2801 – LECTURE 14 – THE NEUROMUSCULAR JUNCTION
- The synapse between somatic motor neurons and skeletal muscle fibres is an excitatory chemical
synapse, the transmitter is acetylcholine (ACh).
- The postsynaptic membrane contains a receptor for ACh, the nicotinic ACh receptor (AChR).
- The nicotinic ACh receptor is a ligand-gated ion channel, a transmembrane ion channel that is opened
when ACh binds to the extracellular portion of the ion channel protein complex.
TASK OF THE NEUROMUSCULAR SYNAPSE
- Transmit the signal from the nerve to the muscle cell.
- Previous synapses talked about (many synapses on motor-neuron) release a small amount of
neurotransmitter and cause only a small depolarization/excitatory post-synaptic potential.
- The neuromuscular junction however is a relay-type synapse.
- When an action potential comes barreling down the motor axon and reaches the nerve terminal it
releases enough transmitter (a lot) in order to deploarise the post-synaptic cell well above threshold.
- This is important because every time the action potential comes down a nerve, it generates very rapidly
an action potential in the muscle fibre that then propogates down along the length of the muscle fibre,
triggering muscle contraction.
- In order to get reliable control of the muscle, there needs to be this rapid and reliable linkage between
action potentials in the nerve and action potentials in the muscle, so that changes in the
rate/frequency of propogation of action potentials down the nerve, produce matching changes in the
firing in the muscle fibre, producing graded muscle contraction or tetanus.
- The higher the frequency, the greater the contraction generated.
- Therefore we can control the movement of our limbs around joints by the frequency of firing.
- There must be a matching up of pre-synpatic to post-synaptic firing.
The neuromuscular junction is highly tuned up in order to do this. Every action potential produces an action
potential in the post-synaptic cell. It is a high-efficacy relay synapse.
STRUCTURE AND MOLECULAR ORGANIZATION
Plain view (photomicrograph) of a mouse neuromuscular junction.
- Each of these terminal branches (branching of the myelinated axon) spread out over a portion of the
muscle fibre surface. In humans, there is less space between the branches and they are more
compact, but the basic pattern is the same amongst various different mammals.
- Stained with an antibody that labels a protein (syntaxin) that’s involved in transmitter release, in
preparing the synaptic vesicles to release their transmitter.
- This staining shows us where the vesicles are being prepared for transmitter release.
- Immediately underneath each of these branches is staining for the ACh receptors, which are lined up
underneath the nerve terminal that sit on the surface of the nerve muscle cell in little gutters that are
lined with ACh receptors.
- Immediately when they are released, they fall onto these ACh receptors to activate them.
- The Schwann cell does not myelinate the nerve terminal.
- It loosely sits on top of the nerve terminal, and seems to be important to provide support, though it does
not wrap around it with a myelin sheath.
- The axon membrane sits above this synaptic cleft, and its bare synaptic membrane (presynaptic
membrane) onto which the vesicles dock and release their neurotransmitter into the synaptic cleft, so
that it diffuses a short distance onto the ACh receptors.
- Each of the nerve terminal branches is broken up into many little release sites.
- Within the whole neuromuscular junction/nerve terminal, there may be 1000 different nerve terminals at
which vesicles are docked and ready to release their contents.
- When the action potential comes barreling down the nerve it depolarizes all of these nerve terminal
branches, Ca+2 will diffuse in through voltage gated Ca+2 channels at each of these many sites,
increasing the probability that a synaptic vesicle will be released.
The nerve terminal part is blown up so that we just see the part of the membrane that contains the pre- and
post-synaptic (muscle cell) nerve membranes on either side
- We have the pre-synaptic release site, the active zones (where the synaptic vesicles are docked,
ready to release transmitter).
- There are only a few of these synaptic vesicles ready to go at any given time – they are the ones that
are stuck to the membrane and bound to these proteins, including syntaxin and a calcium sensor.
There are many sites – 1000 different release sites.
- The way the neuromuscular junction seems to work is that the depolarization allows calcium to come
into many sites, and increases the probability that neurotransmitter will be released at least at some of
those sites, enough sites to release enough transmitter to strongly depolarize the post-synaptic cell.
