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Medneuro- neurophysiology (M1-2) > vesicle release and synaptic transmission > Flashcards

vesicle release and synaptic transmission Flashcards

1
Q

History and concepts of NS

A
  • Gerlach (1871)- neurons as protoplasmic networks
  • Sherrington (1897)- the term synapse (from greek- to hold/clasp)
  • Golgi (1906)- the reticular theory - diffuse nervous network- axon & dendrites; Golgi staining- visualisation of neurons, division of cortex into 3 layers
  • Cajal (1906)- the neuron doctrine- definitely defined the neuron theory; vis. of neurons in cerebellum–< different cells have different morphology
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2
Q

Golgi staining

A

blocks of nervous tissue hardened in potassium bichromate–> immersion in silver nitrate solution

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3
Q

What are the functional regions of a neuron?

A

Input–> dendrite–>integration in soma–> conductance through axon-> output along axon terminal

  • different neurons have slight different morphology depending on their function
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4
Q

first NT to be discovered

A
  • Acetylcholine (Ach) discovered by Löwi (1921).

- responsible for communication at the NMJ (Dale 1930)

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5
Q

Active zone of the presynaptic terminal

A
  • the AZ is the release site of NT, it is an e- dense material at the presyn. terminal; highly specialised structures with specific proteins associated w. the AZ.
  • NT receptors on the postsynaptic terminal are located right across from AZ–> close proximity between pre and postsynaptic NT sites
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6
Q

the NMJ

A
  • chemical, excitatory synapse between motor neuron and muscle fibres
  • only 1 axon per group of muscle fibres (–> axon+group of muscle fibres = motor unit)
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7
Q

the discovery of Katz and Fatt

A
  • provided the first insight into the manner of NT release
  • NT is release in quanta
  • each quantum of NT produces a postsyn. potential of a fixed size (quantal postsyn. pot)
  • total postsyn. pot. is the sum of all quantal potentials.
  • recorded from the frog’s NMJ and observed spontaneous EPSP of 0.5 mV –> miniature endplate pot. (minis)
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8
Q

minis

A

miniature endplate potentials;

  • like the endplate potentials, the signal decreases with distance.
  • represent the responses to small quanta of NT that are spontaneously released
  • 1 mini is the response to synchronous release 5000 Ach molecules
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9
Q

How many Ach receptors are activate in order to generate a mini of a live 0.5 mV?

A

1 Ach receptor produces current of ca. 0.3 microV–> mini involves opening of ca. 2000 receptors

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10
Q

Quantal release hypothesis

A
  • spontaneous release occurs rarely and the release of NT has a fixed size.
  • small postsyn. signal (mini EPSP/IPSP)
  • spontaneous, infrequent, but accelerated and synchronised by AP invading the terminal
  • now or integral no. of the amount of NT- 0.5, 10, 15 mV
  • 1 event=5000 NT molecules
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11
Q

what cell organelles account to NT quanta?

A

synaptic vesicles

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12
Q

characteristics of SV

A
  • each vesicle stores 1 quantum of NT and releases its entire content into the synaptic cleft (all or non manner)
  • SV release NT at AZ
  • the AZ contains a cloud of SVs
  • SV diameter: 40-50 nm
  • some SV are docked to membrane at AZ and are the first to be released. the rest are located closely.
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13
Q

what ion generates the EPSP and leads to NT release?

A

Ca2+ - discovered by Katz & Miledi (1967), recorded from the giant ganglion of the squid

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14
Q

What experiment could be done to test which ion is responsible for NT release? (how presynaptic AP triggers NT release)

A

a) Na+ channels were blocked with TTX–> depol. of the presynaptic terminal with direct current can trigger NT release even after Na+ channels are blocked –> Na+ not responsible for NT release
b) K+ channels were blocked w. TEA (at the same time Na+ were also blocked) –> injection of depol. current to the presynaptic terminal–> EPSP were normal sized (normal NT release–> neither Na+ nor K+ trigger NT release
c) Ca2+ influx triggers NT release; even when Na+ & K+ channels were blocked–> postsyn. depol. varies w. the amount of presyn. inward current (more Ca2+ more EPSP)

