Frequency-dependence of short-term synaptic plasticity at prelimbic L2/3 excitatory synapses.

(A) In utero electroporation following injection of the plasmid (CAG-oChIEF-tdTomato) into the ventricle of an embryo (E17.5). (B) Representative images showing specific expression of oChIEF-tdTomato in L2/3 pyramidal cells after IUE. The red fluorescence of tdTomato clearly visualizes oChIEF-expressing cell bodies in L2/3 and axons in L5. Scale bar: 1 mm, 100 μM, 50 μM from left to right. (C) Recording schematic showing photostimulation of oChIEF-expressing axon fibers of transfected PCs (filled triangles) and a whole-cell recording from non-transfected PCs (empty triangles) or FSINs (empty circles). A collimated DMD-coupled LED was used to confine the area of excitation (typically 3–4 μm in diameter) to a small region (blue circle) near the soma. (D, E) Representative traces for EPSCs averaged over 10 trials at each frequency (left) and average amplitudes of baseline-normalized EPSCs (right) during 20-pulse trains at frequencies from 5 to 40 Hz at PC-PC (D; n = 12, 9, 21, 10 cells for 5, 10, 20, 40 Hz, respectively) and PC-FSIN (E; n = 8, 9, 10, 10) synapses. Each data point was normalized to the average of the first EPSC. (F) Baseline-normalized amplitudes of steady-state EPSCs (EPSCss; black symbols) and synaptic efficacy (EPSCss × f; red symbols) as a function of stimulation frequency (f) at PC–PC synapses. EPSCss was measured from the average of last 5 EPSCs from the 20-pulse trains (n = 12, 9, 21, 10). (G) Paired pulse ratio (PPR) as a function of inter-spike intervals (n = 25, 9, 25, 22, 43 for 200, 100, 50, 25, 20 ms ISI, respectively) at PC-PC synapses. (H) EPSCss and synaptic efficacy at PC-FSIN (n = 8, 9, 10, 10). (I) PPR at PC-FSIN (n = 8, 9, 10, 15, 34). Gray symbols, individual data.

Delayed facilitation results from slow activation of Ca2+-dependent vesicle replenishment at a constantly high vesicular fusion probability

(A) Representative traces (left) and mean baseline-normalized amplitudes (right) of EPSCs evoked by 30-pulse trains at 40 Hz in control (n = 21; black) and in the presence of 50 μM EGTA-AM (n = 14; green). (B) Mean values for the first EPSC amplitude (EPSC1, left) and PPR (right) from the experiments displayed in (A). Gray symbols, individual data. (C) Plot of rate constants for short-term facilitation (kSTF) as a function of stimulation frequency (fstim), showing a linear relationship. The linear regression line (black) is shown fitted to kSTF values, estimated from Figure 1. (D) Representative EPSC traces (left) and average of baseline-normalized EPSCs (right) evoked by 12-pulse stimulation at 5 Hz, followed by 40 Hz 7-pulse train (n = 12). Note that slowly developing facilitation was converted to rapid facilitation after strong PPD. (E) Mean values for PPR at 40 Hz. The baseline PPR was reproduced from Figure 1G and the PPR during 5 Hz train was calculated as (13th EPSC) / (12th EPSC). Gray symbols, individual data. All statistical data are represented as mean ± S.E.M.; n.s. = not significant; **, P<0.01; unpaired t-test.

