BSc BioSci / BSc/MSci Chem w Pharm Year 3 Dr Austen Spruce

Molecular/Integrative Pharmacology
Electrophysiological Methods

Outline - Revision

• Revision: Membrane potential (Vm ) – ion movement

via leak channels; equilibrium potential; driving force
Electrophysiological Methods • Revision: Action Potential – roles of voltage-gated
channels
• Revision: Synaptic physiology – neurotransmitters,
receptors, synaptic potentials
Dr Austen Spruce

Electrophysiological Methods Ion Conductances During Action Potential

Membrane Potential ENa
• Unequal ion distribution 30 +55
o Chemical and Electrical forces
VM
o Equilibrium Potential for ion (Eion)
20 gNa 0 (mV)
 Driving Force (measured in mV – difference between Voltage-gated
membrane voltage [Vm ] and Eion) VM channels
• Selective membrane permeability/conductance to ions gK
10
o Gated and non-gated (leak) ion channels

Action Potentials
Change in conductances (opening/closing of ion channels) -65

Voltage-gated sodium and potassium channels -80

Opened by depolarisation EK AHP
1-2 msec

Assessment of Ion Channel Properties Conductance vs. time

20
Voltage-gated K+ and Na+ channels
Conductance (g). E.g. for K+: -20
Current (can be
-60
measured)
+20
IK
gK  0 mV
Vm  EK  Driving Force on
ion gK -20
• Better measure than current of channel function (≡ No. of -40
open channels) +20
• Driving force variable eliminated 0 mV
• For voltage-gated channels, -20
gNa Adapted from Fig. 9-6.
g dependent on time and voltage -40 Principles of Neural Science

time
1 msec

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BSc BioSci / BSc/MSci Chem w Pharm Year 3 Dr Austen Spruce
Molecular/Integrative Pharmacology
Electrophysiological Methods

Synaptic Physiology
• Neurotransmitter acting on ligand-gated ion channels Advanced Electrophysiological Methods
leads to synaptic potential
• Example: excitatory transmission via entry of Na+ into
postsynaptic cell Outline
EPSP (excitatory post-synaptic • Contribution of channels other than voltage-gated
potential) Na+ and K+ channels to electrical activity – Calcium-
• Many receptor types! activated potassium channel
Fig 5.15. Neuroscience: Exploring the
• Methods used to study channel behaviour and/or
Brain. 3rd Ed. electrical activity:
– Voltage clamp (technical variant – patch clamp)
– Current clamp
– Extracellular recording

Ion Conductances During Action Potential Slow After-Hyperpolarisation (AHP)

Role of Ca2+-activated K+ channels
ENa
30 +55
• In some neurones, Action Potentials (APs) have much
VM longer AHPs

20 gNa 0 (mV) • Initial AHP (1 ms) – delayed closing of V-gated K+

Voltage-gated channels
VM channels
gK • Larger, longer AHP (10’s of ms) - Ca2+-activated K+
10 channels (current is abbreviated to: IK,Ca)
– K+ channel opened by binding of intracellular Ca2+
-65 • Source of Ca2+? (since, resting [Ca2+]i too low)

-80 – Voltage-gated Ca2+ channels

EK AHP
1-2 msec

Physiological Contribution of Ca2+-Activated

Ca2+-Activated K+ Current Generates Slow
K+ Current (IK,Ca)
Afterhyperpolarisation (AHP)
• During Action Potential (AP), voltage-gated Ca2+ channels
open  Ca2+ entry
– Opens the Ca2+-activated K+ channels (IK,Ca channels) – Slow AHP
slow; little contribution to repolarisation of AP
– outward K+ current (“Extra” gK) – prolonged channel
opening
  hyperpolarisation after action potential
Fast AHP
• slow afterhyperpolarisation (AHP)
• With multiple APs
50 ms
•    intracellular Ca2+    IK,Ca  AHP with each Action
• i.e. larger “slow AHP” Potential
Fig. 5.11 Hille. Ion Channels of Excitable Membranes, 3 rd Ed.

