In the last video, we saw that GABA is a main inhibiting neurotransmitter
of the mammalian brain.
GABA acts via two distinct mechanisms.
At the synapse. GABA activates GABA-A receptors,
ligand gated ion channels permeable to choride,
and here it drives a hyperpolarization.
GABA also acts extra-synaptically upon GABA-B receptors.
Here the GABA-B receptor is a seven transmembrane protein.
It's metabotropic, it activates G proteins,
and downstream of that it activates a Potassium conductance,
also causing hyperpolarization.
There are two distinct mechanisms for GABA inhibition,
and in this video we're going to take a closer look
at the GABA-A mediated inhibition.
The GABA-A conductance is a chloride conductance.
Chloride reverses at around -18 mV,
so typically, when a pre-synaptic GABAergic neuron
fires an action potential, it propagates down its axon,
releases GABA, it acts upon GABA-A receptors
and causes a hyperpolarization in the post-synaptic neuron.
That's the fast hyperpolarizing action of the GABA-A receptor.
In this lesson we're going to look in more detail
at the fast inhibition mediated by GABA-A conductances
and see a little bit more about the specific details of how it works.
The GABA-A receptor is a chloride channel.
GABA binding to the GABA receptor
increases the open probability of the receptor,
so in the absence of GABA, if we are recording with an electrode
looking at the single channel, then the openings and closings
of this receptor, it'll be closed.
If then you add GABA to the recording pipette,
you'll see that the open probability
increases and we have single channel currents
that come with a conductance of something like 10 to 30 pS.
So GABA increases the open probability of the GABA-A receptor.
It's of course also interesting to note
what the time course of activation of the GABA currents are,
and in particular, when we're thinking of synaptic release of GABA,
we're thinking about brief pulses of GABA
that may well reach 1 millimolar concentrations.
So, in the same way that we thought about analyzing the AMPA receptors,
we can also record the currents evoked by brief GABA applications
onto patches of membrane containing the GABA receptor.
And so, we can have a laminar flow electrode
which has either no GABA or contains maybe one millimolar GABA.
We mount this onto a piezo device
and we can then give brief one millisecond pulses of GABA to our patch of membrane
containing GABA-A receptors.
We can then measure the resulting current under voltage climb,
so we keep the membrane potential clamped,
and so we measure the current flowing through the GABA receptor.
Here you can see a typical recording like this.
One millisecond pulse of GABA almost immediately activates
the GABA conductance. Increased open probability chloride flux,
and we get an outward current. The GABA disappears.
It's only a one millisecond pulse, but the current remains on
for some ten to 20 milliseconds.
That's the duration of the GABAergic conductance
that's mediated by a single one millisecond pulse of GABA.
So it's a fast conductance that's mediated by the GABA-A receptor,
but it's interesting to note that this is still much slower
than the AMPA currents that are mediated by glutamate
at glutamatergic synapses, so if you do the equivalent experiment
and last week this is what we looked at,
one millisecond pulse of glutamate
activate an AMPA conductance and that lasts maybe only two ms.
The GABAergic conductance here is five to ten times lower
than the AMPA conductance of the glutamatergic synapses.
So GABAergic inhibition is fast,
but it's much slower than the AMPA mediated excitation.
Let's see how those conductances, then,
are reflected in post-synaptic membrane potential changes.
So the GABA currents are chloride currents.
They therefore reverse at the chloride reversal potential,
something like -75 mV.
The GABA conductance is basically linear,
so we can draw a linear current-voltage relationship
so this is a current evoked by a GABA pulse
and this is a membrane potential of which that current is being recorded.
The equivalent AMPA current would look something like this,
wherein a pulse of glutamate will cause a current
and here it reverses close to zero mV.
For GABA, it reverses close to -75 mV
and the interesting range of membrane potentials, physiologically,
is something around -75 to -40 mV.
That's where the membrane potential of a neuron typically is.
Here we have an outward current for GABA
and we have inward currents for AMPA
so inhibition and excitation.
If you now think about the synaptic situation,
we might imagine having a tight small cell that we can consider as iso-potential.
We have a GABAergic synapse that's made here.
An action potential invades, releases GABA,
and activates the GABA-A currents
and we then measure here in the Voltage clamp mode
we measure the current flowing through the GABA receptor.
