Summary C&M Neuroscience
! don’t forget to prepare article for exam!
1. Introduction & basic concepts
Basic knowledge
2. Recording bio-electricity and history
Passive membrane properties
It will take time for the membrane to charge (=delay) & how quickly the membrane can respond
is how quickly the neurons can respond.
• Membranes need a power source (~battery) =
electromotive force = unequal distribution of
ions (eg. K+, Na+)
• All ions are being kept away from their
equilibtrium potential (Na+/K+ ATPase pump)
• Nernst equation (simplified at physiological
temperatures):
o R = universal gas constant; F = faraday constant
• Equilibrium/reversal potential: for each ion when an ion channel opens, it encourages
the membrane potential to move towards its equilibrium potential!
, • Ena+ = +60 mV; EK+ = -100 mV; ECl-= -75 mV; ECa2+ = +120 mV (around these values)
• The equilibrium potentials changes sign if the charge of the ion is reversed or if the
direction of the gradient is reversed, and it falls to zero when there is no gradient
• and I = V/R
• If you change the voltage across the membrane, you will change the driving force, and
the current will increase if you change the voltage away from the reversal potential: so
the further away from RP of a certain ion, the higher the current through the ion channel!
IV relationships for real ion channels
• No ionic current at membrane potentials more negative than E1. Therefore the
conductance there is zero, and the channels must be closed.
• Positive to 0mV: the IV relations are steep (almost straight lines). Here the conductance
is high and the channels must be open.
• In the intermediate voltage range, between E1 and 0 mV, the current is smaller than
expected from the maximal conductance (dashed lines), and hence only some of the
channels are open.
• Over a narrow voltage range the conductance changes smoothly from fully off to fully on.
Historical thinking on bioelectricity
• Paradigm shift: “overshoot”
o First intracellular recordings (H&H)
o Scientists try to generate the best model of reality with knowledge or theories
they have at that time, but at a certain moment in time a paradigm shift happens
where most of what was known or thought important, is changed and the focus
on research questions change.
Hodgkin & Huxley
• Isolated giant axons of squid: they did measurements and
experimental manipulations and modelling of these axons.
They created a mathematical model that fully describes AP
generation
,• They confirmed that the inside of a neuron is indeed
negative => resting potential = negative!
• But the AP goes beyond 0mv = overshoot! (this observation
was different than the previous theories that were out
there (Bernstein)) = paradigm shift
• H&H experiment: replacing the internal axoplasm of
squid axon to see if intracellular composition of axon is
important to create an AP
o Result: axon membrane could still generate AP
(normal sized) even after most of the axoplasm had been removed and replaced
with a solution of potassium salts!
• H&H experiment: radioactive labelling of K+ and Na+ ions and measuring their fluxes
into and out of axons
o influx of Na+ is one of the dominant ions over the lipid bilayer!
• During rest energy is needed for efflux : sodium pump: DNP = inhibitor: the sodium pump
stops once its fuel is removed! => ATP drives the sodium pump (CN = inhibitor of ATP
synthesis)
o The sodium pump contributes a little to the resting membrane potential: when
blocking the sodium pump (with ouabain), it creates a small shift in the
membrane potential. Also sodium extrusion is coupled to potassium uptake,
because when experimentally removing K+, hyperpolarisation does not occur =>
Na+/K+ ATPase pump
• Directionality: inward rectification of K+ ions
o K+ flows more easily inwards than outwards
o K+ inward rectifier channels (= Kir channels) are a type of ion channels in the
membrane that help stabilise the cell’s resting membrane potential by allowing
K+ to flow in when the membrane is near its resting voltage, but preventing K+
loss during depolarisation!
• Neurons are very permeable for K+ and Aps are dependent on Na+!
, Wheatstone Bridge circuit
• To measure membrane permeability (resistance) during AP: Wheatstone Bridge circuit is
a direct observation of conductance increase during the AP (passive observation)
• Voltage clamp = active control
• The Nernst equation was eventually proved quite valid, but with a small addition: also
sodium is important for the resting membrane potential, not only K+!
Conclusions
• Current understanding of basis for neuronal excitability is largely a result of experiments
performed mid 20th century
• H&H were pivotal in developing and using novel technologies and driving concepts
forward:
o They made the first intracellular recordings from axon: demonstrated ‘overshoot’
of APs beyond 0mV suggesting an active process instead of a short circuit.
o Reducing extracellular [Na+] reduces AP height suggesting an increase in
permeability of Na+ underlies the upstroke of the AP!
3. Hodgkin & Huxley & voltage clamp recordings
The action potential
• Intracellular recordings of spikes in the squid
giant axon showed that the membrane
overshot 0 mV. This could not be explained by
a short circuit of the membrane, but rather
suggested an active process.
• Following the action potential, the membrane
rapidly repolarizes and undershoots the
resting membrane potential, termed the after-
hyperpolarisation. This suggests that changes
in membrane permeability contribute to
repolarisation.
• Reducing the extracellular Na concentration
reduces action potential height, suggesting that
an increase in permeability to Na underlies the
upstroke of the AP.
• The membrane potential never reaches Nernst
potential for Na, in part because the membrane
remains permeable to other ions.
H&H changed the field
• Voltage clamp (VC) technique: squid acon IV
• Identification Na+, K+ currents isolation by subtraction
• Detail IV relation kinetics K+
• Inactivation Na+
• Mathematical model
! don’t forget to prepare article for exam!