The voltage-gated calcium channels are in the pre-synaptic membrane.
- Calcium diffuses in because the concentration gradient is very strong.
- The concentration in the blood and space between cells is about 1 – 2 mM. Inside the cell it is 1000th or
10 000th of that, creating a very strong chemical driving force.
- Most of the time, none of these cells are open. It is only when depolarization comes that many of them
open coordinately and calcium can diffuse in.
-
In between is the synaptic cleft, which is extracellular space.
The neurotransmitter is ACh, so each of these synaptic vesicles contains ~5000 molecules of ACh.
There are several hundred of these vesicles above each release site, ready to release their content.
The synaptic cleft contains basement membrane, which contains acetylcholinesterase (AChE).
When the ACh is released, the vesicle fuses with the membrane, the ACh diffuses out and it only has to
travel a distance of about 50nm before it activates the receptors.
- Even as it diffuses across there, the AChE receptors will start degrading the ACh, breaking it down and
hydrolyzing it into choline and asterase.
- It is only the ACh that manages to get past the acetylcolinesterase that will activate the receptors.
- The entire process is about controlling the release of neurotransmitter in a very controlled and precise
way, so that when the membrane depolarizes, the Ca 2+ channels open the gates, calcium diffuses into
this little area of the membrane, and triggers the exocytosis of the vesicle.
- On the post-synaptic cell side, the Schwann cell can be seen sitting loosely above the nerve terminal
(with vesicles within).
- The whole thing sits within a gutter (a dip within the post-synaptic cell membrane).
- The gutter contains ACh receptors (ligand-gated ion channels that control the response to ACh) and
membrane infoldings.
-
When the cation permeable channels open, sodium can diffuse in and potassium can diffuse out.
More sodium diffuses in than potassium does out because of the electrical driving force of the cytoplasm
being more negatively charged than the extracellular fluid (summation with chemical driving force inwards
is very strong).
*every year puts a question or two, testing whether you understand this into the test
- When ACh binds, the protein changes the conformational shape of the gate and opens the pore up.
- The gate opens as a result, and this opening allows sodium to diffuse in, resulting in a net inward
movement in of negative charge, resulting in depolarization.
- The muscle cell has arranged itself to put the voltage-gated sodium channels right next to the ligandgated cation channels in the fold membranes.
- When the ligand-gated channels open, the voltage-gated sodium channels are very close to them in
the membrane infoldings, making that area of the membrane very sensitive to depolarization.
- This is the trigger zone, because this is where the voltage-gated sodium channels are most
concentrated.
- This area is most sensitive, and the Hodgkins cycle is more likely to get going here because the sodium
channels are packed together in the membrane.
- The opening of one depolarises the membrane more, to open the rest of them.
- By pulling ligand-gated channels together into clusters, and pulling the voltage-gated channels together
into a neighbouring bit of membrane, the muscle cell and this synapse has tuned up the sensitivity of
the post-synaptic cell to ACh to make it a really efficient responder to the ACh released, so it will
produce the maximum current into the ligand-gated ion channels, and that current will be efficiently
used to start the Hodgkin’s cycle in neighbouring post-junctional folds.
- Once the Hodgkin’s cycle starts here, it produces a great big all-or-none action potential depolarization
that depolarizes muscle fibre, and then propagates through continuous propagation down along the
length of the muscle fibre.
- This is about getting the muscle fibre activated rapidly, so that we can produce a coordinated
contraction throughout the sarcomeres along the muscle fibre length.
This is basically how we think the neuromuscular junction works.
THE ENDPLATE POTENTIAL (EPP) AND MINIATURE ENDPLATE POTENTIALS (MEPPS)
- We want to study the function, not just the structure of the synapse. To do this, we need to record the
membrane potential of the cell.
- An electrode would be pushed into the muscle cell near the synapse (where the acetylcholine receptors
are).
- We might then stimulate the nerve (as in sciatic nerve prac), trigger action potentials in the nerve, which
propagate down the nerve, and cause transmitter release at the terminal.
