Synaptic transmission and the vesicle cycle

key functional roles of chemical synapse

• Neural computation - integration of many input +/-

• Exhibit plasticity - development, learning and memory

• Act as targets for drug action - neurotransmitter synthesis, release, receptors, uptake, degradation to produce a broad range or complex series of effects

• inc functional flexibility

6 criteria of chemical synapse neurotransmitter

• synthesised and stored in the pre synaptic neuron

• released upon stimulation of pre synaptic neuron upon calcium dependent depolarisation

• located at regions in levels sufficient to evoke physiological responses

• must reproduce physiological effects when applied exogenously

• transmitter recognition and signal transduction mechanisms

• transmitter removal mechanisms

how is dales principle challenged

• challenged by co existence and co release of small molecule transmitter and peptides by interneurons eg GABA and enkephalins

• and more than one small molecule transmitter in some projection pathways eg L glutamate and dopamine

small synaptic vesicles characteristics 

• smaller diameter

• found in synapse active zones

• located close to calcium channels 

• contain small neurotransmitters 

• single AP

• constitutive - local vesicle recycling 

large dense cored vesicles characteristics

• larger diamater

• non specific locations

• release peptides

• found distant to calcium channels

• repetitive AP activity

• regulated control

why is the concentration of large dense cored vesicles lower

• relative proximity to the voltage gated channels

• only seen when there is a sustained AP

evidence for full fusion collapse cycling

• slam freezing of neuromuscular junction after electrical stimulation of motor neuron

• sections were visualised at different times after electrical stimulation

• activity led to increase in membrane surface area → vesicle recycling

docking step 1

• synaptic vesicles only dock at active zone

• presynaptic area adjacent to signal transduction machinery

• active zones differ between neurons by vesicle number

priming step 2

• priming - ready for release

• maturation of synaptic vesicle

• made competent to release transmitter

• requires ATP

• conformational change in proteins that drive release

fusion exocytosis step 3

• full fusion of synaptic vesicle and presynaptic terminal membrane

• requires calcium

• calcium sensor protein

• fusion induces exocytosis - takes 1ms

endocytosis step 4

• triggered by inc intracellular calcium

• involves cytoskeletal protein lattice formation from clathrin monomers

  • this helps to pinch off membrane with clathrin coated pits

• takes about 5 seconds

• ATP dependent

recycling step 5

• mechanism to conserve synaptic vesicle membrane via endosome

• decoating of clathrin coated pits is also atp dependent

• vesicles refill with transmitter

  • atp dependent

which steps of vesicle cycling require ATP?

• recycling

• endocytosis

• priming

kiss and run model of cycling

• full vesicle fusion may not be required

• neurotransmitter leaks out of small fusion pores

• recycled intact

  • no need for clathrin coated vesicles via endosome 

functional evidence for kiss and run model 

• flickering capacitance changes instead of up stepping capacitance

  • capacitance dependent on surface area

classical vs kiss and run cycling in terms of speed and capacity and frequency stimulation

• kiss and run - fast recycling and low capacity, favoured at low frequency stimulation

• whereas classical is slow, high capacity, favoured at high frequency stimulation

which pathway of cycling for glutamate release in hippocampus

• kiss and run

vesicle associated proteins

• synaptobrevins VAMP

• synaptotagmins

plasma membrane associated proteins 

• SNAP-25

• syntaxins

• therefore they are t-snares

snares for release

• synaptobrevin - single transmembrane spanning

• t snare

  • syntaxin - single transmembrane spanning

  • SNAP-25 - anchored to membrane by S-acylation

what domain is important for maintaining tight connection to the cell membrane

• syntaxin regulatory domain

What is the Ca2+ sensor?

• synaptotagmin

• found on vesciles

• binds to SNARE pins in absence of Ca2+ - during priming

• binds to phospholipids in C region in presence of Ca2+

• Ca2+ binding may cause synaptotagmin to pull vesicle into membrane

Why must SNAREs disassociate?

• to allow internalisation of empty vesicles

• re docking of another vesicle

• involves NSF - ATPase which binds to the SNARE-pin complex to facilitate disassociation

release machinery in docking

• trimeric complex of synaptobrevin/syntaxin/SNAP-25

• coiled coil quaternary structure

when do snares form a tighter complex

• during priming

priming of release machinery 

• ATP dependent

• assisted by Munc18 binding to syntaxin Habc domains

• zippering formation of SNARE pins

when does synaptogamin bind to SNARE pins

• absence of calcium

• during priming

when does synaptogamin bind to phospholipids

• in presence of calcium

• binds to c region