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In today's video we're going to be discussing some the commonly used
techniques in electrophysiology.
By using electrodes you can measure membrane voltages
or currents across the membrane, this allows you to listen in on neuronal
activity
these techniques have allowed scientists uncover some other mysteries at the
brain.
After watching this video
should be able to do the following: first,
to understand how intracellular and extracellular recording techniques
can be used to record action potentials; and second, should be able to understand
how patch clamping
can be used to measure current to individual ion channels
Hodgkin Huxley made the first intracellular recording have an action
potential in 1939.
They used a squid giant axon and you can see the original trace
right here. They chose the squid
because as the name implies it's axons are giant
the one at the used here had a diameter about 500 millimeters.
You can see to use at the axon because Hodgkin Huxley use mirrors so that they
could see both the front and side view of their electrode.
They used a pulled glass pipette shown here,
to go inside the axon, there, they measure the potential difference between
inside and outside
that we talked about before
unfortunately soon after these seminal recordings were made
world war 2 broke out, Hodgkin and Huxsley turned their scientific attentions to
technologies that would help the British war effort
following the end of World War Two Hodgkin and Huxsley
again returned there attention to the action potential.
They completed a series a voltage experiments
where they kept the membrane voltage clamped at a certain level and measured
ion currents.
They published a series of four papers in 1952
laying on a mathematical model for the action potential the developed a series a
four differential equations.
One shown here which models the ionic
current across the membrane overtime. It's also shown
in diagrammatic form below this equation should look fairly familiar to you from
the last video.
Basically they've broken down current across the membrane
into four parts: Capacitive current, shown here
and here which is just current going across the membrane; Leakage current
or current,
going through your leak channels; sodium current shown here in here;
and your potassium current the other three equations
determine the facotrs n and then H
over time. Those factors are multiplied by the conductance of
potassium and sodium and they stand for the relative open probabilities
the voltage-gated potassium and sodium channels over time now
what's really interesting to note is that they had no idea that these will
voltage gated channels exist at the time
but looking at the different ionic currents
overtime in the action potential they were able to tell that gating of the
potassium
channels or potassium very was less complicated than that of the sodium
so basically the n term represent the open probability
of the voltage gated potassium channel the n term
refers to the open probability
of the activation gate of the voltage-gated sodium channel
and the h channel models what happens with the inactivation gate
the true test
how well the equation model would happen in real life could
only be done once you solve them all over time so Huxley spent about three
weeks using hand-cranked calculator
member this was before computers got to solve these
and what he found was remarkably similar to what they had measured in the real
animal
so if you look at this trace
the solid black line here
is what they measured in their squid axon
that dotted line
was what they got when they solve all equations and you can see it was
remarkably close
there's a little bit of difference on on falling piece of the action potential
but again remember they didn't even know that the ion channels existed so this was
a remarkable accomplishment and in fact
Hodgkin Huxley as well as Eccles when the 1963 Nobel Prize for their
discoveries concerning
the ionic mechanisms involving excitation
and inhibition in the peripheral and central portions and the nerve cell
membrane
these days we use computers to stimulate neurons and neural networks
one very popular program in research Is Neruon
Neurons takes the Hodgkin Huxley equations and refine them based on our current
knowledge of the ion channels and other factors in the membrane
Neurons is what the program that the two virtual labs that we're going to do you
are based
both neurons in action as well as a SWIMMY lab
so when you're getting frustrated with Neurons in Action and SWIMMY
just remember at least you're not using a hand cranked calculator
intracellular recording
still a popular electrophysiological technique you can use pulled glass
electrodes to measure intracellularly
in axons at their large enough or in cell body or soma as shown here
intracellular recording requires precise placement
of your electrode and often requires a microscope alternately
you can measure action potentials using extracellular recording
you can take a pulled glass electrode or wire electrode
and place it into an area of the brain and require the activity going on around
depending on your tip size you can record just a single
neuron or multiple neurons or what they call field potentials
which just a bunch of neurons firing at once
in my own research I use an extracellular recording technique
I use a pulled-glass electrode to suck up the cut end of a nerve
I can then record the sensory firing coming back from multiple
axon's in that nerve a raw trace is shown here
I can then use software that sorts the different neuron
firings by their shape so this one is just a small
firing shape and this one is a large one that in that way I can look at individual
neurons
and their activity
another commonly used electrophysiological technique is patch clamping
using patch clamping you can measure individual ion channel
you take a pulled glass pipette with very very small tip diameter
and place it on the endge of the cell membrane then place a little bit of
section through the electrode
to suck up a small bit membrane that has one or a few ion channels
in that way you can record the current individual ion channels
in 1991 Neher and Sakmann won the Nobel Prize for developing patch clamping
in for the discoveries about the function of single ion channels that
they made using patch clamping
there are a few variations on the patch clamping technique
the one we just talked about is the on cell technique for you sucked up
on a small patch of membrane and record the current going through one or a few
ion channels if you then pull up your pipette
you can break off just a small little patch membrane this is called an inside-out
patch
the inside part of the
cell is facing the outer or extracellular bath solution
and the outsiderof the cell is facing the inside your pipette tip
if you go back to the on cell method and placed a lot of sections on the cell
you can cause it hole in that membrane and
that allow you to use whole-cell recording is very similar to
intracellular recording
if from this whole cell set up you then pool a little bit so that you break up
parts in the memory these parts will then reform
and shown here and now you have an outside patch
the outside part of the membrane is facing your extracellular bath solution
and the inside part is facing your pipette tip
here we have a typical patch-clamp recording of current going through a
single channel
if you current is up here the channel is in a closed state
if it's down here it in an open state as you can see
the channel will open and current will flow for variable amounts of time
time is here on the x-axis
sometimes the channels opens and closes quite rapidly
we can use patch clamping understand a little bit more of what it means
for the voltage-gated channels to open at threshold.
In biology is never simple as everything is open
once you hit exactly the threshold voltage.
Threshold is really just voltage that most
voltage-gated channels are likely to be open but these channels open for variable
amounts of time
and they have what's called probabilistic opening.
Using a patch of membrane we are going to record
sodium and potassium current, so sodium current
is going to be shown in red here and it's modeled
as a negative an inward current
with potassium is in blue and it's a positive
outward current. So inner patch we have just one sodium
in one potassium channel if we depolarize a membrane potential to threshold
three separate times you see that we don't get exactly the same thing
each time. It's because these channels have probabilistic opening
so in two of the three you do have sodium current flowing
for the voltage-gated sodium channel to
open near the beginning of
the depolarization which is what we talked about would happen
and you see that in general the potassium currrent is flowing
towards the end ifthe depolarization phase and usually is open for a little bit
longer,
but you can see in some them their traces you've got sodium
flowing near the end the depolarization phase got potassium current flowing
early you got no sodium current at all here
with just one channel it doesn't do the exact same thing
every single time, if we then put
in our patch a thousand sodium and a thousand potassium channels
the ionic current starts to look like what we saw before
so you've got sodium current flowing at the beginning it opens and closes fast
so once you dear all thousand those that looks
like the sodium current flow that we saw before
same thing with potassium tends open later stay open longer
ok, so just to reiterate the quick on and off
changes in sodium conductance that we see mirror
the quick ionic current flow of sodium
we see measured here through our patch and similarly this
long on and slow start
conductance changes and potassium also mirrors
the currents that we can measure
no neurons were harm in the making of this video