 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 TYPES OF INTRACELLULAR RECORDING ELECTRODES Patch-clamp electrode ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings patch • Different methods of patch-clamp recording 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 TYPES OF INTRACELLULAR RECORDING ELECTRODES Patch-clamp electrode J. Malmuvioand R. Plonsey, Bioelectromagnetism: Principles and Applications of Bioelectric and BiomagneticFields. New York: Oxford University Press, 1995. ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings BASICS OF INTRACELLULAR RECORDINGS • Compromise: the smaller the electrode tip, the easier to penetrate into the cell but also the higher the electrode impedance and therefore the electrode’s sensitivity to noise • Both current and voltage can be measured • Only current can be injected • Extracellular reference electrode for voltage measurements 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings BASICS OF INTRACELLULAR RECORDINGS • Schematic of intracellular recording arrangement 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings CURRENT CLAMP • Injecting current into a cell through the recording electrode • Recording the membrane potential • Constant current injection, membrane potential free to vary • Used to study how a cell responds, when electric current enters • Cell can be excited or inhibited • Obtained values:• Membrane capacitance • Membrane resistance • Action potential threshold • Importance: understanding neuronal response e.g. to neurotransmitters that act by opening membrane ion channels 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings CURRENT CLAMP 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 Injecting current (Is), recording membrane potential (Vm) ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings CURRENT CLAMP IN PRACTICE • Single electrode recording • Current injected and voltage measured on the same electrode • Electrode has serial resistance (Re) and parasitic capacitance (Ce) • Injected current flows through the serial resistance and charges the parasitic capacitance • The recording circuit and the electrodes have DC offset voltage error • Consequence: the whole circuitry is measured, not only the membrane • To avoid this: serial resistance, parasitic capacitance and DC offset have to be compensated • Compensation carried out outside the cell 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 currentclamp Current clamp circuit ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 CURRENT CLAMP IN PRACTICE Experimental setup Replacement diagram ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings CAPACITANCE COMPENSATION • Variable amplifier at the output of unity gain amplifier • Drives a current-injection capacitor connected to the input • Ideal setting of variable amplifier: this injected current is exactly equal to the current that passes through the parasitic capacitance (Ce) to ground • Consequence: recording bandwidth increases • Risk: if the amplifier gain is increased past the ideal setting, the input signal will be overshot by the injected current, the circuit will oscillate and destroy the cell 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings CAPACITANCE COMPENSATION 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 Compensation carried out outside the cell: schematic and replacement diagram Replacement diagram of compensation ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings SERIAL RESISTANCE COMPENSATION • Technique called bridge balance • Goal: generate a signal proportional to the product of the microelectrode current and the microelectrode resistance • This signal then subtracted from the amplifier output • Consequence: instantaneous voltage step in recorded signal due to ohmicvoltage drop across microelectrode after current step eliminated • Origin of name: originally subtraction was achieved by Wheatstone bridge, now by operational amplifier circuits 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings SERIAL RESISTANCE COMPENSATION Replacement diagram of compensation 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 Rbbvaried until voltage step from recorded signal (Uout) eliminated ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings DC OFFSET COMPENSATION Replacement diagram 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 Set Rdcto make Uoutzero –remember, compensation is carried out outside the cell ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings VOLTAGE CLAMP 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 Membrane voltage (Vm) kept constant, measuring injected current (Is) ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings VOLTAGE CLAMP • Membrane voltage kept (clamped) at a constant value • Injected current that is needed to keep the constant membrane voltage recorded • Used to measure how much ionic current crosses the membrane at a given voltage • Obtained value: Current flowing through the membrane independent of membrane capacitance Importance: voltage dependency of ion channels can be determined 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 Voltage clamp circuit voltageclamp.jpg ElectrophysiologicalMethodsoftheStudyoftheNervous-andMuscularSystem:MethodsofIntraandExtracellularMicrorecordings VOLTAGE CLAMP 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 clamp.jpg http://life.nthu.edu.tw/~g864264/Neuroscience/min/Voltage.html, Dale Purves,1997,Neuroscience,Sinauer Associates, Inc., P.53. Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings TYPES OF EXTRACELLULAR RECORDING ELECTRODES Micropipettes • Pulled from glass pipettes (like intracellular electrodes) • Filled with electrolyte solution, e. g. sodium chloride solution • Used for single cell recordings • Electrode impedance: 5-15MOhm • Relatively high electrode impedance among extracellular electrodes 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings TYPES OF EXTRACELLULAR RECORDING ELECTRODES Single microwireand microwirearray • Sharpened metal wire • Coated with insulator material, except for tip • Different types of metal used: platinum, gold, tungsten, iridium, stainless steel • Lower impedance than glass micropipette electrodes • Used for single unit, multi unit and field potential recordings • Arrays with several microwiresbuilt to record more cells simultaneously • Precise location of each electrode of the array in the brain cannot be determined 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings TYPES OF EXTRACELLULAR RECORDING ELECTRODES Single microwireand microwirearray 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 16wire_electrode_array.jpg http://www.thomasrecording.com/en/cms/upload/document/singleelectrodes.pdf http://commons.wikimedia.org/wiki/File:16wire_electrode_array.jpg Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings TYPES OF EXTRACELLULAR RECORDING ELECTRODES Tetrode • Tetrode: four metal microelectrodes in close proximity in the same insulator coating to record single cell activity • Advantage of tetrode: each of the four electrodes records a little bit different spike waveform of the same cell, this makes it easier to separate the cell’s activity from other cells and background • Improvement: heptode –seven microelectrodes for even better single unit isolation 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings TYPES OF EXTRACELLULAR RECORDING ELECTRODES Tetrode 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 Tetrode.gif http://www.ibiscanada.com/THOMAS_page.html Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings TYPES OF EXTRACELLULAR RECORDING ELECTRODES Microwiremultielectrode • Many (more than 10) microwiresin one common insulator coating • Used for single unit, multi unit and field potential recordings • Able to record activity of e. g. more cortical layers simultaneously 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 Electrode tip Microwires(24 in total) Insulator coating Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings •Metal electrode contacts in silicon substrate • Allows precise electrode size and spacing design with high reproducibility • Much higher electrode count than in metal microwirearrays possible while electrode array size remains smaller • Precise location of each electrode contact in the brain determinable • Linear and 3D arrays can be built • Used for single unit, multi unit and field potential recordings 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 33 TYPES OF EXTRACELLULAR RECORDING ELECTRODES Silicon-based multielectrodes http://www.neuronexus.com/catalog2011.pdf Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings TYPES OF EXTRACELLULAR RECORDING ELECTRODES Silicon-based multielectrodes 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 34 IMG_0489 P. J. Rouscheand R. A. Normann, "Chronic recording capability of the Utah IntracorticalElectrode Array in cat sensory cortex," J NeurosciMethods, vol. 82, pp. 1-15,1998 http://www.neuronexus.com/catalog2011.pdf L. Grand, Development, testing and application of laminar multielectrodesand biocompatible coatings for intracorticalapplications, PhD dissertation, 2010 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings PROBLEMS OF EXTRACELLULAR CELL ACTIVITY DETECTION • Many cells in the proximity of the electrode • Signal amplitude very low: the extracellular medium conducts currents well thus the activity of a single cell spreads rapidly in all directions • The detected waveform depends on the electrode position • Consequence 1: low noise, high gain amplifiers needed to amplify low amplitude signals while keeping