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15
Q

process of Ca2+ dependent SV release

A
  • Microdomains w. high [Ca2+] form in cytosol near open Ca2+ channel –> trigger SV release.
  • concentration of Ca2+ outside is 1.8 microM
  • -> AP in AZ opens a fraction of Ca2+ channels–> Microdomains (high Ca2+) organise around open channel
  • -> Ca2+ influx –> SVs that are docked to membrane at AZ can fuse
  • -> NT release (SV sense changes in [Ca2+] and are activated due to their close proximity to Ca2+ channel)
  • -> Facilitation: release of a 2nd set of SV (further from channel) due to increase of residual intercell. Ca2+ from 0.1 to 0.11 nanoM
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16
Q

what SVs have the highest release probability?

A

Sv at 50 nm are close to Ca2+ channel and have the highest release prob. due to the close proximity to the channel.

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17
Q

release probability of SVs is dependent on:

a. intercellular Ca2+ concentration
b. Na+ concentration
c. extracellular Ca2+ concentration
d. distance of SV from Ca2+ channel
e. presence of Ca2+ chelators (EGTA/BAPTA)
f. all of the above

A

a, d, e

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18
Q

Ca2+ chelators

A

2 Ca2+ chelators that inhibit release probability:
- EGTA- slow chelator (takes time to bind to Ca2+), only competed w. SVs that are far away from channel, loosely coupled to Ca2+ channel (due to the distance)

  • BAPTA- fast chelator, binds to tightly coupled SVs, located close to Ca2+ channel, closer SVs are affected by BAPTA
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19
Q

The hallmarks of NT release process

A

speed, efficiency and flexibility

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20
Q

the SV cycle

A
  1. NT uptake- SV filled w. NT by active transport
  2. formation of reserve pool - Filled SV form a cluster
  3. docking- SV dock to membrane at AZ
  4. priming - ATP dependent priming reaction
  5. fusion- Ca2+ triggered fusion –> SV discharging content
  6. endocytosis- e.g. clathrin mediated..
  7. alternative to (6)- direct recycling or via endosome
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21
Q

mechanisms of SV retrieval after NT discharge

A
  1. reversible fusion pore- The SV doesn’t fuse with the membrane completely, instead NT is released through fusion pore–>kiss and stay/kiss and run; the fastest way reusing SV; predominant at low to normal release rates.
  2. Clathrin mediated endocytosis- cltathrin coated pits after NT release; predominant at normal to high release rates.
  3. bulk retrieval- only for high release rates
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22
Q

Kiss and run

A

a reversible fusion pore mechanism. after NT discharge through fusion pore–> closure of fusion pore–> SV goes back to reserve pool

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23
Q

Kiss and stay

A

a reversible fusion pore mechanism. after NT discharge through fusion pore–> closure of fusion pore–>SV stays on membrane

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24
Q

Clathrin coat endocytosis

A

Excess membrane is retrieved through endocytosis by Clathrin coat pits around the SV and detach it from membrane. these pits are found throughout the axon terminal except for at AZ.

Process: Nucleation (membrane invagination=pits)–> cargo selection–>coat assembly (clathrin around pit)–> scission (separation of coated SV from membrane)–> uncoating

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25
Q

Bulk retrieval

A

Excess membrane reentrers the terminal by budding from uncoated pits. these pits are found primarily at AZ.

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26
Q

vesicle pools

A

a. ready release pool (RRP)- available immediately but depletes rapidly after high stimulation
b. recycling pool- maintains release during moderate stimulation
c. reycycling pool- (majority of SVs within clusters), constitute depot of SVs

27
Q

What methods can be used to study SV release?

