1. Recruitment of New Sites of Synaptic Transmission During the cAMP-Dependent Late Phase of LTP at CA3–CA1 Synapses in the Hippocampus 
Vadim Y. Bolshakov, Hava Golan, Eric R. Kandel, Steven A. Siegelbaum 
Neuron 
Volume 19, Issue 3, Pages (September 1997)
DOI: /S (00)

2. Figure 1 Sp-cAMPS-Induced L-LTP
(A) The effect of Sp-cAMPS on field potentials. Superimposed recordings of field potential EPSPs (fEPSPs) before and 2 hr after Sp-cAMPS application in the absence (left) or presence (right) of anisomycin.
(B) Averaged data illustrating the effect on field potentials (measured as initial slope) when Sp-cAMPS (50 μM) is applied in the absence (closed circles) or presence (open circles) of anisomycin (20 μM). Bars indicate periods of application of compounds. Error bars, SEM; n = 9 for each group.
(C) Rp-cAMPS blocks the action of Sp-cAMPS. Open circles, Sp-cAMPS applied in the presence of Rp-cAMPS (100 μM); closed circles, Rp-cAMPS applied 2 hr following application of Sp-cAMPS; n = 7 for each group.
(D) Paired pulse facilitation (PPF) of compound EPSCs under basal conditions (circles) and after induction of L-LTP with Sp-cAMPS (squares). Ca2+ elevation to 5.0 mM under basal conditions occludes PPF (triangles). Two stimulating pulses were separated by a variable interval. Response to the second pulse was normalized by response to the first pulse. Numbers of experiments are given next to each time point. Twenty events at each time interval were averaged.
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3. Figure 2
Comparison of the Effects of E-LTP and L-LTP on EPSC Amplitude Histograms Obtained from Unitary Synapses
(A) Example of an EPSC histogram under basal conditions.
(B) After inducing E-LTP (same cell as in [A]). The smooth curves show fits with two Gaussian functions.
(C) Comparison of basal and E-LTP EPSC histograms using density estimate plots.
(D) EPSC histogram from a slice that had been pretreated with Sp-cAMPS. Four Gaussian functions were used to fit the histogram over a range of 4 to −12 pA.
(E) EPSC histogram from experiment in which Sp-cAMPS was applied in the presence of anisomycin. Two Gaussian functions are fit to the data.
(F) Density estimate comparison of two EPSC amplitude histograms shown in (D) and (E).
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4. Figure 3 Summary of Effects of L-LTP on EPSC Amplitude Histograms
(A–C) Density estimate plots comparing six representative experiments under basal (control) conditions (A) and after exposure to Sp-cAMPS in the absence (B) or presence (C) of anisomycin.
(D) Summary box plots for EPSC data under each condition showing quantal amplitude, q, and potency, p (mean of the successes). The line within the boxes marks the median and the box boundaries indicate the twenty-fifth and seventy-fifth percentiles. The bars denote the tenth and ninetieth percentiles. Mean values for q in basal (control) slices (n = 17), slices treated with Sp-cAMPS (n = 15), and slices treated with Sp-cAMPS plus anisomycin (n = 6) were (in pA): −3.8 ± 0.8, −4.0 ± 0.7, and −3.8 ± 0.3, respectively (no significant difference). Mean values for p in the same three conditions were (in pA): 4.0 ± 0.8, 6.2 ± 2.2, and 4.7 ± 1.0. p was significantly larger after Sp-cAMPS compared to control (t = 4.31; p < 0.001).
(E) Box plots for fraction of failures, f, and coefficient of variation (CV) of EPSC successes. Mean values for f in control, Sp-cAMPS and Sp-cAMPS plus anisomycin were 0.59 ± 0.14, 0.17 ± 0.05, and 0.59 ± 0.07, respectively. f was significantly lower after treatment with Sp-cAMPS compared to control (t = 1.14; p < ). Mean values for CV of EPSC successes in the same three conditions were 31.5 ± 5.2%, 46.5 ± 14.7%, and 28.4 ± 5.5%. CV was significantly greater in Sp-cAMPS than in control (t = 3.96; p < 0.001).
(F) Number of Gaussian components required to fit successes. Each symbol represents a separate experiment. Data are shown for control conditions (group on the left; n = 17), after pretreatment with Sp-cAMPS (middle group; n = 15), and Sp-cAMPS plus anisomycin (group on the right; n = 6).
(G) Mean fractional area under each Gaussian component fit to successes. Error bars, SD.
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5. Figure 4
Maximum Likelihood Fit of EPSC Histogram Using Monte Carlo Simulations for Evaluating Significance
Data shown for a slice treated with Sp-cAMPS.
(A–C) Best fits of EPSC successes with two (A), three (B), or four (C) Gaussian components (an additional Gaussian component is used to fit the peak at 0 pA, corresponding to the failures).
