Lecture 13

Question 1

Katz and Miledi’s experiment to test how APs trigger Neurotransmitter release

Answer 1

• 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)

Question 2

Are Na+ and K+ currents required for pre- synaptic secretion of neurotransmitters?

Answer 2

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

Question 3

Temperature and NT release

Answer 3

Higher temperature = faster NT release

Low temp causes a pre-synaptic delay

Question 4

Ca2+ influx specifically though Cav2 type calcium channels

Answer 4

drives pre-synaptic secretion of neurotransmitters

Question 5

Rodolfo Linás and his colleagues were the first to provide direct evidence for pre-synaptic Ca2+ currents

Answer 5

• 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)

Question 6

By using the voltage clamp to mimic pre-synaptic action potentials, Linás was able to generate normal-looking EPSPs

Answer 6

• 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!

Question 7

Linás et al also showed that Ca2+ influx occurs at discreet locations in the pre-synaptic terminal…

Answer 7

• 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

Question 8

Pre-synaptic injection of Ca2+ chelator BAPTA….

Answer 8

BAPTA, a fast chelator, blocks synaptic transmission

Question 9

Pre-synaptic injection of Ca2+ chelator EGTA….

Answer 9

EGTA, a slower chelator, does not block synaptic transmission

Question 10

Cav channels must be positioned very close to the site of neurotransmitter release

Answer 10

Within 100 nm

Question 11

Exocytosis

Answer 11

Fusion of pre-synaptic vesicles with the cell membrane

this fusion is driven by a series of proteins

Question 12

SNARE Protiens

Answer 12

mediate Ca2+-dependent fusion of pre-synaptic vesicles with the cell membrane

Question 13

SNARE proteins

Component: Synaptotagmin

Answer 13

Synaptotagmin is the Ca2+ sensor protein

• Via Ca2+ binding C2 domains

Question 14

SNARE proteins

Component: Core SNARE Complex

Answer 14

Synaptobrevin, Syntaxin, and SNAP-25 form the “core SNARE complex”

Question 15

v-SNARE proteins

Answer 15

SNARE proteins that are tethered in vesicles

• Synaptotagmin and synaptobrevin

Question 16

t-SNARE proteins

Answer 16

SNARE proteins tethered the cell membrane (i.e.. the target, thus the “t” in “t-Snare”)
• SNAP-25 and syntaxin

Question 17

Munc 18

Answer 17

Prevents Syntaxin from interacting with other SNARE proteins

Dissociates, then re-associates in order to promote formation of 4 alpha helix structure

Question 18

4 Alpha Helix Structure

Answer 18

Synaptobrevin → 1 helix
Syntaxin → 1 helix
SNAP-25 → 2 helices

Pulls the vesicle closer to the cell membrane (referred to as vesicle priming)

Question 19

Complexin

Answer 19

Stabilizes the vesicle in its primed state in order to prevent vesicle fusion

Question 20

Pore formation –> exocytosis

Ca2+ influx and Ca2+ binding to _________ causes a conformational change allowing it to interact with the core SNARE complex and displace _____________

Answer 20

Ca2+ influx and Ca2+ binding to synaptotagmin causes a conformational change allowing it to interact with the core SNARE complex and displace complexin

Question 21

Pore Formation

Answer 21

vesicle fuses with cell membrane, resulting in exocytosis

Question 22

Pre-synaptic scaffolding protein RIM

Answer 22

mechanism by which Cav2 channels associate with pre- synaptic structures for exocytosis in Drosophila and rodents

might reflect an evolutionarily conserved mechanism….

Question 23

Mini ESPSs

Answer 23

tiny, spontaneous synaptic events

Each about the same amplitude of ~1 mV (unless there is summation)

Question 24

Evoked EPSPs

Answer 24

Much larger than mini EPSPs

• 50 mV to 70 mV (evoked) vs. 1 mV (mini)

Question 25

Fatt and Katz

Answer 25

Determined that neurotransmitters are released in packets Added extracellular Mg2+ and reduced extracellular Ca2+ in order to reduce the amplitude of evoked ESPSs Found that the evoked potentials were stepwise, indicative of summing of individual events. Thus determined that neurotransmitters are released in packets