Lecture 3 - Post synaptic events in neurotransmission Flashcards
What is the process of neurotransmitter release from the synapse?
1 - as an action potential goes through the synapse, membrane depolarisation opens the voltage-gated Ca2+ channels
2 - the in crease in Ca2+ tiggers synaptic vesicles to fuse with the plasma membrane
3 - The releases neurotransmitter diffuses across the synaptic cleft and interacts with the specific post synaptic receptors (either ligand gated ion channels or G protein coupled receptor)
4 - Neurotransmitter can also interact and activate presynaptic autoreceptors, and spillover into adjacent synapses
5 - Neurotransmitter is then eaither degraded or taken up by glia or presynaptic transporters
6 - The synaptic vesicle membrane is then retreived for recycling by endocytosis
7 - Neuropeptides are released from dense cored granules
What molecule is the trigger for neurotransmitter release?
Ca2+ increase
What is the size of the synaptic cleft and how is it held together?
~50nm
held together by many structural proteins
What are the two types of posy synaptic receptor and what are their properties?
Ligand gated ion channel -causes ion fluxes -depolarise the membrane G protein coupled receptor -slow -can undergo signalling -can also impact on ion flux
What must the synaptic vesicles be recycled?
-cell has a limited number of vesicles (~100)
therefore must be recycled via the synaptic vesicle cycle
How was it experimentally shown that neurotransmitter release is coupled to Ca2+ influx in the presynaptic terminal?
- used a voltage clamp technique on a squid giant axon that terminated on a muscle
- found out that potassium and sodium channels were not important by blocked with TEA (K) and TTX (Na)
- stimulated the pre with an influx of Ca2+ current
- this lead to depolarisation in the muscle
- concluded that depolarisation in the post synaptic membrane was due to presynaptic calcium levels
What is the structure of voltage gated Ca2+ channels?
-structure similar to Na+ and K+ channels
-like Na+ channels, Ca2+ are monomeric (evolutionarily younger than K channels)
-4 subunits
-
What are the features of Ca2+ channels involved in synaptic vesicle release?
- resting Ca2+ in the presynapse is ~0.1μm (compared to 1.3 mM in the synaptic cleft)
- level required to trigger synaptic vesicle release is 5-10μm
- release sites are clustered around Ca2+ channels
Why is calcium a good trigger for neurotransmitter release?
Because of the difference in concentration inside and outside of the cell
Extracellular concentration ~1.3 mM
Presynapse concentration ~0.1μm
When stimulated and get a calcium influx, pretty much a digital signal, as there is a massive change very quickly in the concentration (often increases from 0.1μm to 200μm when only 5-10μm is needed)
Either ‘on’ or ‘off’
What is MEPP?
Mini End Plate Potential
How was it shown that neurotransmitter release is quantal?
-recorded from squid giant axon at the outer muscles
-stimulated the axon, and recorded from the post synaptic cell (the membrane potential), measured the end plate potential (EPP)
-discovered that without stimulation they were measuring constant, spontaenous MEPP, which had a constant amplitude of 0.4mV
-when the axon was stimulated weakly (Ca2+ dependent), they discovered that they got EPP in multiples of 0.4mV
=corresponds to synaptic vesicle volume, showing that neurotransmitter release was digital (either released or not)
-also removed all the calcium from the synaptic cleft (from 1.3 mM to 0) and found that the EPPs stopped - however that the MEPPs did not stop, showing that these happen via a calcium independent process
= suggesting vesicles are constantly ready to be released and spontaenously fuse and are released from the plasma membrane
What is the Ca2+ sensor (discovered by Tom Sudhof) in the presynapse, and what are the fusion machinery?
Sensor
-Synaptotagmin
Fusion
-SNARE proteins
What is the process of the synaptic vesicle cycle?
1 - NT uptake
2 - Translocation of the neurotransmitter vesicle to the presynaptic membrane
3 - Docking of the neurotransmitter vesicle via SNARE proteins
4 - Priming prefusion (requires ATP)
5 - Ca2+ influx leads to Fusion Exocytosis and the release of the neurotransmitter into the synaptic cleft
6 - Endocytosis of presynaptic membrane, vesicle membrane coming back in again
7 - Translocation away from the membrane
8 - Endosome fusion with the early endosome
8 - budding off the early endosome
10 - NT uptake
How was the synaptic vesicle cycle studied in the past and more recently ?
Heuser and Reese (1973), Ceccarelli (1972)
-used electron microscopy to visualise the synapse and pre/post
Viewed:
-lots of mitochrondria (ATP dependent process)
-release sites much darker
-docked vesicles ready to release
-defined amount of neurotransmitter in the vesicle
More recently - fluorescent techniques combined with electrophysiology have allowed the synaptic vesicle to be analysed in real time
How was the structure of synaptic vesicles visualised by Tim Roffman?
Mass spectrometry analysis