A
  • FM dyes )optical monitoring of SV recycling)

- PHlurin- protein tags

28
Q

FM dye method

A
  • partially lipophilic dyes (invisible in aqueous solution–> fluorescence increase almost 100 fold when they partition into membrane)
  • because of the charged head group of FM, they cannot cross the lipid membrane–> fluorescence is limited to the outer membrane and recycling SVs
    a. FM dye uptake- labels cells stimulated–> exocytosis–> dye uptake by SV during endocytosis
    b. dye release- stimulation of loaded synapses releases dye from SV–> fluorescence decreases due to repartitioning of dye into aqueous solution
29
Q

pHlurine- protein tags

A
  • pH sensitive GFP, that can bind to the luminal domain of synaptic proteins (e.g. synaptobrevin)
  • pH of the lumen of SV is normally acidic (low)–> pH luring fluorescence quenched
  • SV fuse to plasma membrane–>luminal tag is exposed to the alkaline pH of the extracell. environment–> fluorescence increases
  • after fusion & endocytosis- pHlurine fluorescence is requenched (due to reaccidification of SV)
30
Q

classes of NT-release proteins

A
  • NT transporters- actively pump NT into SV using proton-gradient across SV membrane
  • exocytotic proteins
  • recycling proteins
31
Q

what proteins are involved in NT release (table from slides)

A
  • proton pump- generating electrochemical gradient of protons
  • vesicular transmitter transporter- NT uptake into SV
  • VAMP/synaptobrevin- component of SNARE-complex, acts in a late, essential step for SV fusion
  • synaptotagmin- CA2+ binding, possible trigger for fusion & a component of SV-docking at release site via interaction w. SNARE complex & lipid, promotes clathrin med. endocytosis by binding AP-2 complexes
  • Rab3- role in regulating SV targeting and availability
  • synapsin- tether SV to actin cytoskeleton
  • cysteine string protein- promotes reliable coupling of AP to exocytosis
32
Q

How do SVs fuse (process)

A

a. mobilisation of SVs from reserve pool:
- synapsins are peripheral membrane proteins that are bound to SV. ATP and actin filaments–> Ca2+ influx–> synapsin phosphorylate and released from SV–>SV migration to AZ

b. tethering, docking and priming:
- Munc18 binds to closed syntaxin1–>syntaxin opens (Munc18 stays bound but switches binding to interact w. SNARE complex)
- a protein complex w. 3 multi domain proteins (RIM, RIM-BP, Munc13) mediates the docking and priming od SV, and recruit Ca2+ to docked SVs:
- RIM binds to Rab3 and Rab27 –>SV docking
- RIM binds to Munc13–> activation of Munc13–> binds to syntaxin1–>conformational switch of syntaxin–>SNARE-complex partially assembled.
- complexin binds to SNARE complex–> increases its priming (zippering of SNARE brings membrane closer)
- SNARE/SM complex- substrate for Ca2+–>Ca2+ binding to synaptotagmin–>fusion pore opening–>transforms SNARE from trans to cis–>cis SNARE can be disassembled.

33
Q

How does RIM/Rab3 activates Munc13?

A

By converting it from a homodimer to heterodimer

34
Q

what are Munc18 and Munc 13?

A

SM proteins that prepare the SNARE proteins for assembly of SNARE complex; both bind to syntaxin1.

  • Munc18 binds first to closed syntaxin1–>opens it
  • Munc13 (binds late) catalyses the opening of syntaxin1, and the position switch of bound Munc18 on syntaxin1 to interact w. SNARE proteins.
35
Q

What are SNARE proteins

A
  • SNARE- Soluble NSF attachment protein receptor
  • mediate SV release
  • v-SNARE (vesicle)- synaptobrevin/VAMP
  • t-SNARE (target/membrane)- Snataxin and Snap25
36
Q

Synaptotagmin

A
  • structure: a single membrane spanning domain, a short, intraluminal domain & a large cytoplasmic domain consisting of 2 C2 domains connected by a linker
  • synaptotagmin is a sensor for Ca2+ (and thus for NT release)
  • synaptotagmin triggers SV exocytosis (by Ca2+ binding)
37
Q

Synaptotagmin:

a. is a component of the SNARE-complex
b. is a Ca2+ sensor for NT release
c. is involved in the priming of SVs
d. is activated by Munc13
e. triggers SV exocytosis

A

b & e

38
Q

Priming process

A
  • I priming- Ca2+ influx triggers binding of synaptotagmin to partially zippered SNARE complex w. Munc 13 and 18 bound to it–>synaptotagmin pulls SNARE complex to plasma membrane (PM)
  • II priming- arrest after positioning of SV onto PM –> Ca2+ influx pulls SV closer via synaptotagmin-mediated cross linking –> SNARE assembly (full opening of syntaxin, displacement of Munc18 & binding of complexin)
39
Q

what is complexin?