The action potential causes release of GABA
and we can then see the synaptic conductances
here at different membrane potentials.
At -100 mV we have an inward current.
At -75 mV no current flow,
and at positive potentials to the red,
to the reversal potential there of -50 mV
we have an outward current,
and it's linear and symmetrical around the reversal potential
with the time course of around ten ms
just as we saw for the isolated receptor
with a one ms pulse of GABA.
So that's the synaptic GABA conductance
that tries to bring the membrane potential
to around -75 mV and has a time course of 10-20 ms.
If we now think about this situation in current clamp mode,
where we're now measuring the membrane potential
and we let the membrane potential swing freely
depending upon the conductances that are activated,
we see that there's an inhibitory post-synaptic potential
that has a time course of a little bit longer
than the GABA conductance, the GABA currents themselves.
And that's because, of course, we have the membrane time constants
that we need to discharge the capacitor
after we've charged with our currents,
we need to think about this and additional filtering
because of the capacitance of real cells.
Now, in this case,
when we're thinking about the GABAergic conductance
I've indicated that we're adding 100 pA to the current
of the cell that we're recording from.
So at resting membrane potentials, maybe we're sitting close
to the reversal potential for chloride at -75 mV,
so as we stimulate an action potential in the pre-synaptic cell,
and the post-synaptic cell is at -75 mV,
we're not going to see a response.
We're here at the reversal potential, there's no current flow,
and so there's no change in membrane potential.
We need to depolarize the cell in order to see the inhibitory
post-synaptic potentials.
We can do that by injecting 100 pA of current,
so here if we inject 100 pA of current
we'll charge the membrane of the post-synaptic cell.
Maybe we depolarize it to -50 mV
and now, when we stimulate
an action potential in the pre-synaptic cell,
we get the hyperpolarizing inhibitory post-synaptic potential
that lasts some tenths of milliseconds.
Now, it's interesting to note that even though
the membrane potential of cells is often close
to the chloride reversal potential
and therefore you actually don't record an inhibitory post-synaptic potential
when GABA is released,
there is nonetheless an impact upon the integration
of the post-synaptic cell in terms of, say,
how excitatory conductances are integrated in that neuron.
That comes about because there's a change in the overall membrane resistance.
I'll illustrate that with a simple example here.
We can imagine for example a glutamatergic excitatory conductance
coming into the dendrites of a cell.
That then causes current flow into the dendrite
that'll then go towards the soma,
so there'll be conductances from glutamate
that mediate current chart flow via the AMPA and NMDA receptors
that contribute to changing the membrane potential of the soma.
At the soma we can imagine that there are GABAergic synapses
that are present here, a chloride conductance
that maybe is reversing at exactly the same
as the membrane potential of that cell.
Nonetheless, activating this GABA conductance
will change the impact of that glutamate.
Why is that?
Let's think about the electrical equivalent of the cell soma.
We have current coming into the soma from the dendrites.
That'll contribute to charging the membrane,
the capacitance of the cell,
and also, of course, some of that current
will flow across the leak conductances
that are present in the plasma membrane.
That current, of course, in the end
goes out into the extracellular space
where there's zero mV potential by definition.
So we have a capacitor of the soma,
and we have resistance of the soma.
What we can do with the GABAergic conducting send
is to add on an additional conducting pathway here
so the GABAergic conductor here can then be added
and it changes the total resistance of the soma.
By changing the so-called input resistance of the soma,
that then changes the integrative properties
of how currents flowing into that soma affect membrane potential.
We can think about the equations that govern
the membrane potential changes here
that we've already dealt with in our previous weeks of study,
so the potential here across the cell
is equal to the current flow times the resistance,
and then we have the time course for those changes
that are the charging of the capacitor here
and the membrane time constant is equal to the resistance times the capacitance.
That's the exponential charging function here.
So if we have a square current pulse flowing into the cell soma,
that causes a change of membrane potential,
a charging of the capacitor. You turn the current off
and the capacitor discharges,
so this is the Current
and this is the Voltage.
Now, let's imagine that we put the GABAergic inhibition on.
The change here is a decrease in the membrane resistance.
We've added more conductance to the membrane of the soma.
