learning objectives 1. overall objectives - principles that underlie different electrical recording...
TRANSCRIPT
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LEARNING OBJECTIVES
1. Overall objectives- Principles that underlie different electrical recording techniques- Physiological and biophysical information the techniques provide
2. Extracellular recording and multi-electrode arrays - spiking (all-or-none) information, neural codes conveyed by individual
neurons and by groups of neurons
3. Intracellular recording - measurements of input resistance, synaptic input, and synaptic integration
4. Patch-clamp recording (cell-attached; whole-cell; inside-out patch; outside-out patch) - measurements of input resistance, synaptic input, synaptic integration;
characteristics of voltage-gated ion channels and single ion channel events
Biophysics 6702 Patch Clamp TechniquesStuart Mangel, Ph.D.
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EXTRACELLULAR VS. INTRACELLULAR RECORDING
Extracellularly and intracellularly recorded voltages are in the microvolt and millivolt ranges, respectively.
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Maintaining the resting membrane potential
Vm = lnRTF
pK[K+]o + pNa[Na+]o + pCl[Cl-]i
pK[K+]i + pNa[Na+]i + pCl[Cl-]o
The Goldman-Hodgkin-Katz (GHK) Equation:The steady state membrane potential for a given set of ionic concentrations inside and outside the cell and the relative permeability of the membrane to each ion
extracellular
intracellular
ENa = +56Na+ (150)
EK = -102K+ (3)
ECl = -76Cl- (120)
ECa = +125Ca2+ (1.2)
Na+ (18) K+ (135) Cl- (7) Ca2+ (0.1 µM)Na+,K+-ATPase
-60 to -75 mVNSCC
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Measuring EM
• Measure the potential difference between two electrodes using a D.C. amplifier
• Expected value of the membrane potential is in millivolts (not microvolts), so the gain does not need to be as high
INTRACELLULAR RECORDING
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Intracellular Recording
• When a fine-tipped electrode penetrates the membrane of a cell, one observes a sudden change in the measured potential to a more negative value.
• Typical problems– High impedance μE– Damage when cell
penetrated
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Wheatstone Bridge
• Used to measure an unknown resistance
• Discovered by Hunter Christie, 1833
• Popularized by Charles Wheatstone
MEASURING THE INPUT RESISTANCE
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BALANCING THE BRIDGE
• R1 = Fixed R• R2 = Variable R• R3 = Fixed R• R4 = Unknown R
?
To get R2/R1 = R4/R3,
adjust R2, so that there is
no current across B, CR4 = (R2/R1)·R3
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CALCULATING THE INPUT RESISTANCE OF A CELL
• Balance the bridge before entering the cell
• After impaling the cell,
the bridge is “out of balance” by the R value of the cell
• I is known, measure V, and calculate R using Ohm’s Law (V = IR)
• R = V/I
0 100 200 300 400 500-100
-80
-60
-40
-20
0
Mem
bran
e P
oten
tial (
mV
)
Time (Arbitrary Units)
APPLY DRUG
“Balanced” “Out of Balance”
Did R increase or decrease?
Did channels open or close?
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PATCH-CLAMP RECORDING
• Neher and Sakmann, Nobel Prize, 1991• Tremendous technical breakthrough that improved the signal
to noise ratio of electrical recordings• Record from whole cells or from a small patch of cell
membrane, so only a few ion channels (or one) can be studied• High resistance (in giga-ohms) and high mechanical strength
of the seal between the glass electrode and the cell membrane enable one to observe very small currents.
• The diameter of the tip of patch electrodes can be larger than that of fine-tipped intracellular microelectrodes (1.0 micron vs. 0.05 microns), so that the resistance of patch electrodes is lower (e.g. 5 MΩ vs 200 MΩ). The lower resistance of patch electrodes makes voltage clamping easier.
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Patch clamp recording configurations
Electrode
Glass pipette
Ion channel
Plasma membrane
Cell-attached
Inside-out Outside-out
Whole-cellsuction
pull pull
Perforated-patch antibiotics
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SUMMARY OF ADVANTAGES AND DISADVANTAGES OF PATCH CLAMP CONFIGURATIONS
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THE VOLTAGE CLAMP
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THE ACTION POTENTIAL
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Voltage clamping reveals the ionic currents that underlie the action potentials observed in squid axons
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Activation and Inactivation PropertiesIonic Selectivity
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Reversal potentials for synaptic currents
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Inhibitory actions of GABA synapses result from the opening of ion channels
selective for Cl-
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SODIUM CHANNEL CURRENTS RECORDED FROM CELL-ATTACHED PATCH
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Properties of ACh-gated channels
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Single open ACh-gated channels behave as simple resistors.
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Extracellular Mg2+ ions block NMDA channels under physiological conditions.
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SUMMARY OF ADVANTAGES AND DISADVANTAGES OF PATCH CLAMP CONFIGURATIONS
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Questions:
Stuart Mangel, Ph.D.ProfessorDepartment of Neuroscience The Ohio State UniversityCollege of [email protected]
Readings:Kandel, Schwartz et al., Principles of Neural Science, 2013, 5th Ed., Chap. 7, 9, 10
Squire, Berg et al., Fundamental Neuroscience, 2008, 3rd Ed., Chap. 6, 11