A

a co-factor of synaptotagmin; binds to partially assembled SNARE complex and increases its priming and activates pre-fusion SNARE/SM protein complexes

40
Q

method to investigate docking and fusion

A

flash & freeze EM:
Channelrhodopsin–>isolation of the gene (optogenetics)–> insert the DNA s sequence into specific neurons–> high pressure freeze–>EM

41
Q

how can botulinum toxin can be used for investigating release and docking?

A
  • Bot B- cleaves VAMP/synaptobrevin
  • Bot C- cleaves syntaxin and Snap25 (t-SNARE)
  • -> Bot b prevents the release of NT, and Bot c reduces the number of docked SVs
42
Q

what are electrical synapses?

A

Gap junctions-

  • contacts between neighbouring membranes
  • made of hexameric proteins (connexins)
  • when channel opens- ion flow to other side of the membrane (along electrochemical gradient)
  • very common in astrocytes
43
Q

what are chemical synapses?

A
  • communication between cells via transmitter release:
  • AP in presynaptic cell leads to a cascade of events where SVs fuse w. cell membrane–> SV release NT into the synaptic cleft–> released NT bind to specific receptors in the posesyn. cell –> ligand gated ion channels open
44
Q

the enzyme responsible for degradation of Ach

A

Ach-esterase

45
Q

symmetric/asymmetric synapses

A
  1. asymmetric- excitatory, prominent PSD, spherical vesicles, NT- mainly glutamate
  2. symmetric- inhibitory, no PSD visible in EM, pleiomorphic vesicles, NT- GABA/glycine
46
Q

Which of the following statements is wrong?

a. it is possible to estimate the protein composition (% of proteins) of synaptic vesicles
b. SVs number, localisation and morphology can be studied with EM
c. it is possible to obtain synaptosome purified fractions
d. it is possible to study adult mice w. mutations on vesicular associated proteins because non of them is lethal at postnatal stages
e. the synaptic vesicle cycle is determined by time-lapse fast freezing samples after after stimulation of release

A

d

47
Q

the following groups of proteins are involved in vesicular release:

a. synaptotagmin,Snap25, Vamp-2, Munc13 and PSD95
b. synapsin, snap25, Ach esterase, CamKIIA and Munc13
c. synaptobrevin, Vamp2, Rab3 and G-alpha-o1
d. synaptotagmin, Rab3, and vamp2
e. all of the above

A

d

48
Q

Which of the following statement is correct?

a. the life cycle of SV in the bouton is regulated by proteins in the soma
b. the life cycle of SV in the pontoon happens in each bouton by local regeneration independent from the soma
c. the life cycle and regeneration of SV happens both locally in the bouton (for fast, short term release) and remotely in the soma (for long term regulation and regeneration of SV)

A

b

49
Q

what are scaffold proteins? where can they be found (relating to synaptic transmission)

A

scaffold proteins are members of the signalling cascade of cell surface receptor. their function is multifaceted, and they are involved in tethering and localisation of signalling components, and regulation of correct signal transduction among others.

  • Scaffold along the AZ guides and regulate SV positioning, and inside the SV there is also a scaffold that is responsible for the protein-protein contact between SV and AZ (e.g. Rab3?)
  • also on the postsynaptic density (PSD) there are scaffold proteins that are involved in trafficking, anchoring and clustering of excitatory NT-receptors (e.g. SHANK3)
50
Q

Is [Ca2+]i increase a prerequisite for transmitter release? What type of Ca2+-channels?

A

yes. [Ca2+]i is a prerequisite for NT release, since the fusion of SVs is mediated by Ca2+ influx. (see the experiment of Katz & Miledi).
- Ca2+ channels are clustered at AZ and located near docked SV (therefore rapid NT release after Ca2+ influx).
- These Ca2+ channels are N type Ca2+ channels.

51
Q

Where is ATP synthesised in chemical synapses? why is it important for NT release?

