1. Altered Presynaptic Vesicle Release and Cycling during mGluR-Dependent LTD 
Stanislav S Zakharenko, Leonard Zablow, Steven A Siegelbaum 
Neuron 
Volume 35, Issue 6, Pages (September 2002)
DOI: /S (02)00898-X

2. Figure 1
Effect of Induction of LTD on Loading and Unloading of FM 1-43 in Presynaptic Boutons
(A) Mean maximal slope of the fEPSP (mean ± SEM) as a function of time for slices that underwent 5 Hz stimulation for 3 min in the absence (control, closed circles) and in the presence of 10 μM MPEP (open circles).
(B) Protocol showing periods of electrical stimulation, FM 1-43 application, ADVASEP-7 application, and application of D-AP5 (50 μM) or MPEP (10 μM) (durations not drawn to scale).
(C) Images of FM 1-43 fluorescence in a single field in the stratum radiatum of the CA1 region during dye unloading stimulation before (upper three images) and after (bottom three images) induction of LTD (numbers represent time in seconds after start of stimulation during Unload 1). Left: images of FM 1-43 fluorescence taken after loading and just before the start of the unloading protocol (0 s). Middle: images taken 65 s after start of the unloading protocol, a time when the intensity of FM 1-43 fluorescence decreased by ∼50% (t1/2) for control slices (i.e., no LTD). Right: images taken after maximal unloading of FM 1-43 fluorescence using prolonged stimulation (1.5 Hz for 4 min followed by 10 Hz for 2 min).
Neuron  , DOI: ( /S (02)00898-X)

3. Figure 2
Effect of LTD on FM 1-43 Unloading Kinetics in Individual Slices
(A and B) Time course of FM 1-43 unloading from individual puncta in a single slice before ([A], n = 59 puncta) and 1 hr after ([B], n = 55 puncta) induction of LTD.
(C) The mean time course of puncta unloading before and after induction of LTD, averaged over the puncta from panels (A) and (B), respectively. Puncta were divided into two populations, destaining boutons (>50% loss of initial fluorescence intensity following 4 min of stimulation at 1.5 Hz; bottom two curves) and nondestaining boutons (<50% fluorescence intensity loss; top two curves).
(D and E) Frequency histograms of unloading rates (1/t1/2) of individual puncta from eight slices before ([D], n = 502 puncta) and after ([E], n = 435 puncta) induction of LTD with 5 Hz stimulation.
Neuron  , DOI: ( /S (02)00898-X)

4. Figure 3
FM 1-43 Loading and Unloading in Response to the LTD Induction Protocol in the Absence and in the Presence of MPEP
(A1–D1) Effects on FM 1-43 destaining when LTD induction protocol was applied in normal ACSF (absence of MPEP).
(A2–D2) Effects on FM 1-43 destaining when LTD induction protocol was applied in presence of 10 μM MPEP.
(A) Average rate of FM 1-43 unloading (1/t1/2 values) from destaining boutons measured before (closed symbols) and after (open symbols) 5 Hz stimulation ([A1], p < , paired t test). Each symbol represents the average rate of FM 1-43 release for 30–80 boutons from a single slice.
(B) The number of boutons that loaded with FM 1-43 before (closed symbols) and after (open symbols) 5 Hz stimulation ([B1], p < 0.01, paired t test).
(C) The number of nondestaining boutons before (closed symbols) and after (open symbols) induction of LTD ([C1], p < 0.01, paired t test).
(D) Average fluorescence intensity of individual boutons before (closed symbols) and after (open symbols) induction of LTD. Average intensity was calculated by dividing total fluorescence intensity of individual boutons by their area.
Neuron  , DOI: ( /S (02)00898-X)

5. Figure 4
Comparison of Actions of MPEP, D-AP5, and Stimulation Protocols on LTD of fEPSP and Changes in FM 1-43 Loading and Unloading
(A) Average fEPSP slope measured 1 hr after 5 Hz LTD-induction protocol (if not stated otherwise) for slices: without blockers present (ACSF), pretreated with 50 μM D-AP5, pretreated with 10μM MPEP, or control slices that did not receive 5 Hz stimulation (whose fEPSP was measured at corresponding times to other test conditions).
(B) Average rate (1/t1/2) of FM 1-43 unloading for slices under the same conditions as in (A).
(C) Average number of boutons loaded with FM 1-43 under similar conditions.
(D) Average number of nondestaining boutons. For (A)–(D), data were normalized to the corresponding values determined from the initial FM 1-43 measurements before induction of LTD (see Figure 1). Each point represents a separate experiment.
Neuron  , DOI: ( /S (02)00898-X)