noise as low as possible • Consequence 2: the electrode records activity of several different cells so required information has to be extracted from this summed activity with mathematical methods 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 35 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings PROBLEMS OF EXTRACELLULAR CELL ACTIVITY DETECTION 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 36 More than 100 cells in 50µm and more than 1000 cells in 140µm distance from the electrode Multiple channel unit activity recording. Spikes are visible on many channels, more channels recording spikes of the same cell. Sorted cells on c. G. Buzsáki, Large-scale recording of neuronal ensembles, Nature neuroscience, Vol. 7, No. 5. (May 2004), pp. 446-451 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings EXTRACTING INFORMATION FROM RECORDED SIGNAL Filtering • Typically, wideband –0.1Hz-7kHz –signals recorded • Local field potentials in the low frequency range: <50Hz • Multiple and single unit activities in the high frequency range: >500Hz 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 37 Upper trace: local field potential, filtered 0.3-50Hz(scale: 1024 µV) Lower trace: Multiunit activity, filtered 500-5000Hz(scale: 32 µV) Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings EXTRACTING INFORMATION FROM RECORDED SIGNAL Spike sorting • Basicprinciple of spike sorting: the exact recorded waveform depends on the relative position of the electrode and the surrounding cells thus each cell firing will have a different waveform on each electrode • This information can be used to sort the different recorded waveforms in order to isolate the different cells that produced these waveforms • The spike waveform has typical parameters that help in sorting: e. g. peak-to-peak amplitude, width • Sophisticated mathematical methods to find the best sorting parameters 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 38 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings STEPS OF SPIKE SORTING • Filtering data: application of an e. g. high pass filter to get rid of low frequency field potential signals • Spike detection: setting a threshold to separate spikes from noise activity. Threshold has to be chosen carefully to avoid both false positive (detecting noise as spike) and false negative (detecting spike as noise) decisions • Spike storage • Peak alignment: spike peaks have to be aligned to find best sorting parameters • Spike waveform parameterization: finding best spike parameters for sorting, called feature extraction. Traditionally, peak-to-peak amplitude and other waveform parameters were used. Now, mathematical methods, such as principal component analysis (PCA) and wavelet transformation, with better performance are in use • Clustering: grouping of spikes based on feature extraction • Classification check: checking refractory period –no spikes should occur during refractory period 2011.10.07.. TÁMOP–4.1.2-08/2/A/KMR-2009-0006 39 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings rawfiltered.PNG 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 40 FILTERING DATA Uppertrace: raw, widebanddata, includinglocal fieldpotentialand mulitunitsignals Lowertrace: highpassfiltered data, showingmultiunitfiringsignals Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings SPIKE DETECTION • Manually set threshold for spike detection, section of a 500Hz high pass filtered recording • Threshold set quite low, thus detection will contain few false negative (detecting spike as noise) but more false positive (detecting noise as spike) errors • Threshold also can be set automatically: usually a multiple (3-5 times) of the standard deviation of the signal. However, high firing rate and spike amplitude can rise this way the threshold undesirably high. Therefore, refined methods using signal median value can be used (see QuianQuirogaet al 2004) 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 41 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings FEATURE EXTRACTION • Feature extraction: finding best spike properties to perform spike sorting based on these properties • Traditionally: apparent spike parameters, such as peak-to-peak amplitude, width and energy (square of the signal) were used • These characteristics however proved to be non-optimal for spike sorting • Now, the most used method is principle component analysis (PCA). This method selects the 2 or 3 most characterising (principal) components of the spike vectors, along the maximum variance of the data. However, this is not necessarily the direction of best separation • To overcome this, wavelet transformation can be used. Wavelet transformation is a time-frequency decomposition of the signal. It’s advantage is that very localized shape differences can