- We can monitor the way in which the membrane potential changes when the nerve has been stimulated
to release transmitter.
- Typically we would see some very small miniature endplate potentials (MEPPs) occurring randomly
in time.
- Even if the nerve is not being stimulated, we can occasionally see these mini’s.
- When the nerve is being stimulated, we see much bigger potentials that would normally result in an
action potential.
- We know now that inward current through ACh receptors produces the Endplate Potential EPP) (so
called because the post-synaptic cell is called the endplate).
- EPP is a large amplitude depolarisation that occurs briefly in the postsynaptic membrane following
stimulation of the presynaptic nerve.
- In order to study the function of the post-synaptic AChR we need to prevent the muscle action potential.
- If [Ca2+] is lowered below physiological levels, the action potential from the muscle stops working and
the amplitude of the EPP becomes sub-threshold and graded. With no muscle action potential, the
opening of the AChR channels in response to transmitter release can then be measured.
- Normally when you stimulate the nerve there is a certain delay between when the stimulus artefact is
seen and the stimulus was applied to the nerve (takes time for action potential propagation), then you
would normally see a muscle action potential following shortly after the nerve action potential, and
massive depolarization. Decreasing the [Ca2+], the action potential fails and instead a much smaller
depolaraisation occurs (EPP).
- The EPP sometimes fails to occur at all, or may range between slightly bigger or smaller.
- Even when there is no stimulus to the nerve, there are spontaneous mEPP. These are very small –
approximately 0.5mV, much too small to cause an action potential. They occur spontaneously or
randomly in time.
VESICLE HYPOTHESIS FOR QUANTAL RELEASE
- Plotting the amplitudes of mEPP, shows a normal distribution (despite slight variation, seem to
represent a single population of depolarization events).
- This gives idea that there is a quantum – or fixed amount – released, and from that the idea has been
evolved that that quantum is what is contained in a synaptic vesicle, a minimum unit of release of
transmitter.
- The distribution of the amplitudes of the EPP did not show a normal distribution.
- The amplitude of the EPP depends upon [Ca2+]o. At physiological [Ca2+]o is (~2mM) EPP amplitude
~70mV. If [Ca2+]o is lowered, the amplitude of the EPP is smaller and release becomes ‘hit or
miss’(often ‘failures’).
- When it does occur EPP amplitude is in multiples of the mean amplitude of the spontaneously occurring
MEPPs.
- The idea has emerged that maybe the EPP is the summing together of mEPP’s, each of which
represents the release of a single quantum (e.g. two quanta released at the same time – twice the
depolarization).
- The quantal hypothesis describes that the anplitude of the MEPP is thought to reflect a minimum unit
for release of ACh.
- The EPP is thought to be the sum of multiple MEPPs (quantal events) occurring in synchrony.
- At normal [Ca2+]o (~2mM), more than 100 quantal events add together.
- When the nerve action potential invades the nerve terminal branches, the resulting coordinated influx of
calcium at many transmitter release sites results in simultaneous release of multiple small packets
(quanta) of ACh.
- Each quantum opens approximately 2000 AChR channels, creating quantal inward currents.
- The summation of the quantal currents results in an EPP.
Quantal synaptic transmission is the idea that:
1. ACh is released in equal sized packets from many release sits in a probabilistic fashion.
2. The probability of release is positively regulated by calcium influc into the nerve terminal.
The vesicle hypothesis proposes that the quanta of transmitter are stored within synaptic vesicles in the
nerve terminal. All synaptic vesicles are of roughly the same capacity, so may contain about the same amount
of ACh (one quantum). The quantum of transmitter in a vesicle is all released at once, producing a MEPP by
opening a uniform number of AChR channels to depolarize the postsynaptic cell by a consistent amount.
Graded endplate potentials are the summer results of coincident exocytosis of multiple vesicles.
It converts the all-or-none action potential of the nerve to a graded release of transmitter to a graded postsynaptic potential (which normally reaches threshold) to produce the all-or-none muscle action potential.