6. Figure 5
Effect of Inhibition of Protein Synthesis on LTD of the fEPSP and Changes in FM 1-43 Loading and Unloading
(A) Mean fEPSP as a function of time for slices that were stimulated at 5 Hz for 3 min in the presence of 20 μM anisomycin.
(B and C) Frequency histograms of puncta unloading rates (1/t1/2) from five slices before (B) and after (C) 5 Hz stimulation in the presence of anisomycin (no significant difference; p > 0.05, Kolmogorov-Smirnov test).
(D) Average rate of puncta unloading before and after 5 Hz stimulation in the presence of anisomycin (no significant difference; p > 0.05, paired t test).
(E) Average number of boutons loaded with FM 1-43.
(F) Average number of boutons that failed to destain after unloading.
Neuron  , DOI: ( /S (02)00898-X)

7. Figure 6
Activity-Dependent Quenching of FM 1-43 Fluorescence in Nondestaining Boutons by FM 4-64
(A) Experimental protocol showing periods of stimulation and FM 1-43, ADVASEP-7, and FM 4-64 application (durations not drawn to scale).
(B) Images of fluorescence in a field in stratum radiatum of CA1. Left: images from a control slice obtained after 1 hr incubation in ACSF without induction of LTD. Right: images from a slice obtained 1 hr after induction of LTD. Top: FM 1-43 fluorescence remaining after Unload 1 (two trains of 10 Hz stimulation for 3 min), showing nondestaining boutons. Bottom: FM 1-43 fluorescence of nondestaining boutons that remained after the second round of stimulation in the presence of FM 4-64 (Unload 2, 10 Hz for 3 min). Arrows show all puncta whose fluorescence intensity decayed by more than 50% during Unload 2 (presumably due to quenching by FM 4-64). Scale bar 3 μm.
(C) Time course of FM 1-43 fluorescence decay during Unload 2 for 12 representative puncta in a single slice after induction of LTD. Five puncta showed a quenching of their FM 1-43 fluorescence by FM 4-64 whereas seven puncta did not.
(D) Comparison of mean time course of FM 1-43 release from destaining boutons during Unload 1 (closed squares; averaged from 89 puncta from 4 slices) with the time course of quenching of FM 1-43 fluorescence by FM 4-64 for nondestaining boutons during Unload 2 (open circles; averaged over 39 puncta from 8 slices). The more rapid rate of dye release (and quenching) in this experiment compared to that in previous figures is due to the more rapid rate of stimulation during these unloading protocols (10 Hz versus 1.5 Hz in previous experiments). Data in (D) and (E) are from slices after induction of LTD.
(E) Average rate of FM 1-43 release from destaining boutons during Unload 1 compared with average rate of FM 1-43 quenching by FM 4-64 for nondestaining boutons during Unload 2.
(F) Percent of nondestaining boutons that showed a >50% decay of FM 1-43 fluorescence intensity during Unload 2. Each point represents a separate experiment. Data obtained in the presence (+) or absence (−) of FM 4-64 during Unload 2 from slices in which LTD had (LTD) or had not (control) been induced 1 hr previously.
Neuron  , DOI: ( /S (02)00898-X)

8. Figure 7
Comparison of Effect of LTD Induction on the Rate of FM 2-10 and FM 1-43 Unloading for Destaining Boutons
(A) The mean time course of FM 2-10 unloading from presynaptic boutons before (closed circles) and after (open circles) induction of LTD (n = 4 slices).
(B) The mean time course of FM 1-43 unloading from presynaptic boutons before (closed squares) and after (open squares) induction of LTD (n = 8 slices).
(C) Comparison of the time course of FM 2-10 (solid circles) and FM 1-43 (closed squares) unloading in slices before induction of LTD.
(D) Comparison of the time course of FM 2-10 (open circles) and FM 1-43 (open squares) unloading in slices after induction of LTD.
(E) Average rates of destaining for boutons loaded with FM 1-43 and FM 2-10 before and after induction of LTD by 5 Hz stimulation (*p < 0.05).
Neuron  , DOI: ( /S (02)00898-X)

9. Figure 8 Models for Altered Synaptic Vesicle Cycling during LTD
(A) Before LTD. From left to right, a primed synaptic vesicle loaded with transmitter (green dots) and FM 1-43 (red hexagons) fuses with the presynaptic terminal via a transient fusion pore that then rapidly expands to full fusion, rapidly releases soluble transmitter, more slowly releases FM 1-43, and is retrieved by slow endocytosis.
(B) After LTD. Two models for altered release: (1) switch to fusion pore mode; (2) switch to rapid endocytosis. In model 1, a primed vesicle filled with transmitter and FM 1-43 fuses with the presynaptic membrane via a nonexpanding fusion pore, releases transmitter relatively slowly through the fusion pore, and terminates release through closure of the fusion pore, preventing the escape of FM In model 2, transmitter is released as in (A) (before LTD), but the vesicle is retrieved through rapid endocytosis, preventing the escape of dye.
Neuron  , DOI: ( /S (02)00898-X)