ELECTROPHYSIOLOGICAL METHODS OF THE STUDY OF THE NERVOUS- AND MUSCULAR

Teljes szövegt

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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.

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ELECTROPHYSIOLOGICAL METHODS OF THE STUDY OF THE NERVOUS- AND MUSCULAR

SYSTEM

LECTURE 6

METHODS OF INTRA- AND EXTRACELLULAR MICRORECORDINGS

Az ideg- és izomrendszer elektrofiziológiai vizsgálómódszerei

(Intra- és extracelluláris mikroelvezetések módszerei )

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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

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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

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COMPARISON OF INTRACELLULAR AND

EXTRACELLULAR RECORDING ELECTRODES – ADVANTAGES AND DISADVANTAGES

Extracellular

• Technically easy

• Low signal amplitude (10-500µV)

• Low electrical noise amplifiers needed

• Multiple channel (10-200) recordings possible

• Available in freely moving 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 freely moving animals

• Records intracellular processes directly

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TYPES OF INTRACELLULAR AND EXTRACELLULAR RECORDING ELECTRODES

Extracellular

• Micropipette

• Single microwire

• Tetrode and microwire multielectrode

• Silicon-based multielectrodes

Intracellular

• Micropipette

• Sharp microelectrode

• Patch-clamp electrode

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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

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• Diameter of tip: 0.03-0.06µm

• Length of neck: 6-14mm

• Electrode impedance: 30-200MOhm

• Filled with: 2M potassium acetate (and Neurobiotin tracer)

ELECTRODES

Sharp microelectrode

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• Connected to preamplifier through a non-polarizable Ag/AgCl electrode because of DC recording

ELECTRODES

Sharp microelectrode

Sharp microelectrode with connecting electrode inside

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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

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• 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)

ELECTRODES

Patch-clamp electrode

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• Different methods of patch-clamp recording

ELECTRODES

Patch-clamp electrode

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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

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BASICS OF INTRACELLULAR RECORDINGS

• Schematic of intracellular recording arrangement

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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

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CURRENT CLAMP

Injecting current (Is), recording membrane potential (Vm)

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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

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CURRENT CLAMP IN PRACTICE

Experimental setup Replacement diagram

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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

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CAPACITANCE COMPENSATION

Compensation carried out outside the cell:

schematic and replacement diagram

Replacement diagram of compensation

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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

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Replacement diagram of compensation

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DC OFFSET COMPENSATION

Replacement diagram

Set Rdc to make Uout zero – remember, compensation is carried out outside the cell

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VOLTAGE CLAMP

Membrane voltage (Vm) kept constant, measuring injected current (Is)

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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

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VOLTAGE CLAMP

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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

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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

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ELECTRODES

Single microwire and microwire array

http://www.thomasrecording.com/en/cms/upload/document/singleelectrodes.pdf http://commons.wikimedia.org/wiki/File:16wire_electrode_array.jpg

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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

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ELECTRODES

Tetrode

http://www.ibiscanada.com/THOMAS_page.html

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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

Microwires Insulator coating

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• 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

ELECTRODES

Silicon-based multielectrodes

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ELECTRODES

Silicon-based multielectrodes

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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

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PROBLEMS OF EXTRACELLULAR CELL ACTIVITY DETECTION

G. Buzsáki, Large-scale recording of neuronal ensembles, Nature neuroscience, Vol. 7, No. 5. (May 2004), pp. 446-451

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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 0.3-50 Hz (scale: 1024 μV) Lower trace: Multiunit activity, filtered 500-5000 Hz (scale: 32 μV)

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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

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• 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

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FILTERING DATA

Upper trace: raw, wideband data, including local field potential and mulitunit signals

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• 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)

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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

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• 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

CLUSTERING

B Dombovári, K Seidl, S Herwik, T Torfs, L Grand, R Csercsa, O Paul, HP Neves, P Ruther and I Ulbert (2011). Electrophysiological recordings

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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,

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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: 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

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Interface chamber

Microscope

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Interface chamber

ACSF in Reference electrode Brain slices ACSF out

Thermometer

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Submerged chamber

Microscope

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Submerged chamber

Microscope

ACSF out

Brain slices Intracellular patch electrode ACSF in

Thermostat (set to 32-35 °C)

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IN VITRO RECORDING TECHNIQUES

• Schematic of in vitro recording arrangement

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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?

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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, 4th 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, 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.htm 1995 http://life.nthu.edu.tw/~g864264/Neuroscience/min/Voltage.html

http://www.neuronexus.com

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