learning objectives 1. overall objectives - principles that underlie different electrical recording...
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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.
EXTRACELLULAR VS. INTRACELLULAR RECORDING
Extracellularly and intracellularly recorded voltages are in the microvolt and millivolt ranges, respectively.
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
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
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
Wheatstone Bridge
• Used to measure an unknown resistance
• Discovered by Hunter Christie, 1833
• Popularized by Charles Wheatstone
MEASURING THE INPUT RESISTANCE
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
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?
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.
Patch clamp recording configurations
Electrode
Glass pipette
Ion channel
Plasma membrane
Cell-attached
Inside-out Outside-out
Whole-cellsuction
pull pull
Perforated-patch antibiotics
SUMMARY OF ADVANTAGES AND DISADVANTAGES OF PATCH CLAMP CONFIGURATIONS
THE VOLTAGE CLAMP
THE ACTION POTENTIAL
Voltage clamping reveals the ionic currents that underlie the action potentials observed in squid axons
Activation and Inactivation PropertiesIonic Selectivity
Reversal potentials for synaptic currents
Inhibitory actions of GABA synapses result from the opening of ion channels
selective for Cl-
SODIUM CHANNEL CURRENTS RECORDED FROM CELL-ATTACHED PATCH
Properties of ACh-gated channels
Single open ACh-gated channels behave as simple resistors.
Extracellular Mg2+ ions block NMDA channels under physiological conditions.
SUMMARY OF ADVANTAGES AND DISADVANTAGES OF PATCH CLAMP CONFIGURATIONS
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