Low baseline occupancy of release sites and its increase during facilitation and post-tetanic augmentation

(A, B) Left, Representative EPSCs evoked by 5 and 40 Hz train stimulation (black, 5 Hz; purple, 40 Hz). Right, Variance-mean plots of EPSCs amplitude from averaged EPSCs recorded at PC-PC (A; n = 12, 17 for 5 and 40 Hz, respectively) and PC-FSIN (B; n = 8, 10) synapses. The data were fitted using multiple-probability fluctuation analysis (MPFA). Error bars are omitted for clarity. The 1st EPSC of 5 Hz train (broken line) was used to estimate the resting level of pocc. Filled circles were measured from post-tetanic augmented EPSCs (A, n = 12, 12, 9; B, n = 7, 7, 7 for 0.1, 0.2, 0.5 s IBIs, respectively). (C-E) Post-tetanic augmentation (PTA) experiments at PC-PC (top) and PC-FSIN (bottom) synapses. (C) Representative traces for EPSCs evoked by double 40 Hz train stimulations separated by 0.5 s. (D) Mean baseline-normalized amplitudes of EPSCs evoked by double 40 Hz trains at different inter-burst intervals (IBIs, 0.1, 0.2, 0.5, 1, 2, 5, 10 s). Upper, PC-PC synapse (n = 12, 12, 21, 11, 11, 16, 11 from short to long IBIs, respectively). Lower, PC-FSIN synapse (n = 10, 9, 8, 9, 9, 7, 9). (E) PTA time course. The baseline-normalized amplitudes of 1st EPSC from the 2nd train were plotted as a function of IBIs.

Pharmacological experiments reveal specific molecular mechanisms underlying vesicle loading processes

(A-C) (a) Representative EPSC traces (left) and mean baseline-normalized EPSCs (right) evoked by double 40 Hz train stimulations separated by 0.5 s inter-burst interval (IBI) in control and in the presence of 5 μM U73122 (Aa, n = 7, orange), 20 μM OAG (Ba, n = 12, cyan) or 100 μM dynasore (Ca, n = 10, blue). (b) Mean values for baseline EPSCs (EPSC1, left) and augmentation (right) from the experiments shown in corresponding a panel. (Da) Representative EPSC traces evoked by paired pulses (left) and mean values for baseline EPSC amplitude (middle) and PPR (right) before and after applying 20 μM LatB (n = 6). (Db) Representative EPSC traces (left, upper) and average of normalized EPSCs (left, lower) evoked by double 40 Hz train stimulation separated by 0.5 s in control (n = 21) and in 20 μM LatB conditions (n = 16; pink). Right, Mean values for augmentation in control and LatB conditions. Gray symbols, individual data. All statistical data are represented as mean ± S.E.M., *, P<0.05; **, P<0.01; ***, P< 0.001; unpaired or paired t-test; n.s. = not significant.

STF at both types of local excitatory synapses is abolished by Syt7 KD

(A, B) Representative EPSC traces (left) and mean baseline-normalized amplitudes of EPSCs (right) evoked by 20-pulse trains at 5 to 40 Hz. STP was measured at PC-PC (A; n = 10, 9, 12, 9) and PC-FSIN (B; n = 9, 8, 10, 9) synapses, in which presynaptic Syt7 transcripts were depleted (Syt7 KD). Syt7 KD pyramidal cells are indicated as black triangles on the top. For comparison, STP in WT synapses is reproduced from Figure 1 (dotted lines). Same frequency color codes were used as in Figure 1. (C) Baseline-normalized amplitudes of steady-state EPSC (EPSCss; black symbols) and synaptic efficacy (EPSCss × f; red symbols) as a function of stimulation frequency (f) at PC-PC synapses. EPSCss was defined as the average of last 5 EPSC amplitudes from 20-pulse trains. (D) PPR as a function of inter-spike intervals (n = 10, 9, 12, 18, 26). (E) EPSCss and synaptic efficacy at PC-FSIN synapses. (F) PPR at PC-FSIN synapses (n = 9, 8, 10, 12, 24). Gray symbols, individual data.

