Katz and Miledi’s experiment to test how APs trigger Neurotransmitter release
- Used the squid giant neuron-neuron synapse used for escape response
- TTX application slowly shut off Nav channels
- Post-synaptic APs resulting from pre- synaptic stimulation dropped off first
- From this point on, a plot EPSP vs pre- synaptic AP amplitude showed that stronger pre-synaptic APs elicit stronger EPSPs
- The same was true for “simulated” pre- synaptic APs (i.e. depolarizing current injection)
Are Na+ and K+ currents required for pre- synaptic secretion of neurotransmitters?
Na+ and K+ currents are not required for pre- synaptic secretion of neurotransmitters
- All you need is depolarization
- The amplitude of EPSPs (and hence the amount of NT release) depends on the amplitude of pre-synaptic depolarization
Temperature and NT release
Higher temperature = faster NT release
Low temp causes a pre-synaptic delay
Ca2+ influx specifically though Cav2 type calcium channels
drives pre-synaptic secretion of neurotransmitters
Rodolfo Linás and his colleagues were the first to provide direct evidence for pre-synaptic Ca2+ currents
- Na channels blocked with TTX, K channels blocked with TEA vv
- Voltage clamp depolarization of pre-synaptic neuron (-18 mV → activates HVA Cav2 channels) led to Ca2+ influx
- An EPSP followed shortly thereafter
- Depolarization to +60 mV → no Ca2+ current, no EPSP
* Why? Ca2+ reversal potential (Vm = ECa) so no driving force for Ca2+ entry - However, upon repolarization, pre-synaptic Ca2+ influx though already open channels generated an EPSP
* Before channels have a chance to close (de-activate), lots of Ca2+ gets in because driving force is high (Vm ≠ ECa)
By using the voltage clamp to mimic pre-synaptic action potentials, Linás was able to generate normal-looking EPSPs
- Direct proof that Na+ and K+ currents don’t play a direct role in synaptic transmission!
- Their effect on Vm and Cav2 channels activation is what matters
- Accounted for the missing portion of the synaptic delay!
Linás et al also showed that Ca2+ influx occurs at discreet locations in the pre-synaptic terminal…
- Used Ca2+ indicator dyes that change fluorescence upon Ca2+ influx
- Pre-synaptic regions that lit up were highly localized (Ca2+ micro-/nano-domains)
- Why? Endogenous Ca2+ chelators and exchangers/pumps prevent Ca2+ from getting far
- Cav channels must therefore be positioned closely to the site of neurotransmitter release for Ca2+ to be effective
Pre-synaptic injection of Ca2+ chelator BAPTA….
BAPTA, a fast chelator, blocks synaptic transmission
Pre-synaptic injection of Ca2+ chelator EGTA….
EGTA, a slower chelator, does not block synaptic transmission
Cav channels must be positioned very close to the site of neurotransmitter release
Within 100 nm
Exocytosis
Fusion of pre-synaptic vesicles with the cell membrane
this fusion is driven by a series of proteins
SNARE Protiens
mediate Ca2+-dependent fusion of pre-synaptic vesicles with the cell membrane
SNARE proteins
Component: Synaptotagmin
Synaptotagmin is the Ca2+ sensor protein
• Via Ca2+ binding C2 domains
SNARE proteins
Component: Core SNARE Complex
Synaptobrevin, Syntaxin, and SNAP-25 form the “core SNARE complex”
v-SNARE proteins
SNARE proteins that are tethered in vesicles
• Synaptotagmin and synaptobrevin
t-SNARE proteins
SNARE proteins tethered the cell membrane (i.e.. the target, thus the “t” in “t-Snare”)
• SNAP-25 and syntaxin
Munc 18
Prevents Syntaxin from interacting with other SNARE proteins
Dissociates, then re-associates in order to promote formation of 4 alpha helix structure
4 Alpha Helix Structure
Synaptobrevin → 1 helix
Syntaxin → 1 helix
SNAP-25 → 2 helices
Pulls the vesicle closer to the cell membrane (referred to as vesicle priming)
Complexin
Stabilizes the vesicle in its primed state in order to prevent vesicle fusion
Pore formation –> exocytosis
Ca2+ influx and Ca2+ binding to _________ causes a conformational change allowing it to interact with the core SNARE complex and displace _____________
Ca2+ influx and Ca2+ binding to synaptotagmin causes a conformational change allowing it to interact with the core SNARE complex and displace complexin
Pore Formation
vesicle fuses with cell membrane, resulting in exocytosis
Pre-synaptic scaffolding protein RIM
mechanism by which Cav2 channels associate with pre- synaptic structures for exocytosis in Drosophila and rodents
might reflect an evolutionarily conserved mechanism….
Mini ESPSs
tiny, spontaneous synaptic events
Each about the same amplitude of ~1 mV (unless there is summation)
Evoked EPSPs
Much larger than mini EPSPs
• 50 mV to 70 mV (evoked) vs. 1 mV (mini)
