1. Volume 40, Issue 1, Pages 151-165 (September 2003)
Presynaptic Activation of Silent Synapses and Growth of New Synapses Contribute to Intermediate and Long-Term Facilitation in Aplysia 
Joung-Hun Kim, Hiroshi Udo, Hsiu-Ling Li, Trisha Y Youn, Mary Chen, Eric R Kandel, Craig H Bailey 
Neuron 
Volume 40, Issue 1, Pages (September 2003)
DOI: /S (03)

2. Figure 1
Localization of Synaptophysin-eGFP at Sensory Neuron Varicosities
(A) A sensory cell expressing synaptophysin-eGFP is injected with Alexa-594 prior to imaging. (A1) A phase contrast image reveals the initial segment and major axons of the postsynaptic motor neuron L7. (A2) The axonal arbor of a sensory neuron in contact with the motor neuron is labeled with Alexa-594. (A3) Most sensory neuron varicosities are enriched in synaptophysin-eGFP. (A4) The merged image of Alexa-594 (red) and synaptophysin-eGFP (green) indicates the localization of synaptophysin-eGFP at the presynaptic sensory neuron varicosities (yellow) in contact with the major processes of the postsynaptic cell.
(B) The enlarged images show synaptophysin-eGFP puncta overlying two varicosities (left panel). (B1) Alexa-594, (B2) synaptophysin-eGFP, and (B3) the merged images are shown. Two distinct varicosities (asterisks) and neurites (white arrow head) are illustrated. Note that synaptophysin-eGFP completely fills the large Alexa-594-defined varicosity on the right but only partially fills the smaller varicosity on the left. Sensory neurons expressing eGFP alone display a more diffuse staining pattern, which appears similar to Alexa-594 (right panel).
(C) Long-term facilitation is not affected by the expression of synaptophysin-eGFP in sensory neurons when compared to either the group that was injected with the specific DNA construct but does not express or the group that was not injected with any DNA construct (unpaired Student's t test). Shown are representative recordings of EPSPs from a sensory-motor neuron coculture expressing synaptophysin-eGFP before and 24 hr after 5 × 10 μM 5-HT and a histogram of the mean changes in EPSP amplitude of each group.
Neuron  , DOI: ( /S (03) )

3. Figure 2
5-HT-Induced Enrichment of Synaptophysin-eGFP in Sensory Neuron Varicosities
(A) Two new varicosities enriched in synaptophysin-eGFP are formed 24 hr after 5 × 10 μM 5-HT treatment.
(B) Two preexisting but empty varicosities joined by a short intervaricose segment that are labeled by Alexa-594 but that display little synaptophysin-eGFP become enriched in synaptophysin-eGFP 24 hr after 5-HT treatment. (B1) Alexa-594, (B2) synaptophysin-eGFP, and (B3) the merged images are presented at −3 hr and 24 hr for (A) and (B).
(C) An empty varicosity (C1) and a varicosity only partially filled with synaptophysin-eGFP (C2) are observed −3 hr and 24 hr without 5-HT treatment. The empty varicosity remains free of synaptophysin-eGFP at 24 hr, and the partially filled varicosity is not enriched further in the same time frame. Only merged images of Alexa-594 and synaptophysin-eGFP are shown.
(D) The total population of sensory neuron varicosities are grouped after binning according to the mean pixel intensity of GFP. 5-HT treatment increases the number of varicosities highly enriched in synaptophysin-eGFP (top panel). The percentage of varicosities in the 70%–100% enrichment group significantly increases after 5-HT treatment (paired Student's t test, compared to that before the 5-HT treatment). Control cultures that received no 5-HT do not show any significant change in enrichment of synaptophysin-eGFP between −3 hr and 24 hr (p > 0.2) (bottom panel). A total of 247 varicosities were analyzed (157 for the 5-HT-treated group and 90 for control).
Neuron  , DOI: ( /S (03) )

4. Figure 3
Fine Structure of Synaptophysin-Enriched and Synaptophysin-Empty Sensory Neuron Varicosities
(A) Two presynaptic varicosities from a sensory neuron expressing synaptophysin-eGFP and injected with Alexa-594 are shown in the inset. The merged image of Alexa-594 (red) and synaptophysin-eGFP (green) indicates that the sensory neuron varicosity at the top (arrowhead) is highly enriched in synaptophysin-eGFP (yellow), whereas the sensory neuron varicosity at the bottom (asterisk) displays virtually no synaptophysin-eGFP expression. At the ultrastructural level, the sensory neuron varicosity that was identified by Alexa-594 and heavily labeled with synaptophysin-eGFP (arrowhead in inset) is also found to be filled with synaptic vesicles.
(B) By contrast, the empty sensory neuron varicosity that was labeled with Alexa-594 but which displayed little synaptophysin-eGFP expression (asterisk in inset) is found to contain a markedly reduced complement of synaptic vesicles (asterisks), even though it displays mitochondria, cytoskeletal elements, and other components of the presynaptic compartment.
Scale bar, 1 μm (A and B); 10 μm (inset).
Neuron  , DOI: ( /S (03) )

