Neuromuscular Junction

The Structure of the Neuromuscular Junction

nerve terminals have discrete NT release sites in array with muscle NT receptor sites

the sites of NT release = active zones; opposite an enfolding in the muscle cell surface

basal lamina is between the nerve terminal and muscle surface and binds AChE (acetylcholine esterase)

the muscle membrane opposite the active zones has an electron dense specialization (site of acetylcholine receptors)

terminal branches are arrayed in clusters in mammals rather than strung out over the muscle surface as in other vertebrates (frogs)

the axon lies in a trough that is thrown into folds

Synaptic Vesicles, Exocytosis, and Membrane Recycling

vesicle hypothesis = acetylcholine release is effected by fusion of synaptic vesicles with presynaptic membrane

the nerve terminal has capacity to synthesize acetylcholine as well as synaptic vesicles, and does not depend on deliver of neurotransmitter from the neuron

vesicle recycling = model for local packaging and release of neurotransmitter

synaptic vesicles fuse with presynaptic membrane at the active zone and flatten out into it

at a remote site, membrane is recovered and processed into new synaptic vesicles

vesicle-associated proteins may be recovered by coated vesicles

proton pump Þ pumps H⁺ into vesicle

ACh transporter (pH regulated) Þ doesn’t work in membrane, but works in vesicle

membrane itself may be recovered in larger, uncoated invaginations

Active Zones and Voltage-Gated Ca⁺⁺ Channels

synaptic vesicles release neurotransmitter only at specific sites (active zones)
active zones are created by local collections of voltage-gated Ca⁺⁺ channels in presynaptic membrane
AP opens voltage-gated Ca⁺⁺ channels located in active zones near synaptic vesicles, which leads to rapid localized increase in calcium concentration in microdomain Ca⁺⁺ binds to proteins on vessicle and triggers release

Basal Lamina and AchE

terminates activity of acetylcholine
interposed between nerve terminal and muscle in synaptic cleft bound to the basal lamina

The Acetylcholine Receptor

actually permeable to Na⁺, K⁺, Ca⁺⁺; but at normal resting potentials, primarily an inward Na current and small inward Ca current
receptors located at the crest’s of the post synaptic folds
5 subunits: 2 are identical which bind 2 acetylcholine molecules
ligand gated channel

End-Plate Potentials, Miniature End Plate Potentials, and "Saturated Disks"

EPP = depolarization of muscle cell resulting from stimulus-evoked transmitter release

Represents the summed effect of NT released from many vesicles as they fuse with the presynaptic membrane at the active zones (typical EPP = 25mV)
The greater the influx of Ca⁺⁺, the more NT released, and the greater EPP generated

paired pulse facilitation

Ca⁺⁺ influx from 1^st AP adds to the Ca⁺⁺ influx from 2^nd AP since it doesn’t have enough time to get rid of all the Ca⁺⁺

MEPP = small (0.5mV), spontaneous EPP recorded in the absence of nerve stimulation

Since EPP only changes in MEPP-sized increments, this implies acetylcholine released in quanta (quantal hypothesis)

It is possible to record single small stimulus-evoked EPPs (0.5mV) by decreasing the amount of Ca⁺⁺ in fluid

Will not produce graded potentials under conditions of low extracellular Ca⁺⁺
Evokes either no response (0mV), or a single potential of 0.5mV

Hence, MEPPs are believed to be the depolarization of the muscle membrane caused by the action of the acetylcholine released by a single synaptic vesicle which spontaneously fused with the presynaptic membrane – this is the vesicle hypothesis

Quantal nature of the small EPPs

Fusion of no vesicles = no EPP
Fusion of one vesicle = MEPP (0.5mV)
Fusion of three vesicles = EPP with an amplitude 3x the MEPP (1.5mV)

10-20% of 10,000 molecules of ACh released by a single vesicle bind to AChE in the cleft

the remaining NT binds to AChR in a patch of about 0.3m m² in area = the disk of AChR saturated with agonist NT

saturated disk model: vesicle releases enough ACh to bind all AChE and AChR in 0.1 ms

NT can reach AChR in 15ms after vesicle fusion

other definitions:

quantal content: number of vesicles released
quantal size (MEPP amplitude): amplitude of response to one quantum
MEPP frequency: MEPPs/time

