PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

Transcription

1 PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY 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 TÁMOP /2/A/KMR

2 Peter Pazmany Catholic University Faculty of Information Technology ELECTROPHYSIOLOGICAL METHODS OF THE STUDY OF THE NERVOUS- AND MUSCULAR SYSTEM Az ideg- és izomrendszer elektrofiziológiai vizsgálómódszerei LECTURE 6 METHODS OF INTRA- AND EXTRACELLULAR MICRORECORDINGS (Intra- és extracelluláris mikroelvezetések módszerei ) DOMONKOS HORVÁTH, GYÖRGY KARMOS TÁMOP /2/A/KMR

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 TÁMOP /2/A/KMR

4 COMPARISON OF INTRACELLULAR AND EXTRACELLULAR RECORDING TECHNIQUES 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 TÁMOP /2/A/KMR

5 COMPARISON OF INTRACELLULAR AND EXTRACELLULAR RECORDING ELECTRODES ADVANTAGES AND DISADVANTAGES Extracellular Intracellular Technically easy Technically complicated Low signal amplitude (10-500µV) High signal amplitude (1-100mV) Low electrical noise amplifiers needed Low electrical noise amplifiers needed Multiple channel (10-200) recordings Only few (1-4) channel recordings possible possible (with separate electrodes) Available in freely moving animals Unavailable in freely moving animals Unable to record intracellular Records intracellular processes processes directly directly TÁMOP /2/A/KMR

6 TYPES OF INTRACELLULAR AND EXTRACELLULAR RECORDING ELECTRODES Extracellular Intracellular Micropipette Single microwire Tetrode and microwire multielectrode Silicon-based multielectrodes Micropipette Sharp microelectrode Patch-clamp electrode TÁMOP /2/A/KMR

7 TYPES OF INTRACELLULAR RECORDING ELECTRODES 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 TÁMOP /2/A/KMR

8 TYPES OF INTRACELLULAR RECORDING ELECTRODES Sharp microelectrode Diameter of tip: µm Length of neck: 6-14mm Electrode impedance: MOhm Filled with: 2M potassium acetate (and Neurobiotin tracer) TÁMOP /2/A/KMR

9 TYPES OF INTRACELLULAR RECORDING ELECTRODES Sharp microelectrode Connected to preamplifier through a non-polarizable Ag/AgCl electrode because of DC recording Sharp microelectrode with connecting electrode inside TÁMOP /2/A/KMR

10 TYPES OF INTRACELLULAR RECORDING ELECTRODES 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 ideal for 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 TÁMOP /2/A/KMR

11 TYPES OF INTRACELLULAR RECORDING ELECTRODES Patch-clamp electrode 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 Neurobiotin tracer) TÁMOP /2/A/KMR

12 TYPES OF INTRACELLULAR RECORDING ELECTRODES Patch-clamp electrode Different methods of patch-clamp recording J. Malmuvio and R. Plonsey, Bioelectromagnetism: Principles and Applications of Bioelectric and Biomagnetic Fields. New York: Oxford University Press, TÁMOP /2/A/KMR

13 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 TÁMOP /2/A/KMR

14 BASICS OF INTRACELLULAR RECORDINGS Schematic of intracellular recording arrangement TÁMOP /2/A/KMR

15 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 TÁMOP /2/A/KMR

16 CURRENT CLAMP Injecting current (Is), recording membrane potential (Vm) TÁMOP /2/A/KMR

17 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 Current clamp circuit TÁMOP /2/A/KMR

18 CURRENT CLAMP IN PRACTICE Experimental setup Replacement diagram TÁMOP /2/A/KMR

19 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 TÁMOP /2/A/KMR

20 CAPACITANCE COMPENSATION Compensation carried out outside the cell: schematic and replacement diagram Replacement diagram of compensation TÁMOP /2/A/KMR

21 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 ohmic voltage drop across microelectrode after current step eliminated Origin of name: originally subtraction was achieved by Wheatstone bridge, now by operational amplifier circuits TÁMOP /2/A/KMR

22 SERIAL RESISTANCE COMPENSATION Replacement diagram of compensation Rbb varied until voltage step from recorded signal (U out ) eliminated TÁMOP /2/A/KMR

23 DC OFFSET COMPENSATION Replacement diagram Set Rdc to make U out zero remember, compensation is carried out outside the cell TÁMOP /2/A/KMR

24 VOLTAGE CLAMP Membrane voltage (Vm) kept constant, measuring injected current (Is) TÁMOP /2/A/KMR

25 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 Voltage clamp circuit TÁMOP /2/A/KMR