5. Figure 4
Time Course of 5-HT-Induced Synaptophysin-eGFP Enrichment in Sensory Neuron Varicosities
(A) Clustering of synaptophysin-eGFP into preexisting empty varicosities begins almost immediately (0.5 hr) after 5 × 10 μM 5-HT. Accumulation of synaptophysin-eGFP at these varicosities is completed within 3–6 hr and remains relatively stable for 24 hr. The time course of the mean enrichment of empty varicosities after 5-HT treatment (11 varicosities, n = 4) and representative time-lapse images of the filling of a preexisting empty varicosity are shown at each time point (−3, 0.5, 3, 6, 12, 18, and 24 hr) before the start and after the completion of five pulses of 10 μM 5-HT treatment (90 min).
(B) The time course of the mean enrichment and the change in the number of new varicosities are shown at the same time points as in (A) after normalization with the maximum number of new varicosities (29, n = 4) at 24 hr. The number of new varicosities was determined with Alexa-594 labeling alone. Most of the new varicosities are formed between 12 and 18 hr after 5-HT treatment. Time-lapse images indicate that synaptophysin-eGFP accumulates in a preexisting axonal swelling and is followed by the apparent splitting and subsequent division of this swelling into two new synaptophysin-enriched varicosities between 12 and 18 hr after 5-HT treatment. These two varicosities and their synaptophysin complement are still present 24 hr after 5-HT treatment.
(C) The protein synthesis inhibitor anisomycin blocks the filling of preexisting empty sensory neuron varicosities with synaptophysin-eGFP 3 hr after 5-HT treatment. Histograms represent the mean of the percentage of preexisting empty varicosities that are filled with synaptophysin-eGFP when examined 3 hr after 10 μM 5-HT in the presence (20 varicosities) or the absence (19 varicosities) of 20 μM anisomycin (n = 5 cultures for both; unpaired Student's t test). An empty varicosity, labeled by Alexa-594 (red) but displaying no synaptophysin-eGFP expression, 3 hr before and 3 hr after 5 pulses of 10 μM given in the presence of 20 μM anisomycin. Only merged images of Alexa-594 and synaptophysin-eGFP are presented for (A)–(C).
Neuron  , DOI: ( /S (03) )

6. Figure 5
Time Course of Facilitation and Synaptophysin-eGFP Enrichment Following the Selective Induction of Facilitation in the Intermediate-Term Time Domain
(A) Facilitation and clustering of synaptophysin-eGFP into preexisting empty varicosities (−3, 0.5, 3, and 24 hr) are indicated following the selective induction of facilitation in the intermediate-term time domain isolated from long-term facilitation by the reduced 5-HT protocol (4 × 10 nM 5-HT). Only intermediate-term facilitation is induced without the onset of long-term facilitation (left panel, ANOVA and Neuman-Keuls' test). The mean percentage of preexisting empty varicosities filled at the indicated time points are shown (right panel, ANOVA and Neuman-Keuls' test). Representative time-lapse images of two preexisting empty varicosities are presented before the start and after the completion of four pulses of 10 nM 5-HT (top images at −3, 0.5, and 24 hr; bottom images at −3, 3, and 24 hr). Only merged images (yellow) of Alexa-594 and synaptophysin-eGFP are presented. Although the reduced 5-HT protocol clearly induces synaptophysin enrichment during the intermediate time domain (0.5 hr and 3 hr), this clustering of synaptic vesicles does not persist and is not evident at 24 hr. Note that EPSP amplitude and the enrichment of synaptophysin-eGFP were analyzed in the same dishes (one set of culture dishes, n = 5 were tested at −3, 0.5, and 24 hr for EPSP and −3, 3, and 24 hr for enrichment whereas the other set, n = 7 at −3, 0.5, and 24 hr for enrichment and −3, 3, and 24 hr for EPSP).
(B) Neither actinomycin-D nor anisomycin blocks the enrichment of synaptophysin-eGFP in empty sensory neuron varicosities when intermediate-term facilitation is isolated from long-term facilitation by the reduced 5-HT protocol. Histograms represent the mean percentage of preexisting empty varicosities that are filled with synaptophysin-eGFP when examined at 0.5 and 3 hr after four pulses of 10 nM 5-HT given in the presence of 50 μM actinomycin-D or 20 μM anisomycin or the absence of any inhibitor (p > 0.05, ANOVA).
Neuron  , DOI: ( /S (03) )