(D) Best fit of successes using cubic transform of Gaussian distribution (see Experimental Procedures).
(E–H) Pair-wise comparisons of various null (H0) and test (H1) distributions using Monte Carlo simulations. (E) shows two (H0) versus three (H1) Gaussian components; (F) shows three versus four Gaussians; (G) shows four versus five Gaussians; (H) shows cubic transform of Gaussian distribution (H0) versus four Gaussian components (H1). Closed circle shows the log-likelihood ratio, −2[L(H0) − L(H1)]exp, for each pair of H0 and H1 distributions to fit experimental data. The continuous curve plots cumulative histograms for −2[L(H0) − L(H1)]i values, obtained from fits of H0 and H1 to 100 simulated distributions generated from the H0 distribution by Monte Carlo sampling (see Experimental Procedures). Four Gaussian components provide a better fit than one, two, or three Gaussian components or a cubic Gaussian at a confidence level of α < Five Gaussian components (H1) provide a slightly greater L value compared to four components (H0) but at a level of α < 0.62 (G).
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6. Figure 7 Effects of Ca2+ Elevation on EPSC Amplitude Histograms
The cartoon at the bottom left shows a potential model for enhanced quantal content during L-LTP due to an increase in the number of hypothetical clusters of postsynaptic receptors. If each cluster of receptors could switch on and off independently, then release of a single vesicle could generate EPSCs whose amplitude varies in a quantal manner.
(A and B) EPSC histograms from a slice pretreated with Sp-cAMPS in normal (2.5 mM) and elevated (5.0 mM) Ca2+, respectively.
(A) Successes adequately fit with three Gaussian components in normal Ca2+.
(B) Four Gaussian components are required for adequate fit in elevated Ca2+. Insets illustrate several consecutive EPSCs.
(C) Comparison of density estimate histograms in 2.5 and 5.0 mM Ca2+ during L-LTP for four separate experiments. Top traces show the experiments in panels (A) and (B).
(D) Cumulative histograms of EPSC amplitudes in 2.5 and 5.0 mM Ca2+ during L-LTP. For each cell, successes in both 2.5 and 5.0 mM Ca2+ were normalized by median successful EPSC amplitude in 2.5 mM Ca2+. Error bars, SD.
(E) Same comparison of effect of Ca2+ elevation shown for slices under basal conditions (no Sp-cAMPS treatment).
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7. Figure 5
Evidence That Focal Stimulation Recruits Only a Single Presynaptic Input during L-LTP
(A and B) Examples of intensity threshold tests from two separate recordings from slices pretreated with Sp-cAMPS. (A1) and (B1) plot mean EPSCs as a function of stimulating current intensity (12–20 individual EPSCs were averaged). Note the sharp threshold at which the EPSC is evoked for both cases, indicating stimulation of a single presynaptic input. (A2) and (B2) show EPSC amplitude histograms associated with these two experiments. Histograms show low failure rate and broad success distribution fit by multiple Gaussian components, typical of L-LTP data. Fewer events were collected for histograms in these experiments than normal (owing to the fact that the extended threshold intensity test was also applied), so the Gaussian fits are less well defined.
(C) Second test for single input stimulation using rise time of EPSCs.
(C1) Individual examples of a small, intermediate, and large EPSC from the same synaptic connection are superimposed.
(C2) The three EPSCs have been scaled by their peak amplitudes. Note the similar rise time of the three events.
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8. Figure 6 Spectral Analysis Method for Evaluating Multiquantal EPSCs
(A) Cumulative EPSC amplitude distribution for EPSC data shown in Figure 4 (noisy trace). The number of EPSCs with amplitudes less than the value on the x-axis is plotted. Total number of events = 473. A tenth order polynomial provided a unimodal, continuous approximation of the data (smooth curve). We excluded failures in the fitting as well as the extreme high end of the EPSCs (amounting to of the data at both high and low ends of the distribution).
(B) Top traces show the probability density function (PDF) for experimental data (multipeaked curve) and the derivative of the polynomial curve shown in (A) (unimodal curve). Bottom trace shows the difference between the experimental and polynomial PDFs, illustrating the presence of approximately equally spaced peaks, with spacing around −4 pA.
(C) Spectral density plots obtained using a fast Fourier transform of the PDF difference curve shown in (B). Spectral density is plotted as a function of frequency (1/current). Smax is peak spectral density, which occurs at a frequency equal to 1/q, where q is spacing between peaks and equals −3.96 pA. A multi-Gaussian fit to the same distribution yields a similar value for q of −4.15 pA.
(D) Distribution of Smax values for 100 simulated PDFs obtained by random sampling of 473 events from the unimodal polynomial PDF shown in (A). Smax for the experimental data (indicated on the plot) was greater than those from all 100 simulations, yielding a significance of p < 0.01.
