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    Electrophysiological Recording Techniques: Goal of Physiological Recording

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    xixi1y
    This document summarizes various electrophysiological recording techniques used to detect communication signals between neurons in real time. It discusses current and voltage clamp techniques used to measure membrane potential, currents, and conductances. It also describes the instruments, electrodes, and methods used to obtain intracellular recordings from neurons, including obtaining a gigaseal and issues that can arise during recordings.

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    Electrophysiological Recording Techniques: Goal of Physiological Recording

    Uploaded by

    xixi1y
    46 views19 pages
    This document summarizes various electrophysiological recording techniques used to detect communication signals between neurons in real time. It discusses current and voltage clamp techniques used to measure membrane potential, currents, and conductances. It also describes the instruments, electrodes, and methods used to obtain intracellular recordings from neurons, including obtaining a gigaseal and issues that can arise during recordings.

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    Recording Techniques

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    This document summarizes various electrophysiological recording techniques used to detect communication signals between neurons in real time. It discusses current and voltage clamp techniques used to measure membrane potential, currents, and conductances. It also describes the instruments, electrodes, and methods used to obtain intracellular recordings from neurons, including obtaining a gigaseal and issues that can arise during recordings.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    46 views19 pages

    Electrophysiological Recording Techniques: Goal of Physiological Recording

    Uploaded by

    xixi1y
    This document summarizes various electrophysiological recording techniques used to detect communication signals between neurons in real time. It discusses current and voltage clamp techniques used to measure membrane potential, currents, and conductances. It also describes the instruments, electrodes, and methods used to obtain intracellular recordings from neurons, including obtaining a gigaseal and issues that can arise during recordings.

    Copyright:

    Attribution Non-Commercial (BY-NC)
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    of 19

    Electrophysiological Recording Techniques

    Wen-Jun Gao, PH.D.


    Drexel University College of Medicine

    Goal of Physiological Recording To detect the communication signals between neurons in real time (s to hours) Current clamp measure membrane potential, PSPs, action potentials, resting membrane potential Voltage clamp measure membrane current, PSCs, voltage-ligand activated conductances

    Current is conserved at a branch point

    A Typical Electrical Circuit

    Example of an electrical circuit with various parts. Current always flows in a complete circuit.

    Resistors and Conductors


    Summation of Conductance: Conductances in parallel summate together, whether they are resistors or channels.

    Ohm's Law
    For electrophysiology, perhaps the most important law of electricity is Ohm's law. The potential difference between two points linked by a current path with a conductance G and a current I is:

    Representative Voltmeter with Infinite Resistance

    Instruments used to measure potentials must have a very high input resistance Rin.

    Capacitors and Their Electrical Fields


    A charge Q is stored in a capacitor of value C held at a potential DeltaV. Q = C* delta V

    capacitance

    Capacitors in Parallel Add Their Values

    Currents Through Capacitors

    Membrane Behavior Compared with an Electrical Current


    A A membrane behaves electrically like a capacitance in parallel with a resistance.

    B Now, if we apply a pulse of current to the circuit, the current first charges up the capacitance, then changes the voltage
    Response of an RC parallel circuit to a step of current

    The voltage V(t) approaches steady state along an exponential time course:

    The steady-state value Vinf (also called the infinite-time or equilibrium value) does not depend on the capacitance; it is simply determined by the current I and the membrane resistance R:

    This is just Ohm's law, of course; but when the membrane capacitance is in the circuit, the voltage is not reached immediately. Instead, it is approached with the time constant t, given by

    Thus, the charging time constant increases when either the membrane capacitance or the resistance increases. Consequently, large cells, such as Xenopus oocytes that are frequently used for expression of genes encoding ion-channel proteins, have a long charging phase.

    Electrodes
    In circuits, we use wires In biology, nature uses liquids Electrodes are used to transform current flow from electrons to ions

    Silver/Silver Chloride Electrode - AgCl Coating


    Copper wire silver wire Electron (e-) flow

    Cl-

    Ag

    AgCl coating

    Electrode reaction: Ag + ClAgCl + electron (e-) The silver/silver chloride electrode is reversible but exhaustible

    Several points to remember about Ag/AgCl electrodes


    (1) The Ag/AgCl electrode performs well only in solutions containing chloride ions; (2) Because current must flow in a complete circuit, two electrodes are needed. If the two electrodes face different Cl- concentrations (for instance, 3 M KCl inside a micropipette* and 120 mM NaCl in a bathing solution surrounding the cell), there will be a difference in the half-cell potentials (the potential difference between the solution and the electrode) at the two electrodes, resulting in a large steady potential difference in the two wires attached to the electrodes. This steady potential difference, termed liquid junction potential, can be subtracted electronically and poses few problems as long as the electrode is used within its reversible limits; (3) If the AgCl is exhausted by the current flow, bare silver could come in contact with the solution. Silver ions leaking from the wire can poison many proteins. Also, the half-cell potentials now become dominated by unpredictable, poorly reversible surface reactions due to other ions in the solution and trace impurities in the silver, causing electrode polarization.

    Platinum Electrode

    A platinum electrode is irreversible but inexhaustible.