7. Figure 6
Functional Determination of Individual Presynaptic Sensory Neuron Varicosities
(A) Distribution of synPH expression and determination of the functional state of individual sensory neuron varicosities. Raw images of presynaptic varicosities of a sensory neuron expressing synPH before (A1) and after (A2) 200 mM KCl perfusion reveal that some varicosities (white arrowheads) display an increase in fluorescence intensity after depolarization of the sensory neuron whereas other varicosities (white arrows) do not. (A3) A pseudocolor image of the same field of view as that shown in panels (A1) and (A2) obtained by subtracting (A2) from (A1) illustrates the change of fluorescence intensity of synPH (in arbitrary fluorescence units). (A4) The phase contrast image illustrates the major processes of the motor neuron L7. Black arrows and arrowheads indicate the distribution and relative points of contact of the synPH active and inactive presynaptic sensory neuron varicosities (A1–A3) on the initial segment of the postsynaptic target cell.
(B) Enlargement of an individual synPH (+) sensory neuron varicosity shows an increase (54%) in synPH fluorescence intensity upon sensory neuron depolarization induced by 200 mM KCl. Images of synPH (green) and Alexa-594 labeling (red) of the presynaptic varicosity are shown. The fluorescence intensities are illustrated in pseudocolor before (rest) and after (stim) the depolarization of the sensory neuron (in arbitrary fluorescence units).
(C) Coexpression of synPH (green) and synaptophysin-RFP (red) leads to colocalization of the two synaptic vesicle proteins specifically in varicosities as shown in the merged image (yellow). The phase contrast image indicates the loci of the presynaptic sensory neuron varicosities along the major processes of the postsynaptic motor neuron L7.
Neuron  , DOI: ( /S (03) )

8. Figure 7
5-HT-Induced Activation of Silent Presynaptic Varicosities and the Growth of New Functional Synaptic Varicosities
The functional state of individual sensory neuron varicosities as determined before and 24 hr after 5 × 10 μM 5-HT. (A) The merged images (red, Alexa-594; green, synPH) reveal that a preexisting empty varicosity lacking synPH (red) at −3 hr becomes enriched (yellow) at 24 hr after 5-HT treatment. The pseudocolor images before (rest) and after (stim) depolarization of the sensory neuron indicate that there is no significant change in fluorescence intensity at −3 hr (presynaptically silent and not competent for evoked transmitter release) but illustrate a significant increase in fluorescence intensity (presynaptically active and competent for evoked transmitter release) 24 hr after 5-HT treatment. (B) Only sensory neuron neurites are present at −3 hr, but a new varicosity is formed and enriched in synPH (yellow) at 24 hr after 5-HT treatment. The pseudocolor images show an increase in fluorescence intensity, indicating that the newly formed presynaptic varicosity is functional. (C) A preexisting and synPH-enriched varicosity is competent both before and after 5-HT treatment. There is no substantial change in varicosity structure or synPH distribution. The pseudocolor images also indicate that the varicosity is functional at both −3 hr and 24 hr following 5-HT treatment. The pseudocolor scale shows fluorescence intensity of synPH (in arbitrary fluorescence units) for rest/stim panels of (A)–(C).
Neuron  , DOI: ( /S (03) )

9. Figure 8
Model of Time Course and Functional Contribution of Two Distinct Presynaptic Structural Changes Associated with Intermediate- and Long-Term Facilitation
Five pulses of 10 μM 5-HT trigger the clustering of synaptic vesicles to preexisting silent synapses (3–6 hr) and the formation of new synapses (12–18 hr). The resultant newly filled and newly formed synapses are competent for evoked transmitter release, which is likely to contribute to the synaptic enhancement that underlies long-term facilitation. The rapid filling and activation of silent presynaptic terminals at 3 hr suggests that, in addition to its role in long-term facilitation, this modification of preexisting synapses may also contribute to the intermediate phase of synaptic plasticity. Red triangles represent functionally competent release sites (active zones), and empty circles indicate synaptic vesicles.
Neuron  , DOI: ( /S (03) )