Metaplasticity at CA1 Synapses by Homeostatic Control of Presynaptic Release Dynamics  Cary Soares, Kevin F.H. Lee, Jean-Claude Béïque  Cell Reports  Volume 21, Issue 5, Pages 1293-1303 (October 2017) DOI: 10.1016/j.celrep.2017.10.025 Copyright © 2017 Terms and Conditions

Cell Reports 2017 21, 1293-1303DOI: (10.1016/j.celrep.2017.10.025) Copyright © 2017 Terms and Conditions

Figure 1 Homeostatic Strengthening of CA1 Synapses Is Accompanied by a Reduction in the Magnitude of LTP (A) Network activity was suppressed at 6 DIV. sed at 6 DIV by addition of the sodium channel antagonist tetrodotoxin (TTX, 1 μM) to the culture media. (B) Recordings of AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs). Average event traces (≥50 events) were used to quantify the mean mEPSC amplitude for each cell (p = 0.03, Student’s t test; n = 15 cells and 12 cells for control and TTX, respectively). (C) Example experiments in which a pairing protocol (180 electrical stimuli delivered at 3 Hz, while clamping the cell at 0 mV) was used as an induction stimulus for LTP. (D) Normalized summary data for experiments in which a pairing protocol was applied (control: raw amplitudes at t = 35–45 min compared to baseline, p < 0.001, Wilcoxon signed-rank test, n = 16 neurons; TTX: raw amplitudes at t = 35–45 min compared to baseline, p = 0.569, Wilcoxon signed-rank test, n = 15 neurons; normalized control versus TTX amplitudes at 35–45 min, p = 0.004, Wilcoxon rank-sum test). All error bars represent SEM. See Figure S1 for related data. Cell Reports 2017 21, 1293-1303DOI: (10.1016/j.celrep.2017.10.025) Copyright © 2017 Terms and Conditions

Figure 2 Abolishing Network Activity Prevents Synapse Unsilencing in the Developing Hippocampal Network (A) Functional mapping of AMPA- and NMDA-mediated EPSCs by two-photon uncaging of MNI-glutamate. Glutamate uncaging at dendritic spine tips (indicated by the red dots) yielded uncaging-evoked EPSCs (uEPSCs) recorded in voltage clamp at −70 mV (inward traces) and at +40 mV (outward traces). (B) A demonstration for how the AMPA-to-NMDA ratio was calculated (see Results; Supplemental Experimental Procedures). (C) An example uncaging experiment in which two silent synapses (no conductance at −70 mV) are neighbored by two synapses and a nearby “extrasynaptic” dendritic location at which AMPAR-EPSCs were detected. (D) Two separate TTX treatment regimens (early and late) were used to determine how the silent synapse fraction is affected by prolonged network silencing at different stages of development. (E) Quantification of all uncaging AMPA-to-NMDA ratios across slice development and in conditions of network silencing (silent synapse threshold: AMPA/NMDA < 0.25). (F) Percentage of silent synapses measured at each developmental stage and in each treatment condition. (G) The average amplitude of mEPSCs recorded from neurons in the early TTX treatment regimen at DIV 6–7 (control versus TTX: p < 0.001; unpaired Student’s t test). All error bars represent SEM. See Figure S2 for related data. Cell Reports 2017 21, 1293-1303DOI: (10.1016/j.celrep.2017.10.025) Copyright © 2017 Terms and Conditions

Figure 3 Structural and Functional Features of Postsynaptic Hebbian LTP Are Unaltered by a Prolonged Silencing Paradigm (A) Repetitive two-photon uncaging of MNI-glutamate (30 pulses at 0.5 Hz) was used to induce LTP at a single spine of a transfected CA1 neuron. (B) Summary data of normalized mCherry and SEP-GluA1 intensity for all 14 single-spine uncaging LTP experiments in the control condition. (C) Similar to (A), except that the slice was treated with TTX for 3 days prior to imaging. (D) Summary data of normalized mCherry and SEP-GluA1 intensity for all 13 uncaging LTP experiments recorded from TTX-treated slices (control versus TTX: mCherry intensity at stimulated spines 18–28 min post-LTP induction: p = 0.62, unpaired t test; control versus TTX: SEP-GluA1 intensity at stimulated spines 18–28 min post-LTP induction: p = 0.75, unpaired t test). (E) Simultaneous recording of uncaging-evoked EPSCs and corresponding iGluSnFR transients at spines. (F) A single-spine experiment (same as E) from a control neuron in which iGluSNFR signals in response to glutamate uncaging were monitored during an LTP experiment that lasted ∼25 min. (G) Uncaging-evoked EPSCs recorded at the soma for the experiment shown in (F). (H and I) Average normalized amplitude of iGluSNFR signals (H) and uEPSCs (I) from spine experiments in which LTP was induced by 2P-Glu uncaging (n = 8 spines in each condition). (J) Normalized amplitude of uEPSCs in both control and TTX treatment conditions, 12–20 min following LTP induction (p = 0.64, unpaired t test). All error bars represent SEM. See Figure S3 for related data. Cell Reports 2017 21, 1293-1303DOI: (10.1016/j.celrep.2017.10.025) Copyright © 2017 Terms and Conditions