The Neuromuscular Junction is Specialized for Rapid, Repetitive Action

at frog neuromuscular junction, 250 active zones present along the nerve terminal

not all of the active zones are utilized during depolarization
during near maximal sustained activity (10Hz), 50 vesicles are released per depolar
in one sec, 500 vesicles are released = 2 per active zone
even at tetanic rates of stimulation, only 10% of AChR would be used
thus, receptors are only reused once every 10sec; plenty of time for previous load to dissociate and receptors to recover, allowing junction to support sustained, rapid rates of synaptic transmission

amplitude of the EPP is more than enough to trigger an AP (safety zone)

the rate of NT output falls with prolonged stimulation (depression)

due to safety zone, this decreased NT output can still trigger an AP

if coupled with curare (competative inhibitor of AChR), EPP will fall below the safety zone Þ no AP

Post Tetanic Potentiation – mediated by synapsin I

presynaptic AP produces normal postsynaptic AP
tetanus AP produces increased postsynaptic AP, and subsequent postsynaptic AP's will be increased even with normal presynaptic AP
proposed mecahnism for this involves the protein synapsin I:

synapsin I binds to vesicles and to the cytoskeleton
ionized Ca⁺⁺ influx activates Ca⁺⁺-calmodulin dependent protein kinase II, which phosphorylates synapsin I and decreases its affinity for vesicles; the vesicles are then relatively free of the cytoskeleton and can fuse more easily with the presynaptic membrane
during tetanic AP, most of synapsin I is cleaved, so when next AP occurs, the Ca⁺⁺ will not need to be used for synapsin cleaving leaving more Ca⁺⁺ available for vesicle release

Three Types of Vesicles

(1) vicinity vesicles

vesicles in terminal region but not close to the release site

(2) docked vesicles

vesicles moved near the membrane bound with synapsin I
syntaxin moves vessicles from vicinity to docked position

tetanus and botulism toxin both destroy syntaxin thereby keeping vesicles in vicinity form – leads to paralysis with no long term effects

(3) primed vesicles

activated vesicles near the membrane that are ready to fuse (without synapsin)
need energy input to prime the docked vesicles

Synaptic Plasticity at NM Junction

normal AP = ~ 200 vesicles/fiber

evokes all or none muscle AP (EPP threshold)

EPP usually reaches threshold, so NM junct termed relay synapse

Depression

reduction in quantal content
occurs even at low freq
strongest at high quantal content
mechanism: depletion of readily released vesicles

Facilitation

increase in quantal content
strongest at low quantal content (low depression)
only at high freq
mechanism: accumulation of Ca⁺⁺ at nerve terminal

net is usually depression but normally no noticible effect – safety factor (extra ACh and AChR)

synaptic transmission is fast (~20 ms) but muscle contraction slow – leads to summation of signals and tetanic contraction

Drug Effects

AChR antagonist (curare)

nondepolarizing block
effective reduction of quantal size
no effect on quantal content
reduced amplitude of EPP
reduced amplitude of MEPP

AChR agonist (nicotine, succinylcholine)

depolarizing block

high receptor activation

voltage-gated Na⁺ channel inactivation

in immediate area of muscle (active zone) Þ muscle inactivated

quantal size reduced; quantal content normal

Ca⁺⁺ channel blocker

decrease extracell Ca⁺⁺
increase extracell Mg⁺⁺
decreased quantal content
same quantal size and MEPP amplitude

AChE inhibitors

increased timecourse of MEPP and EPP
increase amplitude, quantal size
no effect on quantal content
main effect: increase safety factor
if severe, may yield depolarizing block

Nerve-Muscle Preparation

1:1 stim:contraction until freq (50 Hz) where contractions fuse into tetanic contraction

observed effect is muscle tension; graded responses from stimulation of different # fibers, not graded response of one

low concentration curare

low freq: small amplitude reduction – most EPPs still threshold
high freq: tension decreased, ceases at tetanic – from decreased size (curare) and decreased content (depression)
AChE inhib can help up to a point

succinylcholine less freq dependant than curare; also not helped by AChE inhibition

Mg⁺⁺ reduces contraction slightly at low freq; high freq less depression, so may facilitate