A

ATP is generated in mitochondria and is required for SV fusion and recycling

52
Q

what mechanisms make NT receptors so specific?

A
  • intrinsic transcriptional program at birth directing the pairing of NT and receptors.
  • Adhesion molecules- signalling molecules that direct the localisation of specific molecules onto a specific site. the combination of different adhesion molecules creates the identity of the cell (e.g. neuroligin1 always codes for X-type cell).
53
Q

what is the avg. life span of a synaptic protein?

A

5-8 days

54
Q

what are neurexin and neuroligin?

A
  • Neurexin and neuroligin are bidirectional organisers of developing synapses
  • act as adhesion molecules
  • neuroligin triggers differentiation of presynapse and neurexin triggers the differentiation of postsynapse
55
Q

How is the PSD organised (by scaffold proteins)?

A
  • assembled from families of multi domain scaffold proteins
  • the scaffold proteins (i.e. SHANK) create docking sites for receptor molecules.
  • the scaffold proteins are very stable, and the receptors can be added (docked) or eliminated (removed) easily –> key for plasticity
  • individual scaffold proteins build macromolecular signalling complexes (excitatory synapses can have many complex for different NT, whereas inhibitory are simpler and only have GABA receptors
56
Q

SHANK 3…

a. is a presynaptic, active zone protein
b. is involved in synaptic plasticity
c. senses the Zn2+ influx into dendritic spines
d. assembles a postsynaptic signalling complex
e. essential for the transcriptions of synaptic proteins
f. is a postsynaptic scaffold protein

A

b, c, d & f

57
Q

what is FMRP? how do mutations in FMRP molecules affect synaptic activity?

A
  • FMRP is a protein that is involved in transporting mRNA to dendrites.
  • FMRP in dendrites is involved in regulatory mechanisms of protein expression.
  • loss of FMRP in synaptic sites leads to many synaptic dismorphies, as they control scaffold proteins (e.g. fragile x syndrome)
  • mutations in FMRP molecules affect the abundance of synapses and synaptic transmutation (e.g. in autism- mutations in SAHNK3)
58
Q

what is the relationship between SHANK3 and zinc?

where is the Zinc coming from? what is the effect of zinc deficiency?

A
  • the oligomerisation of SHANK3 is mediated by its C-terminal SAM domain and modulated by Zinc.
  • Zn2+ is released naturally with vesicle opening from presynaptic terminal.
  • if there is a mutation at this site, the cell tends to favour disassembly of PSD (–> Zn2+ sink- sucks the Zn2+ out of SHANK3 and leads to disassembly of synapse);
  • Zn2+ deficiency leads to low Zn2+ content in presyn. vesicles and therefore affects the postsynaptic terminal.
59
Q

what inotropic receptors are there?

A
  • glutamate receptors- most important excitatory receptors (e.g. NMDAR, AMPAR)
  • GABA receptors- most important inhibitory receptors
  • Glycin receptors- inhibitory receptors located in spinal cord, brainstem..
  • Ach receptors- nicotinic Ach receptors (mainly in NMJ)
60
Q

features of ligand gated ion channels

A
  • create a hydrophobic surface in channel to help the selectivity of ions –>channel opens
  • gating property change of channel by changing the subunit of the receptor (cannot happen in Ca2+ channes) –> e.g. NMDAR
61
Q

how can subunit replacement affect synaptic plasticity and learning?

A

subunit replacement can affect the amount of ions that can access the channel.
–> for example: in NMDAR the subunit of the receptor from birth until p15-20 is NR1+NR2b–>from p15 the subunit 2b is replaced by 2a which affects the capacity of the channels (gating property change) and thus affects learning ability of humans

62
Q

what are G-protein coupled receptors?

A
  • non-inotropic receptors
  • associated with G-proteins
  • G-proteins activate various messengers that modify the permeability of a nearby ion channel
  • sit close to inotropic receptors and increase the variability of signals
63
Q

What is Hebb’s rule of synaptic plasticity

A

neurons that fire together wire together, neurons that fire out of sync lose their link
–> defining the ability of the synapse to change its strength, according to the firing pattern and time of neurons

Medneuro- neurophysiology (M1-2) (6 decks)

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