be discerned because wavelet coefficients are localized in time 2011.10.07.. TÁMOP–4.1.2-08/2/A/KMR-2009-0006 42 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings • Grouping of spikes based on the extracted features • Traditionally done manually. Manual clustering however introduces errors and is very subjective • Automatized methods, based on Bayesian decision can be used • Mostly, classification version of expectation-maximization method is used 2011.10.07.. TÁMOP–4.1.2-08/2/A/KMR-2009-0006 43 Clustered spike waveforms CLUSTERING clusters.jpg B Dombovári, K Seidl, S Herwik, T Torfs, L Grand, R Csercsa, O Paul, HP Neves, P Ruther and I Ulbert(2011). Electrophysiological recordings with active microprobe arrays. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings CLASSIFICATION CHECK • To check classification, checking refractory period is an easy tool • Refractory period can visualized by the autocorrelogram of sorted cell firing 2011.10.07.. TÁMOP–4.1.2-08/2/A/KMR-2009-0006 44 E:\ITK\elfiz\kepek\diploma_kepek\HunSi_Nr5_11_ch20_l_xcorr.jpg Autocorrelogram of sorted cell firing. The middle of diagram around zero contains no firing according to cell refractory period. A badly sorted cell autocorrelogram would also contain firing around zero, showing no refractory period because of diagram showing firing of multiple cells. Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings IN VITRO RECORDING TECHNIQUES • Recording from brain slices • Slice preparation steps:• Removing brain tissue • Cutting tissue with vibratomewhile kept in modified artificial cerebrospinal fluid • Typical slice thickness: 350-500 µm • Slices put into a submerged or an interface chamber • Maintaining artificial cerebrospinal fluid (ACSF) flow through chamber • Oxygen level and temperature kept constant in chamber 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 45 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings IN VITRO RECORDING CHAMBERS Interface chamber 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 46 Vibration-free table Extracellular laminar multielectrode Interface chamber Intracellular sharp electrode Microscope Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings IN VITRO RECORDING CHAMBERS Interface chamber 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 47 ACSF in Reference electrode Brain slices ACSF out Thermometer Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings IN VITRO RECORDING CHAMBERS Submerged chamber 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 48 Vibration-free table Submerged chamber Intracellular patch electrode Microscope Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 49 IN VITRO RECORDING CHAMBERS Submerged chamber Microscope ACSF out Brain slices Intracellular patch electrode ACSF in Thermostat (set to 32-35 °C) Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings IN VITRO RECORDING TECHNIQUES • Schematic of in vitro recording arrangement 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 50 invitroschematic Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings REVIEW QUESTIONS • What is the difference between intracellular and extracellular recording? • What are the advantages and disadvantages of these techniques? • What types of electrodes can be used for these techniques? • What is the difference between a sharp microelectrode and a patch-clamp electrode? • What are the different methods of patch-clamp recording? • What is current clamp and what can be measured with it? • What is voltage clamp and what can be measured with it? • What are the different types of extracellular electrodes? • What is a tetrode? • What is spike sorting? 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 51 Electrophysiological Methods of the Study of the Nervous-and Muscular System: Methods of Intra and Extracellular Microrecordings REFERENCES J. A. Stamford (ed.), Monitoring Neuronal Activity, Oxford University Press, 1992 P. Michael Conn (ed.), Electrophysiology and Microinjection, Academic Press, 1991 F. Bretschneider, J. R. de Weille, Introduction to Electrophysiological Methods and Instrumentation, Elsevier, 2006 E. R. Kandel, J. Schwartz, T. Jessell(eds.), Principles of Neural Science, 4thed., Elsevier, 2000 Squire, L.R., Bloom, F.E., McConnell, S.K., Roberts, J.L., Spitzer, N.C., Zigmond, M.J.: FundamentalNeuroscience, 2nd. ed. AcademicPress, 2003. G. Buzsáki, Large-scale recording of neuronal ensembles, Nature neuroscience, Vol. 7, No. 5. (May 2004), pp. 446-451 MalmivuoJ., Plonsey, R.: Bioelectromagnetism, http://www.bem.fi/book/index.htm1995 http://life.nthu.edu.tw/~g864264/Neuroscience/min/Voltage.html http://www.neuronexus.com http://www.thomasrecording.com http://www.ibiscanada.com/THOMAS_page.html 2011.10.07.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 52