Control of voluntary muscle: the neuromuscular synapse Dr Bill Phillips Dept of Physiology

Control of voluntary muscle: the neuromuscular synapse Task of the neuromuscular synapse Structure and molecular organisation The endplate potential (EPP) and miniature endplate potentials (MEPPs) Quantal hypothesis and vesicle hypothesis Synaptic vesicle cycle and regulated exocytosis

Task of the neuromuscular synapse Low efficacy Synapse: Many such inputs are summated by the motor neuron Neuromuscular synapse Relay synapse High efficacy

Structure and molecular organisation: plan view Postsynaptic acetylcholine receptors Presynaptic nerve terminal Last internode Terminal branches 10m

A single transmitter Release site

Nerve terminal branch: Is loosely wrapped around by a Schwann cell that seems to be important for organising the nerve terminal

The presynaptic release site: Many synaptic vesicles are found in clusters in the cytoplasm above the presynaptic “active zone” Some vesicles are docked next to voltage-gated Ca2+ channels Action potential opens these voltage-gated Ca2+ channels leading to a brief influx of Ca2+

Synaptic cleft: Filled with basement membrane that holds pre- and postsynaptic cells together, an open mesh of proteins such as collagen and laminins Acetylcholinesterase (AChE) bound to the basmement membrane hydrolyses acetylcholine (Ach) to choline and acetate.

Postsynaptic membrane: Terminal branches sit within gutters Secondary folds in the membrane contain voltage-gated Na+ channels- where the muscle Hodgkin cycle starts “Lips” of secondary folds contain high and uniform concentrations of nicotinic acetylcholine receptors (AChR) that bind and respond to ACh released by the terminal.

Recording the endplate potential

Subthreshold recordings In normal muscle, every presynaptic action potential results in a postsynaptic muscle action potential The stimulus that triggers the muscle action potential is hidden If the [Ca2+]o is lowered below physiological levels, a sub-threshold endplate potential is recorded.

The endplate potential (EPP) and miniature endplate potentials (MEPPs) EPP is a large amplitude depolarisation that occurs briefly in the postsynaptic membrane following stimulation of the presynaptic nerve. MEPPs are small amplitude depolarisations that occur spontaneously even when the nerve is not firing action potentials The amplitude of the EPP is depends upon [Ca2+]o. If [Ca2+]o is lowered, the amplitude of the EPP declines more and more. The smallest amplitude EPP is of the same amplitude as the spontaneously occurring MEPPs.

Quantal hypothesis and vesicle hypothesis The EPP is thought to be the sum of the many simultaneous MEPPs occuring in synchrony Depolarisation and influx of Ca2+ at many transmitter release sites is though to occur when the nerve action potential invades the nerve terminal branches. This is thought to trigger simultaneous release of small packets (quanta) of acetylcholine (ACh)

Quantal hypothesis proposes that ACh is released in equal sized packets from many release sites in a probabilistic fashion

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) While unproven, the vesicle hypothesis is widely accepted as likely to be true.

Synaptic vesicle cycle and regulated exocytosis Nerve terminals are too far away from their cell soma to rely upon a continuous supply of new synaptic vesicles filled with neurotransmitter. They need to recycle both transmitter and synaptic vesicles

Steps in the synaptic vesicle cycle: 1. Influx of Ca 2+ triggers exocytosis of ~1/10 primed vesicles 2. Empty synaptic vesicle membrane is recaptured (endocytosed) 3. Translocated 4. Empty veswicle fuses with endosomal compartment, acidifies 5. New, reformed vesicle buds from endosome 6. Transporter protein pumps acetylcholine from cytoplasm into vesicle (choline salvaged from synaptic cleft) 7. Vesicle transported to presynaptic membrane 8. Docking/tethering of synaptic vesicle to presynaptic membrane 9. Priming of vesicle- ready for regulated exocytosis.

Overview of steps in neuromuscular transmission The axon terminal contains synaptic vesicles filled with neurotransmitter Arrival of the nerve action potential opens voltage-gated Ca2+ channels in the nerve terminal plasma membrane Ca2+ influx triggers increased probability of vesicle exocytosis ACh diffuses across a synaptic cleft, binds to and activates AChR channels on the postsynaptic membrane of the target neuron The AChR channels opening increases the permeability of the membrane to both Na+ and K+ (monvalent cations). Since the resting membrane potential of the muscle cell is -90mV this results in depolarisation (Why?).