Electrophysiological Techniques

Question 1

How does the Na/K transporter generate electrical activity as a consequence of controlling cell volume?

Answer 1

• we have a lot of protein inside cells but relatively little outside, therefore water wants to move into the cell - cause lysis
• however the Na/K pump moves 3Na out and brings 2K in, reducing the osmotic pressure.
• leads to the creation of electrochemical gradients for Na and K

Question 2

Electrical activity generated by flow/separation of charge

Answer 2

• At rest, K leak channels allow K to leave the cell down the conc gradient
• will flow out until stopped by the -ve charge prevents it
• Gives the RMP
• At rest at 70mV there will be no movement of K+ ions

Question 3

What is Ohms law?

Answer 3

V=IR
V = voltage - potential difference between two points
I = Current between two points
R = resistance to current flow between two points

Question 4

How else can we describe resistance?

Answer 4

The reciprocal of conductnace

• R= 1/G
• when conductance is high, you have low resistance
• I = GV

Question 5

How does Ohms law relate to cells?

Answer 5

I = G V

• V = energy gradient produced by electrochemical gradients
• G = membrane is permeable to ions
• I = flow of charge produced my ion movement

Question 6

What are the 3 main microelectrode techniques?

Answer 6

• EC recording
• IC recording
• Voltage-clamp recording

Question 7

Extracellular recording

Answer 7

• two microelectrodes placed outside very close to surface
• records cell-induced membrane potential between electrodes
• Single cells = single-unit recording
• group of cells = multi-unit recording

Question 8

Uses of EC recording

Answer 8

• record electrical activity in anaesthetised/conscious animals
• Hubel and Wiesel - Nobel prize 1981 - showed specific neurons in visual cortex responded to different visual stimuli
• mapped out visual cortex

Question 9

Intracellular recording

Answer 9

• one microelectrode placed inside, other outside
• records membrane potential across membrane of single cell
• allows manipulation of external solutions (apply drugs etc)
• doesnt alter IC composition of cells

Question 10

Uses of IC recording

Answer 10

• record electrical responses to external stimuli (e.g. NTs)
• measure conductance changes from opening/closing of ion channels
• Can do in vivo, but hard as electrodes are tiny
• usually do in vitro - allows you to put drugs in bathing solution
• change in membrane is caused by current and conductance
• addition of glutamate will cause increase in conductance as ion channels open

Question 11

Voltage-clamp recording

Answer 11

• one microelectrode measures voltage (clamped)
• one applies current to and from cell to keep voltage at clamped level
• uses clever feedback amplifier - measure V and I at the same time
• measures membrane current (will change with voltage)
• The current that is needed to be applied to maintain the right voltage is equivalent to the current that is flowing across the membrane

Question 12

E.g. of Voltage clamping

Answer 12

• Step a nerve cell from -70mV to -20mV - open sodium channels
• cell wishes to depolarise to +50mV (ENaC)
• but amplifier provides current to clamp cell at -20mV
• Current applied - equal to current through opening Na channel

Question 13

Patch-clamp technique - Sakmann and Neher (1976)

Answer 13

• made a patch pipette electrode - blunt end on the outside of the cell - enables them to isolate a small area of membrane - can see the current activity moving through small patch of membrane
• use ACh on the patch pipette - bound to ligand gated channels, measure current produced when channels are opened

Question 14

Sakmann and Neher improvements

Answer 14

• Produced “Giga-Ohm seal” between glass patch pipette and cell membrane
• achieved by heat polishing the tip of the pipette and cleaning the cell membrane from debris, such as connective tissue
• the giga-ohm seal is important as it reduces background noise (can record very small currents) and increases the mechanical stability of patch

Question 15

What are the different configurations of the patch clamp technique?

Answer 15

• inside out - inside of the cell is in the bathing solution
• whole cell - can blow a hole through the membrane, don’t lose the cell, just record the electrical activity of the whole cell
• outside-out - can pull off the patch, breaking the membrane, giving you two pieces of bi-lipid membrane. Because they are in an electrolysed solution, they are going to want to get together and join up. Now have a small patch to record from - like a mini cell

Question 16

Cell-attached patch

Answer 16

• Agonist applied to pipette solution (ligand gated receptors)
• voltage steps applied through pipette (voltage gated channels)
• Agonists applied to EC solution (GPCRs)
• PROBLEMS - RMP cannot be controlled. Cannot control the composition of the IC medium

Question 17

Inside-out patch

Answer 17

• agonists can be applied directly to cytoplasmic surface of cell membrane (activation of 2nd messenger systems)
• can control both the composition of EC and IC media
• PROBLEMS - very reductionist approach - will the channels behave as they do in the body?

Question 18

Whole-cell recording

Answer 18

• agonists applied to pipette solution (2nd messenger)
• agonists applied to EC solution (ligand-gated/GPCR)
• Voltage steps appiled through pipette (VGCC)
• Control composition of both IC and EC media
• PROBLEMS - measure only macroscopic current - not individual channel openings. important IC components may dialysis out of cell