(E) Summary of spectral analysis data for EPSC histograms under basal conditions (closed symbols) and after induction of L-LTP (open symbols). For each experiment, we plot Smax normalized by total variance of the spectra (y-axis) versus the p value for the null hypothesis that each EPSC distribution is unimodal (no multiquantal peaks). The dashed line shows a significance level of 0.05.
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9. Figure 8 Effect of Ca2+ Elevation on Averaged EPSC Histograms
Effects under basal conditions ([A] and [B]) and during L-LTP ([B] and [C]). (A) and (C) show averaged histograms in normal (2.5 mM) external Ca2+. (B) and (D) show histograms in elevated (5.0 mM) external Ca2+. Four histograms from separate experiments were averaged in all examples. Before averaging each histogram, the mean value of the Gaussian component fit to the failures peak was subtracted from the x-axis (current) value associated with each bin. These subtracted values were then normalized by the estimated quantal amplitude (difference in mean value between Gaussian fit to first success peak and Gaussian fit to failures peak).
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10. Figure 10 Spontaneous Miniature EPSCs during L-LTP
(A–C) Examples of mEPSC amplitude distributions under basal conditions (A), after treatment with Sp-cAMPS to induce L-LTP (B), and after treatment with Sp-cAMPS in the presence of anisomycin (C).
(D) Cumulative histogram of mEPSC amplitudes from slices in basal condition and after treatment with Sp-cAMPS. Bars show SEM (n = 9 in both cases).
(E) Effect of Ca2+ elevation on cumulative mEPSC distribution during L-LTP. mEPSCs were first measured in normal (2.5 mM) Ca2+ and then in elevated (5.0 mM) Ca2+. For each cell, mEPSC amplitudes were normalized by median mEPSC value in 2.5 mM Ca2+. Distributions plot the fraction of mEPSCs with amplitudes less than the value given on the x-axis (n = 4). The bottom panel is an illustration of a potential mechanism for increased mEPSC size during L-LTP (right synapse) due to formation of multiple release sites (active zones) in a single presynaptic bouton through growth of perforated synapses.
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11. Figure 9
Comparison of Non-NMDA Receptor and NMDA Receptor EPSC Amplitude Histograms under Basal Conditions
The cartoon at the bottom shows an illustration of a silent synapse model for L-LTP. In basal conditions, one synapse has both NMDA (closed ovals) and non-NMDA (open ovals) receptors. The other synapse has only NMDA receptors and so is silent at the resting potential (left). After induction of L-LTP, active non-NMDA receptors are added to the silent synapse, increasing the number of nonsilent sites of synaptic transmission (right).
(A) Basal non-NMDA receptor EPSC histogram fit by two Gaussian functions. Normal ionic conditions.
(B) NMDA receptor EPSC histogram from the same cell obtained in the presence of CNQX and the absence of Mg2+ (external Mg2+ was washed out for 15 min). Data are also adequately fit by two Gaussian components.
(C) Comparisons of non-NMDA (solid line) and NMDA (dotted line) receptor EPSC density estimate histograms for four experiments.
(D) Effect of Ca2+ elevation on cumulative distribution of successful NMDA receptor EPSC amplitudes. Responses are normalized by median EPSC amplitude in 2.5 mM Ca2+ (n = 4).
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12. Figure 11
Effect of Sr2+ on mEPSCs under Basal Conditions and during L-LTP
(A and C) mEPSC distributions in the presence of extracellular Ca2+ (top graphs) or Sr2+ (bottom graphs) under basal conditions ([A], control) and during L-LTP ([C], Sp-cAMPS).
(B and D) Cumulative mEPSC distributions in the presence of Ca2+ (closed symbols) or Sr2+ (open symbols) under basal conditions (B) or during L-LTP (D).
(E) Summary of mEPSC results showing mean mEPSC amplitudes (left) and coefficient of variation (CV) of mEPSC amplitudes (right) under basal conditions (control; n = 13), after treatment with Sp-cAMPS (Sp-cAMPS; n = 17), and after treatment with Sp-cAMPS in the presence of anisomycin (Sp-cAMPS + Aniso; n = 3). Data also show mean mEPSC amplitude in extracellular Sr2+ from slices under basal conditions (Control + Sr2+; n = 6) and after exposure to Sp-cAMPS (Sp + Sr2+; n = 6). Box plots as in Figure 3. In control slices, mean mEPSC amplitude was −6.56 ± 2.9 pA in Ca2+-containing solution and −6.15 ± 2.74 pA in Sr2+-containing solution (no significant difference; paired t test, p > 0.05; n = 6). During L-LTP, the mean mEPSC amplitude in potentiated slices was significantly greater in Ca2+-containing solution (−8.56 ± 5.93 pA) than in Sr2+-containing solution (−6.88 ± 3.52; paired t test, p < 0.004; n = 6).
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