26 VOLTAGE CLAMP Dale Purves,1997,Neuroscience,Sinauer Associates, Inc., P TÁMOP /2/A/KMR

27 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 TÁMOP /2/A/KMR

28 TYPES OF EXTRACELLULAR RECORDING ELECTRODES Single microwire and microwire array 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 microwires built to record more cells simultaneously Precise location of each electrode of the array in the brain cannot be determined TÁMOP /2/A/KMR

29 TYPES OF EXTRACELLULAR RECORDING ELECTRODES Single microwire and microwire array TÁMOP /2/A/KMR

30 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 TÁMOP /2/A/KMR

31 TYPES OF EXTRACELLULAR RECORDING ELECTRODES Tetrode TÁMOP /2/A/KMR

32 TYPES OF EXTRACELLULAR RECORDING ELECTRODES Microwire multielectrode Many (more than 10) microwires in 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 Insulator coating Microwires (24 in total) Electrode tip TÁMOP /2/A/KMR

33 TYPES OF EXTRACELLULAR RECORDING ELECTRODES Silicon-based multielectrodes Metal electrode contacts in silicon substrate Allows precise electrode size and spacing design with high reproducibility Much higher electrode count than in metal microwire arrays 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 TÁMOP /2/A/KMR

34 TYPES OF EXTRACELLULAR RECORDING ELECTRODES Silicon-based multielectrodes P. J. Rousche and R. A. Normann, "Chronic recording capability of the Utah Intracortical Electrode Array in cat sensory cortex," J Neurosci Methods, vol. 82, pp. 1-15, L. Grand, Development, testing and application of laminar multielectrodes and biocompatible coatings for intracortical applications, PhD dissertation, TÁMOP /2/A/KMR

35 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 TÁMOP /2/A/KMR

36 PROBLEMS OF EXTRACELLULAR CELL ACTIVITY DETECTION G. Buzsáki, Large-scale recording of neuronal ensembles, Nature neuroscience, Vol. 7, No. 5. (May 2004), pp 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 TÁMOP /2/A/KMR

37 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 Upper trace: local field potential, filtered Hz (scale: 1024 μv) Lower trace: Multiunit activity, filtered Hz (scale: 32 μv) TÁMOP /2/A/KMR

38 EXTRACTING INFORMATION FROM RECORDED SIGNAL Spike sorting Basic principle 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 TÁMOP /2/A/KMR

39 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 TÁMOP /2/A/KMR

40 FILTERING DATA Upper trace: raw, wideband data, including local field potential and mulitunit signals Lower trace: high pass filtered data, showing multiunit firing signals TÁMOP /2/A/KMR

41 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 Quian Quiroga et al 2004) TÁMOP /2/A/KMR

42 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 TÁMOP /2/A/KMR

43 CLUSTERING 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 Clustered spike waveforms 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) TÁMOP /2/A/KMR

44 CLASSIFICATION CHECK To check classification, checking refractory period is an easy tool Refractory period can visualized by the autocorrelogram of sorted cell firing 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 TÁMOP /2/A/KMR

45 IN VITRO RECORDING TECHNIQUES Recording from brain slices Slice preparation steps: Removing brain tissue Cutting tissue with vibratome while kept in modified artificial cerebrospinal fluid Typical slice thickness: 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 TÁMOP /2/A/KMR

46 IN VITRO RECORDING CHAMBERS Interface chamber Microscope Vibration-free table Extracellular laminar multielectrode Interface chamber Intracellular sharp electrode TÁMOP /2/A/KMR

47 IN VITRO RECORDING CHAMBERS Interface chamber Thermometer ACSF in Reference electrode Brain slices ACSF out TÁMOP /2/A/KMR

48 IN VITRO RECORDING CHAMBERS Submerged chamber Microscope Vibration-free table Submerged chamber Intracellular patch electrode TÁMOP /2/A/KMR

49 IN VITRO RECORDING CHAMBERS Submerged chamber Microscope ACSF out Thermostat (set to C) Brain slices Intracellular patch electrode ACSF in TÁMOP /2/A/KMR

50 IN VITRO RECORDING TECHNIQUES Schematic of in vitro recording arrangement TÁMOP /2/A/KMR

51 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? TÁMOP /2/A/KMR

52 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, 4 th ed., Elsevier, 2000 Squire, L.R., Bloom, F.E., McConnell, S.K., Roberts, J.L., Spitzer, N.C., Zigmond, M.J.: Fundamental Neuroscience, 2nd. ed. Academic Press, G. Buzsáki, Large-scale recording of neuronal ensembles, Nature neuroscience, Vol. 7, No. 5. (May 2004), pp MalmivuoJ., Plonsey, R.: Bioelectromagnetism, TÁMOP /2/A/KMR