1. Introduction & basic concepts
Basic knowledge
2. Recording bio-electricity and history
Passive membrane properties
It will take time for the membrane to charge (=delay) & how quickly the membrane can respond
is how quickly the neurons can respond.
• Membranes need a power source (~battery) =
electromotive force = unequal distribution of
ions (eg. K+, Na+)
• All ions are being kept away from their
equilibtrium potential (Na+/K+ ATPase pump)
• Nernst equation (simplified at physiological
temperatures):
o R = universal gas constant; F = faraday constant
• Equilibrium/reversal potential: for each ion when an ion channel opens, it encourages
the membrane potential to move towards its equilibrium potential!
, • Ena+ = +60 mV; EK+ = -100 mV; ECl-= -75 mV; ECa2+ = +120 mV (around these values)
• The equilibrium potentials changes sign if the charge of the ion is reversed or if the
direction of the gradient is reversed, and it falls to zero when there is no gradient
• and I = V/R
• If you change the voltage across the membrane, you will change the driving force, and
the current will increase if you change the voltage away from the reversal potential: so
the further away from RP of a certain ion, the higher the current through the ion channel!
IV relationships for real ion channels
• No ionic current at membrane potentials more negative than E1. Therefore the
conductance there is zero, and the channels must be closed.
• Positive to 0mV: the IV relations are steep (almost straight lines). Here the conductance
is high and the channels must be open.
• In the intermediate voltage range, between E1 and 0 mV, the current is smaller than
expected from the maximal conductance (dashed lines), and hence only some of the
channels are open.
• Over a narrow voltage range the conductance changes smoothly from fully off to fully on.
Historical thinking on bioelectricity
• Paradigm shift: “overshoot”
o First intracellular recordings (H&H)
o Scientists try to generate the best model of reality with knowledge or theories
they have at that time, but at a certain moment in time a paradigm shift happens
where most of what was known or thought important, is changed and the focus
on research questions change.
Hodgkin & Huxley
• Isolated giant axons of squid: they did measurements and
experimental manipulations and modelling of these axons.
They created a mathematical model that fully describes AP
generation
,• They confirmed that the inside of a neuron is indeed
negative => resting potential = negative!
• But the AP goes beyond 0mv = overshoot! (this observation
was different than the previous theories that were out
there (Bernstein)) = paradigm shift
• H&H experiment: replacing the internal axoplasm of
squid axon to see if intracellular composition of axon is
important to create an AP
o Result: axon membrane could still generate AP
(normal sized) even after most of the axoplasm had been removed and replaced
with a solution of potassium salts!
• H&H experiment: radioactive labelling of K+ and Na+ ions and measuring their fluxes
into and out of axons
o influx of Na+ is one of the dominant ions over the lipid bilayer!
• During rest energy is needed for efflux : sodium pump: DNP = inhibitor: the sodium pump
stops once its fuel is removed! => ATP drives the sodium pump (CN = inhibitor of ATP
synthesis)
o The sodium pump contributes a little to the resting membrane potential: when
blocking the sodium pump (with ouabain), it creates a small shift in the
membrane potential. Also sodium extrusion is coupled to potassium uptake,
because when experimentally removing K+, hyperpolarisation does not occur =>
Na+/K+ ATPase pump
• Directionality: inward rectification of K+ ions
o K+ flows more easily inwards than outwards
o K+ inward rectifier channels (= Kir channels) are a type of ion channels in the
membrane that help stabilise the cell’s resting membrane potential by allowing
K+ to flow in when the membrane is near its resting voltage, but preventing K+
loss during depolarisation!
• Neurons are very permeable for K+ and Aps are dependent on Na+!
, Wheatstone Bridge circuit
• To measure membrane permeability (resistance) during AP: Wheatstone Bridge circuit is
a direct observation of conductance increase during the AP (passive observation)
• Voltage clamp = active control
• The Nernst equation was eventually proved quite valid, but with a small addition: also
sodium is important for the resting membrane potential, not only K+!
Conclusions
• Current understanding of basis for neuronal excitability is largely a result of experiments
performed mid 20th century
• H&H were pivotal in developing and using novel technologies and driving concepts
forward:
o They made the first intracellular recordings from axon: demonstrated ‘overshoot’
of APs beyond 0mV suggesting an active process instead of a short circuit.
o Reducing extracellular [Na+] reduces AP height suggesting an increase in
permeability of Na+ underlies the upstroke of the AP!
3. Hodgkin & Huxley & voltage clamp recordings
The action potential
• Intracellular recordings of spikes in the squid
giant axon showed that the membrane
overshot 0 mV. This could not be explained by
a short circuit of the membrane, but rather
suggested an active process.
• Following the action potential, the membrane
rapidly repolarizes and undershoots the
resting membrane potential, termed the after-
hyperpolarisation. This suggests that changes
in membrane permeability contribute to
repolarisation.
• Reducing the extracellular Na concentration
reduces action potential height, suggesting that
an increase in permeability to Na underlies the
upstroke of the AP.
• The membrane potential never reaches Nernst
potential for Na, in part because the membrane
remains permeable to other ions.
H&H changed the field
• Voltage clamp (VC) technique: squid acon IV
• Identification Na+, K+ currents isolation by subtraction
• Detail IV relation kinetics K+
• Inactivation Na+
• Mathematical model
