# Synaptic transmission and the vesicle cycle ILO: - the two fundamental synaptic mechanisms by which excitable cells and in particular neurones can affect one another's function: electrical or chemical synaptic communication - how a neurotransmitter supporting chemical synaptic communication is defined and the diversity of types of small molecules that can be defined as being a neurotransmitter - how these are stored in small membranous vesicles, what constitutes the classical vesicle cycle and full collapse vesicle fusion during release - the experimental evidence for this full collapse fusion release model and how the vesicle membrane is recycled - further experimental evidence for an alternative to this model: the "kiss and run" model of vesicle cycling - the proteins that make up the release machinery and how they are readied for release during "docking" and "priming" - what happens to these proteins during the release process and the subsequent retrieval of vesicle membrane following full collapse fusion # Types of synapse - **chemical synapse** - molecules stored in vesicles - molecules diffuse across a gap - relatively slow - unidirectional - majority of synaptic transmission in the nervous system - **electrical synapse** - holes in adjoining cell membranes - linked by channels - gap junctions or connexons - signalling is very fast - bidirectional - direct electrical coupling between cells - electrical synchronisation in the heart - relatively rare in the nervous system - inhibitory interneurons or local networks # Chemical synapse Key functional roles - 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 ## Neurotransmitter 6 criteria :  ### Types  - amino acids - amines - purines - peptides - **dales principle** - neurons release just one transmitter at all of its synapses - 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 ## Vesicles - neurotransmitters are likely to be stored in one type of vesicle ### Types  For LDCVs - concentration is lower because of the relatively proximity to the voltage gated channels - only seen when there is sustained AP in a more global manner rather than restricted to synaptic active zone ### Cycling  1. vesicle is filled with neurotransmitter with appropriate transporter which uses ATP as an energy source to drive against conc gradient and fill the vesicle 2. vesicle collected in to reserve pool, mobilised to active zone for docking 1. atp dependent process 3. primed to be sensitive to calc conc to initiate membrane fusion 1. also atp dependent 4. exocytosis following inc in intracellular conc of calcium 5. vesicle membrane fully collapses into the membrane 6. loss of membrane recovered with endocytosis, calcium dependent with coated pits 1. uncoating requires atp 7. small vesicles become part of endosome, all recycled 8. then pinched off again to start the cycle ### Evidence for full fusion/collapse - slam freezing - rapidly cooling of the neuromuscular junction on a metal block after electrical stimulation of motor neurone axon fibres to initiate acetylcholine release - sections of the presynaptic membrane were visualised at different types after electrical stimulation to follow any changes in presynaptic membrane - activity led to increase in membrane surface area - therefore vesicle recycling ### Step 1 - docking - close association with plasma membrane - synaptic vesicles only dock at active zone - presynaptic area adjacent to signal transduction machinery - active zones differ between neurons by vesicle number ### Step 2 - priming - ready for release - maturation of synaptic vesicle - made competent to release transmitter - requires ATP - conformational change in proteins that drive release ### Step 3 fusion/exocytosis - full fusion of synaptic vesicle and presynaptic terminal membrane - requires calcium - calcium sensor protein - fusion induces exocytosis - takes 1ms ### Step 4 endocytosis - recovery of fused membrane - 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 ### Step 5 - recycling - mechanism to conserve synaptic vesicle membrane via endosome - decoating of clathrin coated pits is also atp dependent - vesicles refill with transmitter - atp dependent ### Kiss and Run Model? - fast recycling and low capacity, favoured at low frequency stimulation - may be majority of glutamate release in hippocampus - whereas classical is slow, high capacity, favoured at high frequency stimulation - full vesicle fusion may not be required - neurotransmitter leaks out of small fusion pores - SSVs recycled intact - and not recycled as clathrin coated vesicles via the endosome Functional evidence - flickering capacitance changes instead of up stepping capacitance - capacitance dependent on surface area ### Targeting vesicles Vesicle associated proteins - synaptobrevins VAMP - synaptotagmins Plasma membrane associated proteins - SNAP-25 - syntaxins ### Snares for release - synaptobrevin - single transmembrane spanning - t snare - syntaxin - single transmembrane spanning - SNAP-25 - anchored to membrane by S-acylation ### Release machinery in the different steps     Syntaxin regulatory domain is important in maintaining a tight connection to the cell membrane Snares form a tighter complex during priming - atp dependent - Habc domains binding assisted by Munc18 - zippering - formation of the SNARE pins 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

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

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