Figure 4 Prolonged Network Silencing Slows Vesicle Replenishment Rate and Renders Synapses More Susceptible to Short-Term Depression (A) Summary data for all experiments in which evoked AMPAR-mediated EPSCs were recorded at 3 Hz (n = 10 cells in each condition). This stimulus frequency was chosen to match that of the LTP induction protocol from Figure 1D. (B) Summary data for a 10-Hz (100-pulse) stimulation experiment in which AMPAR-EPSCs were monitored (n = 13 and 10 cells for control and TTX-treated groups, respectively). (C) Paired-pulse ratio of AMPAR-mediated EPSCs (Vm = −70 mV; p = 0.69, unpaired t test, n = 10 recordings in each condition). (D) Monoexponential fitting of evoked EPSC amplitude recovery following the 10-Hz stimulus train in each condition (B). (E) Plotted is the cumulative EPSC amplitude (normalized to baseline) for the 10-Hz stimulus train (E1). A linear fit of the last 20 stimuli offered two quantifiable metrics (Thanawala and Regehr, 2016): the y intercept, which offers information about the size of the readily releasable pool (p = 0.76, unpaired t test) (E2), and the steady-state slope, which is related to the vesicle replenishment rate (p = 0.01, unpaired t test) (E3). All error bars represent SEM. See Figures S4 and S5 for related data. Cell Reports 2017 21, 1293-1303DOI: (10.1016/j.celrep.2017.10.025) Copyright © 2017 Terms and Conditions

Figure 5 Prolonged Network Silencing Reduces Transmission Fidelity during Repetitive Synaptic Stimulation (A) Probabilistic vesicular release of glutamate onto visually identified iGluSnFR-expressing spines. A brief electrical stimulus (0.1 ms, orange line) was delivered to the slice via a patch electrode positioned proximal to the dendritic arbor of a CA1 cell. (B) 50 consecutive stimuli were delivered at ∼0.1 Hz to estimate a single-spine-release probability. Each row in the intensity-based ΔF/Fo plot represents the baseline-normalized intensity of the iGluSnFR signal from a single line-scan experiment (like in A). Above right: peak amplitudes are depicted in a scatterplot. (C) Estimates of release probability at single spines (p = 0.95, unpaired t test). (D) A subset (5 s) of a prolonged line scan (30 s) at a single spine during which 90 electrical stimuli were delivered at a frequency of 3 Hz. (E) Peak amplitude of iGluSnFR signals from an example 3-Hz stimulation experiment in each condition. The amplitude of failure events were assigned to 0 for presentation purposes. (F) Mean estimates of release probability during each 10-s epoch of the 3-Hz stimulus train (normalized to baseline; same as C). Unpaired t tests were used for statistical analysis (∗ indicates p < 0.05). (G) Mean spine potency (amplitude of only successful release events) during each epoch of the 3-Hz stimulus train, normalized to the mean potency during the baseline period. All cross comparisons between treatment conditions are not significant (p > 0.05, unpaired t test). All error bars represent SEM. Cell Reports 2017 21, 1293-1303DOI: (10.1016/j.celrep.2017.10.025) Copyright © 2017 Terms and Conditions

Figure 6 Failure to Induce LTP in TTX-Treated Slices Can Be Accounted for by a Comrpomised Fidelity during the LTP Induction Phase (A) A graphical depiction of a “reduced” LTP stimulus induction paradigm (orange line) that was designed to match the short-term depression phenotype observed in TTX-treated slices (revised and replotted from Figure 4A). The reduced stimulus induction protocol consisted of (in order) 15 evoked stimuli at 3 Hz, 20 stimuli at 1.5 Hz, 20 stimuli at 1 Hz, and 10 stimuli at 0.5 Hz. (B) A reduced LTP stimulus experiment that mimics the short-term depression phenotype observed in TTX-treated slices was delivered to control neurons and compared to our full LTP stimulus in interleaved experiments (180 stimuli at 3 Hz). Both induction protocols lasted 60 s, during which time the neuron was clamped at 0 mV. The reduced stimulus paradigm failed to potentiate EPSCs significantly (3-Hz stimulus: raw EPSC amplitudes at t = 30–40 min compared to baseline, p = 0.016, Wilcoxon signed-rank test, n = 8 cells; reduced stimulus: raw EPSC amplitudes at t = 30–40 min compared to baseline, p = 0.547, Wilcoxon signed-rank test, n = 8 cells; 3-Hz stimulus versus reduced stimulus: normalized EPSC amplitudes at t = 30–40 min, p = 0.015, unpaired t test). All error bars represent SEM. Cell Reports 2017 21, 1293-1303DOI: (10.1016/j.celrep.2017.10.025) Copyright © 2017 Terms and Conditions