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Key Notes

Neurotransmitter release occurs most commonly by calcium-dependent exocytosis from vesicles, in response to excitation of the axon terminal by action potentials. Non-vesicular calcium-independent release of

glutamate and GABA via transporters can occur under some circumstances.

Transmitter is released in discrete packets, quanta, that correspond to exocytosis from a single vesicle. The spontaneous, random release of a single quantum causes miniature endplate potentials (at the

neuromuscular junction) or miniature postsynaptic potentials (at CNS synapses). Postsynaptic potentials arise from the release of several quanta simultaneously. At central synapses action potentials trigger

neurotransmitter release in only a proportion of occasions.

Calcium imaging shows how Ca2+_{moves in space and real time through}

cells. This reveals that following excitation of the nerve terminal calcium influx is restricted to a small region, but the local concentration reaches 200 mM, sufficient to trigger the exocytosis mechanism for small synaptic vesicles very rapidly.

Amines and peptides are released by high-frequency stimulation, only after an appreciable delay, because large dense-core vesicles are situated some distance from the active zone.

Several linked steps are involved in exocytosis. Recruitment shifts vesicles from a reserve pool into a releasable pool. Binding of vesicle- associated proteins and plasma membrane proteins permits the vesicles to be docked at the active zone in close proximity to voltage-dependent calcium channels. Partial fusion of the vesicle is achieved by priming, mediated by the assembly of a macromolecular complex, and involving the hydrolysis of ATP. The final rapid stage of exocytosis occurs when excitation triggers Ca2+_{influx. Binding of calcium to synaptotagmin}

permits fusion to go to completion.

In the Heuser–Reese cycle vesicles are recycled. Vesicle membrane is coated with clathrin so that it invaginates. Fission of coated vesicle is then triggered by hydrolysis of GTP bound to dynamin. Once in the

cytoplasm the vesicle loses its clathrin coat.

In central synapses, in addition to the Heuser–Reese cycle, a much faster kiss-and-run cycle, with much simpler endocytosis, allows high levels of release to be maintained by a small pool of vesicles.

Vesicular release

Release is quantal

The role of calcium

Exocytosis from large dense-core vesicles

Biochemistry of exocytosis

Endocytosis

Vesicular release Most neurotransmitter release occurs by transmitter-loaded synaptic vesicles fusing with the presynaptic membrane so that the contents of the vesicle are discharged into the synaptic cleft. This is an example of exocytosis. It is trig- gered by the arrival at the nerve terminal of an action potential which causes a transient and highly localized influx of Ca2+_{. After release the vesicle membrane}

is recycled from the presynaptic membrane to form new vesicles by endocy- tosis. The vesicles are subsequently loaded with transmitter via active trans- porters localized in the vesicle membrane.

Under some circumstances non-vesicular, Ca2+_{-independent release of trans-}

mitters, particularly GABA and glutamate, can be seen. This is thought to occur by the reversal of transport mechanisms that normally serve to reuptake trans- mitter from the synaptic cleft back into the nerve terminal.

Release is quantal In vesicular release, neurotransmitter is secreted in discrete packets or quanta. Each quantum represents the release of the contents of a single vesicle, about 4000 molecules of transmitter. At the neuromuscular junction (nmj), ACh released from a single vesicle diffuses across the cleft in about 2 µs, reaching a peak concentration of around 1 mM, activating 1000–2000 nAChR, to give a depolarization of the muscle fiber membrane locally of approximately 0.5 mV. Such events occur randomly and spontaneously under resting conditions and are called miniature endplate potentials (mepps). The endplate potential produced by a single action potential arriving at the motor neuron terminal results from the summation of about 300 quanta being liberated simultaneously from around 1000 active zones, presynaptic membrane regions specialized for transmitter release.

At CNS synapses miniature postsynaptic potentials (mpsps) are seen. They are the equivalent of mepps at the nmj. Miniature endplate potentials are excita- tory or inhibitory, depending on the transmitter, and are due to the release of transmitter from a single vesicle acting on only 30–100 receptors that lie under the active zone of central synapses. Excitatory and inhibitory postsynaptic potentials represent the summation of multiple mpsps, generated by an action potential invading several active zones simultaneously. This happens either because axons branch to form several discrete terminals or because some termi- nals have more than one active zone.

The active zone of many CNS synapses appears to have only one release site. Classical transmitters are imported into vesicles driven by the efflux of H+

via specific transporters. The proton gradient is generated by a vesicular proton ATPase. Peptides are packaged in the Golgi apparatus from which vesicles bud to be transported to the axon terminal, itself incapable of protein synthesis.

Autoreceptors respond to the transmitter released by the neuron in which they are located. They occur at the presynaptic terminal, the soma and dendrites. They regulate neurotransmitter release, synthesis, and neuron firing rate, usually homeostatically.

Related topics Overview of synaptic function (C2) Nerve–muscle synapse (J1)

Calcium channels (C6)

C5 – Neurotransmitter release 65

Refilling

This is known as the one vesicle or one quantum hypothesis. However trans- mitter release does not happen every time an action potential arrives at the presynaptic terminal. Individual active zones behave in an all-or-none fashion because an action potential will either trigger the release of the single quantum or not. The proportion of successes will reflect the probability of release. At central synapses the probability of release varies between different sites and at least at some synapses it also depends on the recent history of the synapse. The arrival of an action potential at a nerve terminal causes an influx of Ca2+

through voltage-dependent calcium channels. Direct evidence for the role of calcium is provided by calcium imaging, a technique which makes visible how Ca2+_{signals spread in time and space through cells. Fluorescent dyes are used}

which on binding Ca2+_{absorb UV light at a different wavelength than they do in}

the unbound state. Neurons are preloaded with the dye and the emission of UV from the dye is observed in response to its excitation by the two distinct absorp- tion wavelengths. This gives a quantitative measure of how the concentration of Ca2+_{changes in the neuron in real time.}

This technique shows that it takes about 300 µs for calcium channels at the active zone to open in response to an action potential. The driving force for calcium entry is extremely high because of the large concentration gradient. The free Ca2+_{concentration at rest in a terminal is 100 nM whilst the external concen-}

tration is about 1 mM. Despite this huge concentration gradient, the presence of diffusion barriers and calcium buffers in the terminal restrict the rise in calcium concentration to within 50 nm of the channel mouth. This region is called a calcium microdomain. The [Ca2+_{] within 10 nm of the channel mouth rises to}

100–200 µM, which matches the half-maximal concentration of Ca2+ _{for gluta-}

mate release. Several overlapping microdomains cooperate to trigger the release of a vesicle in close proximity.

In contrast to small clear synaptic vesicles (SSVs), the mechanism for release from large dense-core vesicles (LDCVs) vesicles takes longer and has a higher affinity for calcium (half-maximal release occurs at about 0.4 µM), because only

a small amount of Ca2+ _{manages to diffuse to the LDCVs, which are some}

distance from the active zone. Hence exocytosis of amines and peptides occurs with a delay of about 50 ms and only in response to high-frequency stimulation of the neuron which causes high levels of calcium influx.

Exocytosis from SSVs involves several linked steps, most of which need calcium. Nerve terminals contain two pools of SSVs. The releasable pool is located at the active zone and can take part in repeated cycles of exocytosis and endocytosis at low neuron firing frequencies. The reserve pool consists of vesi- cles tethered to cytoskeletal proteins, and can be mobilized by repetitive stimu- lation to join the releasable pool. This is called recruitment. Liberation of a

vesicle from the cytoskeleton requires Ca2+_{-dependent phosphorylation of}

synapsin I, a protein which anchors vesicles to actin filaments in the terminal.