1. Biophysics 6702 Patch Clamp Techniques Stuart Mangel, Ph.D.
LEARNING OBJECTIVES
1. Overall objectives
Principles that underlie different electrical recording techniques
Physiological and biophysical information the techniques provide
Experimental advantages and limitations of the techniques
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

2. EXTRACELLULAR VS. INTRACELLULAR RECORDING
Extracellularly and intracellularly recorded voltages are
in the microvolt and millivolt ranges, respectively.

3. Maintaining the resting membrane potential
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
Vm = ln RT F
pK[K+]o + pNa[Na+]o + pCl[Cl-]i
pK[K+]i + pNa[Na+]i + pCl[Cl-]o
extracellular
intracellular
ENa = +56
Na+ (150)
EK = -102
K+ (3)
ECl = -76
Cl- (120)
ECa = +125
Ca2+ (1.2)
Na+ (18)
K+ (135)
Cl- (7)
Ca2+ (0.1 µM)
Na+,K+-ATPase
-60 to -75 mV
NSCC

4. INTRACELLULAR RECORDING
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

5. Intracellular Recording
When a fine-tipped electrode penetrates the membrane of a cell, the measured potential suddenly changes to a more negative value.
One can calculate the input resistance of the cell using Ohm’s Law (V = IR); i.e., measure V, and because I is known, calculate R (= V/I)
Typical problems:
High impedance (μE)
Cell damaged when penetrated

6. 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 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.

7. Patch clamp recording configurations
Electrode
Glass pipette
Ion channel
Plasma
membrane
Cell-attached
Whole-cell
Perforated-patch
antibiotics
suction
pull
pull
Inside-out
Outside-out

8. SUMMARY OF ADVANTAGES AND DISADVANTAGES OF PATCH CLAMP CONFIGURATIONS

9. THE VOLTAGE CLAMP

10. THE ACTION POTENTIAL

11. Voltage clamping reveals the ionic currents
that underlie the action potentials observed in squid axons

12. Activation and Inactivation Properties
Ionic Selectivity

13. Reversal potentials for synaptic currents

14. Inhibitory actions of GABA synapses result from the opening of ion channels selective for Cl-

15. SODIUM CHANNEL CURRENTS RECORDED FROM CELL-ATTACHED PATCH

16. Properties of ACh-gated channels

17. Single open ACh-gated channels behave as simple resistors.

18. Extracellular Mg2+ ions block NMDA channels under physiological conditions.

19. SUMMARY OF ADVANTAGES AND DISADVANTAGES OF PATCH CLAMP CONFIGURATIONS

20. Biophysics 6702 Patch Clamp Techniques Stuart Mangel, Ph.D.
DISCUSSION TOPICS
1. Name some experimental advantages and limitations of the techniques you have used. Which characteristics of the techniques made them experimentally advantageous or limited?
2. Name some experimental advantages and limitations of commonly used techniques in biophysics research. Which characteristics of the techniques made them experimentally advantageous or limited?
3. Are there common characteristics of experimental techniques that limit their usefulness in diverse areas of research? How can the reliability of the data a technique produces be established in spite of its limitations?

21. Questions:
Stuart Mangel, Ph.D.
Professor
Department of Neuroscience
The Ohio State University
College of Medicine
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