It's reversing the same as the resting membrane potential of the cell,
so we can simply just think of it as a change
in the resistance of the membrane.
So we decrease the resistance of that membrane.
That has two impacts, if we look at this equation here.
The first is that the steady-state voltage
is going to become less because that depends
upon the input resistance of the cell,
and the other impact is that the membrane time constant
is going to change, it's going to decrease,
so we've got a decrease in the membrane time constant,
and if we now look at how the membrane potential would change,
it's faster,
so the overall time constant here
is faster than it was, but also,
the overall change in membrane potential is smaller.
So by activating a GABAergic conductance,
even if it's at the same membrane potential
as the actual cell that we're recording from,
and there's no inhibitory post-synaptic potential per se,
that'll still cause an inhibitiion of excitatory glutamatergic inputs
coming in,
so here we thought about steady state injections of currents,
but we'll have the same situation
if we think about glutamatergic inputs coming in,
brief currents, where if we have
at resting conditions an EPSP that looks like this,
then we turn on the GABAergic conductance
and the EPSP gets smaller, and also a bit faster,
because we've changed the membrane time constant
and the input resistance of the cell soma.
So shunting inhibition is another important form of inhibition
in addition to the hyperpolarizing inhibition mediated by charge
flowing through the GABA receptor. Now, GABAergic synapses are made
on a large variety of subcellular compartments.
GABAergic inhibition can occur here on the distal dendrites,
so we have GABAergic input right here in the distal dendritic operizations,
and presumably, a GABAergic input here
will have a large effect upon the nearby
excitatory synapses coming in here,
but might not affect GABAergic synapses occurring on other dendritic branches.
There might be specific inhibition
that's localized to individual dendritic arborizations.
So there could be a high degree of spatial specificity
as to where the GABAergic inhibition might function.
There are also GABAergic inhibitory synapses
all over the basal and more proximal dendrites,
and interestingly, there are also GABAergic synapses
that are directly placed on the soma,
and that's something that you won't find usually
for excited glutamatergic synapses that are sitting on the dendrites,
but not on the soma,
so GABAergic inhibition is different
and prominent on the cell bodies,
where it could affect the input resistance
and the integration of the synaptic current
flowing in from the dendrites
in this divisive way,
by changing the input resistance of the cell soma.
Interestingly, there are also GABAergic synapses
directly on the axon initial segment.
Again, there are no excitatory synapses in this area,
but inhibitory synapses are prominent
here at this site, where action potentials are initiated in most cells.
In GABAergic inhibition, then might not affect dendritic integration at all,
but might simply form an editing function
where it decides whether a cell can output an action potential or not,
independent of what synaptic computations
are occurring in the rest of the cell.
Then, finally, in the spinal cord, but not anywhere else in the mammalian brain,
there are also GABA-A synapses directly here
on the pre-synaptic specializations,
and that only occurs in the spinal cord,
but these synapses at other locations
occur throughout the mammalian brain,
and each one probably performs a slightly different function.
So in this video we've seen that the GABA-A conductance
lasts some 10 to 20 ms and has a reversal potential
somewhere around -75 mV,
depending upon the local chloride concentrations.
The GABA-A conductance can mediate inhibition in two slightly different ways,
both of which, of course, depend upon the chloride conductance
mediated by the ligand gated GABA-A receptor.
The first one is hyperpolarizing inhibition.
This occurs when the membrane potential is depolarized
relative to the chloride reversal potential,
and that's simply a movement then of chloride ions
in negative charge inside the cell,
causing a change in membrane potential.
The other sort of inhibition relies upon a conductance,
on a change in the input resistance of a cell,
so even if the membrane potential is actually sitting
at the same equilibrium potential as chloride,
there can still be a significant impact upon the integration
of excitatory inputs that ligend gated a chloride conductance
will decrease the input resistance of the soma
and the excitatory synaptic input that arrives at the soma
then causes a smaller change in membrane potential
if the GABA-A conductance is open.
So there are multiple different ways in which GABA can cause inhibition,
and furthermore, it's complicated
by the spatial distribution of the GABA-A input
that'll presumably have many different effects
depending upon where exactly the GABAergic synapses are activated.
We'll learn more about the sub-cellular targeting
of GABA synapses in subsequent videos.