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BSc BioSci / BSc/MSci Chem w Pharm Year 3 Dr Austen Spruce
Molecular/Integrative Pharmacology
Electrophysiological Methods

Voltage Clamp
Techniques to Study Ion Channels - Using Intracellular (“Sharp”) Electrodes
Electrode controls intracellular Vm
Voltage Clamp
• Measure current under a controlled voltage
• Current amplitude depends on:
o Number of open channels (determined by properties)
When ions flow
o Driving force (Vm - Eion) – Fixed in V Clamp!
through channels:
• Total ionic current equal and opposite
• Isolate individual currents current is injected (via
E.g. Voltage-gated INa and IK Im ) to maintain
Current vs voltage relationship membrane potential at
the value set by Vm
electrode

Voltage Clamp enables measurement of Sodium and Potassium Current

current 0
0 Vm
Leak current Vm
-60
mV -60
Vm -50 Total ionic current – leak +TTX
outward
-60 plus voltage-gated channels IK
outward Il
Im
Im 0 Im
inward +TEA
Il inward INa
0
1 ms
time
Adapted from Fig. 9-3. Principles of Neural Science Adapted from Fig. 9-3. Principles of Neural Science

Families of Ionic Currents Patch Clamp

• Refinement of method of recording from single cell
Vm step: • Electrode does not pierce cell membrane
60 • For small, fragile (mammalian!) cells
Im IK Most commonly used under voltage clamp
30
0 60 • Two main uses:
-30 1.Record single channel activity
0 -60 cell-attached
Depolarisation causes  IK because more15
channels open and driving force is
Other modes of recording single channel activity:
increasing (Vm – EK) -45  inside-out (chemicals can be applied to cytoplasmic face
msec INa  outside-out (allows manipulation of extracellular
-15 chemicals
2.Rupture membrane patch  “Whole-cell recording”
Depolarisation causes  INa because more channels open, but  Allows measurement of total ionic current
then INa  because of smaller driving force (Vm – ENa)

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BSc BioSci / BSc/MSci Chem w Pharm Year 3 Dr Austen Spruce
Molecular/Integrative Pharmacology
Electrophysiological Methods

Single Channel Recording Patch Clamp – Modes of Recording

Sodium Sodium Cell attached
pipette Whole-cell
Pipette channel channel
tip (closed) (open)
Na+

Neurone
Seal

Cell-Attached
Vm
Voltage step - stimulus
channel open channel closed
I Inside-out Outside-out

Adapted from: Box 4.2. Neuroscience: Exploring the Brain Ion Channels of Excitable Membranes (2001). Hille. Sinauer

Current Clamp Current Clamp Recordings

• Usually achieved using single “sharp” electrodes Voltage recordings

• Voltage is allowed to change and is measured
• Electrode used to “inject” constant current into cell as
well as measure Vm
– Eg, injection of  charge into cell will cause
depolarisation
– V-gated channels open generating a membrane
current  further change in Vm
– Voltage change recorded (Action Potential)

Current injections

Extracellular Recording Hippocampus – Neuronal Connections

• Brain slice
• Uses:
– Assessment of synaptic physiology
– Measurement of neuronal activity
• Stimulating electrode – initiate APs in many presynaptic
axons
• Recording electrode (extracellular) – signal has opposite Stratum radiatum
polarity to that inside cell
• Signals that can be measured include a “sum” of all of the
postsynaptic potentials occurring in cells in the vicinity
– PSPs: A “field recording”
• Intracellular recordings can also be made from neurons in
a slice preparation
Adapted from Fig. 63-9. Principles of Neural Science

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BSc BioSci / BSc/MSci Chem w Pharm Year 3 Dr Austen Spruce
Molecular/Integrative Pharmacology
Electrophysiological Methods

Example of Use of Extracellular Recording Field Recordings

Control of synaptic function by presynaptic
GABAB Receptors Baclofen
• Hippocampus

fEPSP
• Stimulus leads to activation of many synapses (on CA1) 1 mV
• Recording electrode measures: field excitatory postsynaptic
potential (fEPSP) (neurotransmitter = glutamate)
Control 10 ms
• Concentration of calcium in the presynaptic terminals is
measured as well (as expected, Ca2+ increases during
synaptic activation) 75

[Ca2+] nM
• Baclofen (GABAB receptor agonist) is added to slice:
  fEPSP and  presynaptic Ca2+ 70 Baclofen
• Interpretation: Activation of presynaptic GABAB receptors
inhibits entry of Ca2+ and release of transmitter
65
Wu & Saggau (1995) J Physiol 485:649

References

• Membrane Potential:
• Synaptic physiology:
• Voltage clamp:
Molecular and Integrative Pharmacology Canvas site

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