** Remains a hypothesis, but most neuroscientists believe it to be the method
SYNAPTIC VESICLE CYCLE
There is a nerve terminal (neuromuscular
junction) with many of these release sites. At
each of these release sites there is one or more
vesicle docked, ready to release its contents.
Blue represents the voltage-gated calcium
channels. At resting rate potential, it is very rare
for one of the vesicles to release their content. It
does happen sometimes, and that results in
MEPP.
Nerve action potential propagates into nerve
terminal and membrane depolarization opens
voltage-gated Ca2+ channels. Ca2+ diffuses into
terminal ([Ca2+]i ↑ from ~10nM → ~100µM)
simultaneously at ~1,000 release sites in the
terminal.
An increase in the calcium concentration
increases the probability that a vesicle will release
its contents. It is thought a calcium senor protein is
attached to these vesicles (e.g. synaptotagmin),
so that when the calcium binds to it, the vesicle is
more likely to release its content.
A short while thereafter, the increased
concentration of calcium, acting via the calcium
sensor protein, triggers the fusion of the vesicle
with the pre-synaptic membrane. The contents of
the vesicle (ACh) can then diffuse out into the
synaptic cleft. It diffuses a short distance to bind
and react with the AChR. Not all of the release
sites are activated and release transmitter, even
though calcium enters all. This seemingly
inefficient system is in fact advantageous because
it rarely reacts to only one action potential, but a
train of impulses (e.g. 40/s). This requires vesicles
docked and primed in reserve
A short while later those channels open and
sodium can diffuse in and depolarize and
produce the end-plate potential. This rises up
to a peak, before the ACh is broken down by
AChE, so the channels close again and the
membrane starts to repolarise, and the cell
returns to resting potential.
PROTEINS INVOLVED IN TRANSMITTER EXOCYTOSIS
- The SNARE proteins are associated with the synaptic vesicle.
- Coupled with the target membrane SNARE proteins they drag the synaptic vesicle closer to the presynaptic membrane and get it ready/prime it for transmitter release.
- Though there are only a small number ever primed, these proteins are important in this process.
- How they are controlled is not yet fully understood.
- Synaptotagmin is part of the synaptic vesicle membrane.
- It is at least one of the calcium sensors mentioned.
- The calcium channels near by are hooked up to a whole complex of vesicle, SNARE complex, calcium
channels and synaptotagmin.
- Calcium channels are thought to be aligned close the vesicle so there can be this rapid linkage between
depolarization, calcium channel opening, calcium diffusing in and activation of synaptotagmin.
Synaptotagmin has binding sites for calcium, and is thought to be able to increase the probability of
synaptic vesicle exocytosis.
The whole system seems to of evolved as an accurate way to control the release of transmitter.
RECYCLING OF ACETYLCHOLINE
- The reason AChE is thought to be there is to ensure that the ACh doesn’t keep working on these
receptors, and that it is broken down rapidly so that the synapse is ready for the next vesicle to
release its content.
- The ACh has enough time to activate receptors, but then it is broken down by AChE.
- The AChE is stuck to a mesh-work of extracellular matrix - proteins like collagen and fibronectin that
form a network/scaffolding/mesh through which molecules like ACh can easily diffuse through, but
have attached to them enzymes like AChE.
-
If AChE did not function, ACh would continue to activate AChR’s, which could be quite damaging to
the cell.
It would mean that the channels would not close again, so it would not be ready to respond to another
action potential coming down the nerve.
This would result in a loss of the close linkage between nerve and post-synaptic action potential –
post-synaptic activation could not accurately mirror pre-synaptic activation during high-frequency.
ACh is broken down into acetate and choline.
Acetate is a small molecule and is easily manufactured by the nerve terminal Choline requires more
energetic processes, so it must be recycled. Within the pre-synaptic membrane there are secondary
active transporter molecules, which use the concentration of sodium molecules to drag choline
molecules back into the nerve terminal cytoplasm, where it is resynthesised into ACh, before it is
prompted by another secondary transport molecule to return to the synaptic vesicles, so they are
ready for the next action potential some minutes later (it is a slow process)