Lecture 13 Flashcards

1
Q

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

A
  • 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)
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2
Q

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

A

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
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3
Q

Temperature and NT release

A

Higher temperature = faster NT release

Low temp causes a pre-synaptic delay

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4
Q

Ca2+ influx specifically though Cav2 type calcium channels

A

drives pre-synaptic secretion of neurotransmitters

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5
Q

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

A
  • 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)
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6
Q

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

A
  • 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!
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7
Q

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

A
  • 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
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8
Q

Pre-synaptic injection of Ca2+ chelator BAPTA….

A

BAPTA, a fast chelator, blocks synaptic transmission

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9
Q

Pre-synaptic injection of Ca2+ chelator EGTA….

A

EGTA, a slower chelator, does not block synaptic transmission

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10
Q

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

A

Within 100 nm

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11
Q

Exocytosis

A

Fusion of pre-synaptic vesicles with the cell membrane

this fusion is driven by a series of proteins

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12
Q

SNARE Protiens

A

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

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13
Q

SNARE proteins

Component: Synaptotagmin

A

Synaptotagmin is the Ca2+ sensor protein

• Via Ca2+ binding C2 domains

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14
Q

SNARE proteins

Component: Core SNARE Complex

A

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

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15
Q

v-SNARE proteins

A

SNARE proteins that are tethered in vesicles

• Synaptotagmin and synaptobrevin

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16
Q

t-SNARE proteins

A

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

17
Q

Munc 18

A

Prevents Syntaxin from interacting with other SNARE proteins

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

18
Q

4 Alpha Helix Structure

A

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

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

19
Q

Complexin

A

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

20
Q

Pore formation –> exocytosis

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

A

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

21
Q

Pore Formation

A

vesicle fuses with cell membrane, resulting in exocytosis

22
Q

Pre-synaptic scaffolding protein RIM

A

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

might reflect an evolutionarily conserved mechanism….

23
Q

Mini ESPSs

A

tiny, spontaneous synaptic events

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

24
Q

Evoked EPSPs

A

Much larger than mini EPSPs

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

25
Q

Fatt and Katz

A

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