Required reading:

Text:

Delcomyn Chapt. 7

CD:

Interactive exercises

Additional reading:

Shepherd Chapt. 6 and 8

The Physiology of Excitable Cells, Fourth Edition
D. J. Aidley Cambridge University Press, 1998

Role of Ca²⁺ and depolarization in transmitter release

Both depolarization and extracellular Ca²⁺ are prerequisites for release of neurotransmitter

• Blocking an AP in a presynaptic terminal eliminates transmission at that terminal
• Reducing level of extracellular Ca²⁺ also eliminates transmission

Studied synaptic function in squid giant axon, pre and postsynaptic terminals large, accessible to intracellular microelectrodes

Experiment

• Impaled pre and postsynaptic terminals with microelectrodes
• Add tetrodotoxin (TTX) to reduce size of AP without eliminating it
• Stimulate presynaptic neuron and record EPSP in postsynaptic neuron
• EPSP not directly affected by TTX because generated by ligand sensitive channels allowing both K+ and Na+ to cross membrane, not voltage sensitive Na+ channels
• Vary amount of TTX and therefore amplitude of AP

• Small amounts of presynaptic depolarization did not result in detectable EPSP
• For presynaptic depolarizations larger than 45 mV, EPSP dependent on presynaptic potential

• Use larger amount of TTX to completely block AP
• Depolarize terminal by passing current directly via electrode

the greater the depolarization, greater transmitter release

Ca2+ and transmitter release

Ionophoresis

Technique for injecting ions, electrically charged molecules onto neuron

• Put CaCl₂, for example in electrode
• Apply positive current to electrode, force Ca²⁺ ions out onto cell
• By controlling time of onset, duration, and amplitude of current, regulate amount and time of Ca²⁺ release

• Use frog neuromuscular junction, reduce level of extracellular Ca²⁺ at synapse until synaptic transmission abolished
• Use iontophoresis (ionophoresis) to apply controlled amount of ions at precise times

• Transmission successful only when Ca²⁺ made available just before the action potential in the presynaptic axon reached the terminal

• Ca²⁺ ions enter neuron via voltage sensitive Ca²⁺ channels
• AP depolarizes terminal, opens channels, allows Ca²⁺ to diffuse in
• Ions trigger fusion of vesicles with neuronal membrane
• Level of intracellular Ca²⁺ kept low by active removal from cell, by sequestration and binding in endoplasmic reticulum and mitochondria

• Electron microscope studies indicate synaptic vesicles ubiquitous feature of synapses
• Biochemical studies indicate vesicles contain neurotransmitter

Vesicles are site of neurotransmitter storage and release into synaptic cleft

Quantal release

Vesicular hypothesis requires that neurotransmitter be released in discrete packets corresponding to contents of one vesicle, synaptic vesicles of a particular type in neuron are about same size

Miniature end plate potentials (mepps)

Evidence for quantal release from frog neuromuscular junction

• Highly amplified intracellular recordings from muscles at neuromuscular junction
• Minute, spontaneous postsynaptic potentials, amplitude varies in 0.5 mV steps, suggesting release of discrete numbers of quanta

• At neuromuscular junction estimated several hundred vesicles involved in EPP, small variation in size make it impossible to observe effects of adding one or two vesicles

Experiment demonstrating quantal nature of EPP

• Reduce number of quanta in EPP by partially blocking neurotransmitter release with high levels of extracellular Mg²⁺ (Mg²⁺ blocks Ca²⁺ channels)
• If Mg²⁺ levels are high, only a little neurotransmitter is released and release is quantal as shown above
• An estimated 200 quanta involved in normal neuromuscular junction EPP

Mechanism of vesicular release

1) Mobilization of vesicles

• Vesicles bound to cytoskeleton, often actin filaments, and held firmly in place, must be freed for release
• Process requires Ca²⁺ influx

Releasable pool vesicles attached to membrane and ready for release

• Vesicles that are mobilized are not the same ones as those released for given AP, delay between opening of Ca²⁺ channels and release of neurotransmitter only 0.1 to 0.2 msec, not enough time for cascade of biochemical events required to mobilize vesicle

1. release of presynaptic vesicles already docked at membrane
2. mobilization of vesicles from storage pool to releasable pool by promoting docking with membrane, mechanism by which vesicle moves and docks not yet understood

Mobilization mechanism

• synapsin 1 (member of family of proteins that binds with vesicular membrane and cytoskeleton) binds synaptic vesicles to cytoskeleton
• when synapsin phosphorylated, undergoes conformation change, detaches from vesicle membrane
• phosphorylation of synapsin I promoted by several types of protein kinase, some of which activated by Ca²⁺, thus Ca²⁺ influx causes mobilization of vesicle

Docking and release of neurotransmitter

Active zone presynaptic terminal membrane contains:

• specialized proteins for attachment of synaptic vesicles
• voltage sensitive Ca²⁺ channels
• sometimes other channels as well
• synaptic vesicles in active zone aligned in double rows
• 20 to 30 nm from these are double rows of particles embedded in synaptic membrane,evidence these are Ca²⁺ channels

• involves many proteins bound in vesicular membrane, bound to cell membrane of active zone, and free in cytoplasm
• exact role of each protein not yet clear, those in membrane bind in lock and key fashion with those in cell membrane to position vesicle

• N-ethylmaleimide sensitive factor (NSF), Soluble NSF Attachment proteins (SNAPs), wrap arround membrane and vesicular associated proteins and help to stablize association
• In the presence of Ca²⁺ and ATP, promote fusion and release of transmitter

• Several identified vesicular membrane proteins important in docking and fusion, synaptotagmin and vesicular SNAP receptors (v-SNAREs), bind with SNAPs from cytoplasm
• An important v-SNARE is synaptobrevin, also known as Vesicle Membrane Associated Protein (VAMP)

• Target SNAP receptors (t-SNAREs), two important t-SNAREs are syntaxin and Synaptosomal- associated protein (SNAP-25)

Docking occurs by mutual binding of t-SNAREs and v-SNAREs as multiprotein complex shown in figure

Fusion

Fusing of vesicle with active zone membrane and release of neurotransmitter into synaptic cleft

1. Fusion pore Initially small channel forms between interior of vesicle and outside of cell, triggered by Ca²⁺ entry and presence of ATP