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NESC 2570 > Neurotransmitter Release > Flashcards

Neurotransmitter Release Flashcards

1
Q

NMJ to study NT release

A
  • NMJ used in 50s and 60s to study chemical transmission due to it being a large and accessible synapse
  • motor neurons form large presynaptic terminal called end plates
  • stimulation of Motorola nerve generates end plate potentials (EPP)
  • EPP usually elicits AP in muscle
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2
Q

Miniature EPP

A
  • Katz studied changes in muscle membrane potential in the absence of motor nerve stimulation
    • miniature EPP
  • amplitude of mEPP is homogenous (around 0.5mv)
  • mEPP are too big to represent potential change in response to singe acetylcholine receptor opening
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3
Q

Quantal nature of NT release at NMJ

A
  • motor nerve stimulation with low extracellular Ca2+: sometimes no EPP, sometimes very small
  • amplitude of smallest EPP equals that of mEPP
  • larger EPP are multiples of mEPP
  • EPP made up of individual units elicited by exocytosis of a quantum of NTs
  • mEPPs result from the spontaneous, AP independent release of one quantum of NT
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4
Q

Evidence for NT storage in synaptic vesicles

A
  • Katz observed accumulation of small vesicles at presynaptic terminals
  • evidence: acetylcholine enriched in vesicles isolated from brain tissue by density gradient centrifugation (Whittaker)
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5
Q

NT quanta correspond to synaptic vesicles

A
  • freeze-fracture EM of NMJ (Heuser and Reese)
  • NMJ stimulated then frozen and analyzed using electron microscopy
  • vizualization of SV undergoing fusing

-4AP (potassium channel inhibitor) to increase NT release

  • parallel recordings of EPPs to determine quantal content
  • number of fusing synaptic vesicles and quantal content to correlate
  • interpretation: exocytosis of individual synaptic vesicle leads to release of one quantum of NY
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6
Q

NT dependent on Calcium

A

-presynaptic injection of calcium chelators eliminate post synaptic potential

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7
Q

Inhibition of presynaptic VHCC by cone snail

A
  • cone snail toxin causes paralysis
  • omega-conotoxin black specific N-type voltage gated calcium channels
  • calcium channels needed for NT release at NMJ
  • funnel web spider toxin blocks P/Q VGCC which is necessary for NT release at central synapses
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8
Q

Characteristics of VGCC activation

A
  • VGCC activate slowly in response to membrane depolarization
  • delayed opening accounts for synaptic delay
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9
Q

Biogenesis of SV containing small-molecule NTs

A
  • synthesis and uptake of small molecule NTs locally within presynaptic terminals
  • either uptake of NT from extracellular space by plasma membrane transporters
  • or uptake of precursors from extracellular space and local synthesis of precursors
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10
Q

Loading of small molecule NT into SV

A
  • NTs loaded into SV against electrochemical gradient by vesicular NT transporters
  • secondary active transport: antiport of H+
  • H+ gradient created by vesicular proton pump (uses ATP)
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11
Q

Biogenesis of SV containing peptide NTs

A
  • neuropeptides synthesize in the soma (ER->golgi)
  • peptide filled large dense core vesicles are transported along microtubules via fast axonal transport
  • neuropeptides do not undergo reuptake
    • degraded by proteolytic enzymes
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12
Q

Evidence for local recycling of SV

A

-PM surface area needs to be held constant despite SV exocytosis: compensatory endocytosis

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13
Q

SV cycle

A
  • endocytosis complete (10-20s) after exocytosis
  • endocytosis vesicles bypass endosomes, becoming SVs
  • recycled SV associate with PM and become fusion competent in approx 1 min
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14
Q

SV pools

A
  • readily releasable: SV immediately available for release (2-4%)
  • reserve pool: SVs available for exocytosis but not immediate release (20%)
  • resting pool: non-recycling SVs, 80%
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15
Q

Sequence of events leading to NT release

A
  1. Docking: SV come in close proximity to PM
  2. Priming: interaction between proteins in SV and PM
  3. Fusion: of SV with PM is calcium dependent
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16
Q

SNARE complex

A
  • membrane fusion involves SNAREs
  • SNARE complex bring negatively charged membrane into close apposition (energy required)
  • SV membrane: synaptobrevin
  • PM:syntaxin + SNAP25
  • SNARE complex formation involves generation of energetically favourable alpha-helical bundle
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17
Q

Clostridial neurotoxins

A

-responsible for tetanus and botulism are highly specific proteases the block NT release by cleaving SNAREs

18
Q

Munc18

A
  • essential for evoked and spontaneous NT release
  • binds syntaxin in closed conformation, unfolding it so it can interact with other SNARES
  • binds to SNARE complex to facilitate SNARE complex mediated fusion directly
19
Q

NSF and a-SNAP

A
  • disassemble SNARE complexes
  • SNAREs are reused
  • SNARE complexes very stable to chaperone is required to dissociate them
  • NSF is the chaperone
    • uses ATP as energy source
  • NSF binds to complex via adapter protein a-SNAP
20
Q

Synaptotagmin

A
  • calcium sensor for evoked release
  • integral SV membrane protein with SNARE complex
  • binds calcium
  • AP eveoked fast release in mice lack functional synaptotagmin gene
    • spontaneous release unaffected
21
Q

