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 Medicine614-292-5753mangel.1@osu.edu

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