chapter 6 - sv pools

Question 1

6.1 [sv pools] what is the 3 pool model (a)? how does it function (b)? Describe it

Answer 1

• The existance of distinct SV pools was first suggested by electrophysiological experiments: during high-frequency stimulation of presynaptic neurons, the postsynaptic response declined over time = synaptic depression - so the frequency of psr decreases over time - how can it be explained?

Hypothesis: during frequent stimulation, NT quanta are released from a limited pool of “release-ready” SVs, emptying the pool at a rate faster than fresh SVs could replenish those expended.
> Assuming that each AP discharges a similar fraction of SVs, a progressive decrease in release is expected with each stimulus, until a lower steady state level, in which release is perfectly balanced by the slow refilling of the “readily-releasable pool”.
Thus, only a limited fraction of SVs is “release-ready” and additional SVs are recruited to replenish this pool. - It takes longer to refill the docking zone with vesicle that the “BREAK” between stimulations

Readily releasable pool (RRP): consists of SVs that are release-ready
Recycling pool (RP): consists of SVs that can replenish the RRP
> Total recycling pool (TRP)= RRP + RP: groups all SVs capable of undergoing release
Resting pool (R,P): consists of SVs that remain unreleased, even after prolonged stimulation

Question 2

6.2 [sv pools] explain the methods to study sv pools and apply to a given scientific question

Answer 2

methods to distinguish RTP from RtP

1. > TRP can be labeled using strong stimulation in the presence of extracellular FM dye or quantum dots (QDs).
   QDs are nanoparticles of uniform and defined size, which have remarkably stable and bright photoluminescence (or electrodense core).
   To quantify the number of SVs in the TRP, these methods can be combined with EM, using photoconversion. In this procedure FM dye-labeled synapses can be exposed to the chemical 3,3’-diaminobenzidine (DAB), and prolonged photoillumination causes excitation-induced conversion of DAB to an electron-dense product only in dye-containing SVs (exocytosed and then endocytosed- trp pool). Also QDs with an electron-dense core can be used.
   - allows to count vesicles

2
> PHIuorin: GFP variant (pK, = 7.1): fluorescence is quenched at the acidic pH of SVs, and recovered on SV fusion and exposure to the near-neutral extracellular pH - sv have a 3-4 ph, so fluorine doesn’t fluoress but then if exocytosed they do.
> SynaptopHluorin: fusion protein of pHluorin to the lumenal portion of SV proteins.
> upon high-frequency stimulation, all SVs from the TRP will be released. - stimulating the presynaptic nerve (10hz) leads to exocytosis and fluorescence
> Acute application of bafilomycin (BAF) prevents re-acidification of recycled vesicles, thus resulting in fluorescent labeling of these SVs - if we add baf sv will fluoress when exocytosed but if endocytosed again then baf makes sure that sv can’t be re-acidified so sv will keep fluoressing
> Ammonium chloride (NH4CI) neutralizes the acidic interior of R,P vesicles, resulting in unquenching of synaptopHluorin in these SVs - NH4cl makes sure that ph of sv will be neutralized (fluorescence starts)
> measurement of the change in fluorescence intensity during HFS with
BAF and subsequent NH,CI thus allows to identify TRP and R,P
- doesn’t allow to count vesicles

3
> The number of SVs in the RRP (NRRp) can be determined using electrophysiological methods, but also using an osmotic challenge, typically hypertonic sucrose, which induces Ca?+-independent exocytosis of SVs in the RRP

4
> Other tools and methods often used in the study of SV pools: cf. Box 1 in Alabi and Tsien, Cold Spring Harb Perspect Biol, 2012