Synaptotagmin structure

A
  • cytoplasmic c-terminus of synaptotagmin has 2 C@ domains that bind 4-5 calcium ions
  • calcium binding cooperative: affinity for calcium initially low; rises with partial binding of Ca2+
  • C2 domains bind phospholipids in a calcium dependent manner: affinity low in calcium free; high in calcium bound state
22
Q

Synaptotagmin mechanism

A
  • binds to SNARE complex
  • calcium binding leads to additional alectrostatic charges on C2 domains, causing them to bind negatively charged phospholipids in SV and PM
  • membranes now in close apposition
    • energetically favourable to fuse
23
Q

Synaptotagmin properties and implications for NT release

A
  • low initial binding affinity of synaptotagmin for calcium; high calcium extrusion and buffering capacity of neurons means SV fusion only close to open calcium channels in calcium microdomains and nanodomains
  • cooperative binding of calcium leads to super linear dependence of NT release on VGCC
24
Q

Structure of active zone

A
  • docked SVs surrounded by filamentous material: active zone cytomatrix (large protein complex)
  • active zone cytomatrix had modular structure at NMJ and possible CNS synapses
25
Q

Functions and components of active zone cytomatrix

A
  1. Provide docking site for SVs and facilitate priming
  2. Recruit VGCCs
  3. Provide transsynaptic cell adhesion

Components:

  1. Synaptic cell adhesion proteins (neurexins)
  2. Scaffolding proteins (RIM, Munc13)
26
Q

Neurexins

A
  • family of presynaptic cell adhesion proteins
  • bind to families of postsynaptic cell adhesion proteins: neuroligins etc
  • organize pre and postsynaptic specializations with scaffolding proteins such as CASK and PSD95
  • implicated in neuro developmental disorders such as autism
27
Q

RIM

A

-modular scaffolding components with multiple functions:

  1. Tethering of SV via interaction with SV protein Rab3
  2. Support of SV priming via interaction with Munc13
  3. recruitment of VGCCs
28
Q

Munc13

A
  • essential for NT release
  • facilitate priming (SNARE complex formation)
  • Munc13 activity regulated:
    • calcium activates Munc13 and facilitates priming
    • DAG also activates Munc13
29
Q

Sequence of events during clathrate mediated endocytosis

A
  1. Nucleation leads to assemble of a clathrin lattice on patches of PM
  2. Membrane invagination generates clathrin coated pits
  3. Fission of membrane to creat clathrin coated vesicle
  4. Uncoating: disassembly of clathrin coat
30
Q

Nucleation

A
  • adaptor proteins (AP2 and AP180) bind to proteins to be endocytosis
  • clathrin binds to adaptor proteins
  • clathrin oligomerizes
31
Q

Invagination

A
  • during oligomerization, clathrins triskelion structure facilitates invagination
  • has spherical structure when many come together to help invagination
  • integral membrane proteins such as Epson also promote membrane curvature
32
Q

Membrane fission

A
  • dynamin involved in membrane fission
  • inhibition causes arrest of endocytosis at stage of invagination pits
  • oligomerization of dynamin into stacks of rings around membrane stalk initiates budding
  • Dynamin has GTPase activity: GTP hydrolysis causes tightening of rings, pinching off synaptic vesicles
33
Q

Mechanism of uncoating

A
  • Hsc70 is a chaperone needed to disassemble energetically favourable clathrin coat
  • Hsc70 is an ATPase
  • Hsc70 needs adaptor protein: Auxilin to bin to clathrin
34
Q

Actin in SV recycling

A

-2 roles:

  1. Transport of SV from endocytosis sites to active zone
  2. Retention of SV in resting pool to prevent them from reaching active zone
35
Q

Synapsins in SV recycling

A
  • peripheral membrane proteins on SVs
  • synapsins bind actin and may help anchor SV to cytoskeleton
  • membrane association is phosphorylation dependent
    • CaMKII or PKA causes dissociation from SVs so they can go to active zone
  • proposed function: maintenance of reserve pool of SV
36
Q

Stochastic nature of NT release

A

-synaptic transmission occur only with a certain NT release probability: Pr=# responses/# of trials

37
Q

Factors determining NT Pr

A
  • size of readily releasable SV pool (RRP)
  • RRP determined by # of SV that can dock at active zone, and fraction of docked SVs that are primed
  • individual SVs in RRP display different likelihood of AP triggered fusion
    • differences in presynaptic calcium currents
    • differences in spatial coupling of calcium influx and primed SVs
38
Q

Short term plasticity of NT release

A

-in response to repetitive stimulation, NT release can facilitate or depress

  • high Pr synapses often depress
  • low Pr synapses often facilitate
  • many show both
39
Q

Causes of short-term depression

A
  • depletion of readily releasable pool of docked and primed SVs
  • lower open time of Ca2+ channels, so less calcium influx
  • receptor desensitization
40
Q

Short term facilitation

A
  • following initial stimulation, basal calcium levels increase
  • residual calcium can:
    • increase SV docking
    • accelerate SV priming
    • facilitate SV fusion

NESC 2570 (9 decks)

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