    Electrodes and Pipettes


    Reversible electrode silver wire coated with Ag and AgCl forward flow: electrons from wire convert AgCl to Ag atoms and Cl- ions, the Cl- become hydrated and enter solution reverse flow: Ag atoms give up electron and combine with Cl- from solution solution must contain Cl okay for some silver to be exposed if AgCl exhausted, Ag will leak into solution and poison cells Glass micropipettes

    Pipette is critical for recording


    Fabrication - pullers (multi-stage pullers Sutter p-97) - glass with filament - tip size and shape (resistance) - fire polishing ? - sylgard? (bath solution level, dental wax or grease) Intracellular solutions - Osmolarity, pH value, blockers, dyes - clean and no contamination, filtered with Sterile Membrane Filter (0.45 M)

    Getting a recording
    1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Find a health cell Fill a pipette, place in holder and apply positive pressure Put pipette in the bath Get the test pulse running and make sure it works Zero the offset Bridget balance the pipette Position the pipette above the cell Verify positive pressure and advance into the slice Push pipette tip onto cell surface, a small dimple would appear, and then release pressure Apply slight suction and a negative holding potential (-60 mV) If everything is right, you will get a gigaohm seal

    Intracellular solutions
    Standard intracellular (patch) solution: 135 K-Gluconate or K-MeSO4, 10 HEPES, 7 NaCl, 2 Na2-ATP, 2 MgCl2 (pH 7.2 with KOH) Osmolarity - ~10% lower than extracellular (higher is better for lower Rs) Could also add/replace: - GTP (0.3 mM) - Phosphocreatine (10 mM) - Fluorescence dyes or biocytin - Cs instead of Gluconate/MeSO4 to block K+ currents - High Cl to increase inhibitory responses at resting membrane potentials (eg. replace K-Gluconate with KCl)

    Patch clamp setup

    10

    See http://www.olympusmicro.com/primer/techniques/dic/dicconfiguration.html

    Steps to gigaseal

    11

    Surface cleaning is critical for successful recording Use of a cleaning pipette to remove tissue overlying a cell in tissue slices.

    Current Clamp and Voltage Clamp


    In a current-clamp experiment, one applies a known constant or time-varying current and measures the change in membrane potential caused by the applied current. This type of experiment mimics the current produced by a synaptic input. In a voltage-clamp experiment one controls the membrane voltage and measures the transmembrane current required to maintain that voltage.

    12

    Why record with voltage clamp


    Despite the fact that voltage clamp does not mimic a process found in nature, there are three reasons to do such an experiment: (1) Clamping the voltage eliminates the capacitive current, except for a brief time following a step to a new voltage. The brevity of the capacitive current depends on many factors that are discussed in following chapters; (2) Except for the brief charging time, the currents that flow are proportional only to the membrane conductance, i.e., to the number of open channels; (3) If channel gating is determined by the transmembrane voltage alone (and is insensitive to other parameters such as the current and the history of the voltage), voltage clamp offers control over the key variable that determines the opening and closing of ion channels.

    Record Voltage
    Positive potential - a positive voltage at the headstage input with respect to system ground Transmembrane potential, Vm - Vinside relative to Voutside Depolarizing potential - is a positive shift in Vm

    13

    Record Current
    Positive current - flows out of amplifier into the electrode - flows out of the pipette tip into the cell Inward current - flows from the outside surface of membrane to the inside surface Positive and Negative currents and voltages are always based on the headstages perspective

    Typical Voltage-Clamp Experiment

    14

    Glass Microelectrodes and Tight Seals Intracellular Electrode Measurement


    This intracellular electrode is measuring the resting potential of a cell whose membrane contains only open K+ channels. As the seal resistance Rs increases, the measurement approaches the value of EK.

    15

    Good and Bad Seals in single channel recording

    In a patch recording, currents through the seal also flow through the measuring circuit, increasing the noise on the measured current.

    Digitizing Analog Signal


    8 bit = 28 = 256 12 bit = 212 = 4,096 16 bit = 216 = 65,536 8 bit = 278.4 mV 12 bit = 4.88 mV 16 bit = 0.305 mV

    16

    Technical issues
    1. Loosing the seal on break-in (leaky cell; depolarized membrane potential, such as negative (downward) shift, jump in holding current, Series resistance (VC/CC, filtering, voltage-drop/error, compensation), Noise (60 Hz line frequency, grounding, high-frequency noise single channel), Wash-in/wash-out (perforated patch recording: Amphotericin or Gramicidin), Offsets (junction potentials, Ag/AgCl electrodes), Recording issues (amplifier gain, Analog/Digital boards, saturation, sample rates), Space clamp.

    2. 3. 4. 5. 6. 7.

    Visually guided vs. blind?


    Visually guided Expensive Cheaper Blind

    Expensive microscope & camera required, better manipulators (?) Best for cells near the surface Typically lower series resistance Can record from multiple cells Can record from multiple locations on the same cell Can identify cells before recording (morphology, fluorescence)

    Only need dissecting microscope & no camera Can patch deep cells (also in vivo) Typically higher series resistance Difficult to record from multiple cells

    Impossible (?) to record from multiple locations on the same cell Identification only possible after experiment (but can use electrical cues)

    17

    Data analysis
    Phases of the Action Potential

    Relative Absolute refractory period refractory period

    Firing threshold is the point at which the number of activated Na+ channels > inactivated Na+ channels

    Ion Flow During an Action Potential

    18

    Input resistance and time constant decay

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