Syt7 KD synapses exhibit complementary changes in the number of release sites and their vesicle occupancy

(A, B) Left, Representative traces of EPSCs evoked by 5 and 40 Hz train stimulations (black, 5 Hz; purple, 40 Hz). Right, Variance-mean plots of EPSCs amplitude from averaged EPSCs recorded at PC-PC (A; n = 9, 15, 12, 10, 9) and PC-FSIN (B; n = 4, 14, 10, 9, 8) synapses in which presynaptic Syt7 has been knocked down. The data were fitted using MPFA and error bars are omitted for clarity. The mean 1st EPSC amplitude of 5 Hz train (dotted line) was used for estimation of baseline pocc. Broken lines indicate MPFA fits to variance-mean plot of WT synapses reproduced from Figure 2. (C-E) Recovery experiments at PC-PC (top) and PC-FSIN (bottom) synapses in which presynaptic Syt7 was knocked-down. (C) Representative EPSCs evoked by double 40 Hz train stimulations separated by 0.5 s. (D) Mean baseline-normalized amplitudes of EPSCs evoked by double 40 Hz trains at PC-PC (n = 12, 10, 9, 9, 10, 9, 8) and PC-FSIN (n = 10, 9, 10, 11, 8, 9, 10) synapses at different interburst intervals (IBIs). (E) PTA time course. Baseline-normalized amplitudes of 1st EPSC from the 2nd burst were plotted as a function of various IBIs. Dotted lines indicate augmented EPSCs in the WT reproduced from Figure 2.

Recovery of TS vesicles following depletion is accelerated by Syt7

(A-C). Recovery experiments at PC-PC synapses in WT (A) and Syt7 KD (B). Mean baseline-normalized amplitudes of EPSCs evoked by two consecutive 3-pulse 40 Hz trains in WT (n = 16, 13, 11, 11, 11, 9 from short to long IBIs, respectively) and KD (n = 13, 11, 12, 8, 10, 11) synapses at different IBIs (0.1, 0.2, 0.5, 1, 2, 5 s). Inset, representative traces of EPSCs evoked by two consecutive 3-pulse 40 Hz train stimulations separated by 0.5 s. (D-F). Recovery experiments at PC-FSIN synapses in WT (D) and Syt7 KD (E). Mean baseline-normalized amplitudes of EPSCs evoked by two consecutive 3-pulse 40 Hz trains in WT (n = 9, 11, 11, 9, 10, 13) and KD (n = 9, 12, 9, 7, 10, 9) synapses at different IBIs. Inset, representative traces of EPSCs evoked by two consecutive 3-pulse 40 Hz train stimulations separated by 0.5 s. (C, F) Recovery time course. Baseline-normalized amplitudes of 1st EPSC from the 2nd burst were plotted as a function of various IBIs.

Behavioral effects of Syt7 deficiency in L2/3 PCs of the mPFC

(A) Top, Representative images showing bilateral expression of U6-GFP in L2/3 of PCs after IUE at E17.5. Scale bar: 1 mm, 100 μm. Bottom, Schematic of trace fear conditioning and extinction (tone test) protocol. (B) Freezing behavior of control (expressing scrambled shRNA, Scr) and Syt7 KD rats during acquisition of tFC. (C) Freezing ratio during tone tests on following days. Data are shown as average freezing during T1–T4, T5–T8, or T9–T12 (T, trials). The freezing on T1-T4 was significantly lower in KD rats suggesting that formation of trace memory was impaired in Syt7 KD rats (n = 10, 11 for Ctrl and KD, respectively; P = 0.0007, F(1, 19) = 16.43, two-way repeated measures ANOVA; P = 0.0064, 0.0211, 0.0064, 0.0064; Holm-Sidak test). (D) Representative images of c-Fos immunoreactivity in the prelimbic cortex of control or Syt7 KD rats 90 min after tFC acquisition. c-Fos (left, red, Cy5) and GAD67 (right, cyan, Cy3) were immunostained in the same brain slice expressing U6-GFP (middle; green). Scale bar, 50 μm. (E) Exemplar images of c-Fos positive neurons expressing GFP (top) or GAD67 (bottom) in control (left) or Syt7 KD rats (right). Scale bar, 50 μm. (F-H) Effects of Syt7 KD on c-Fos density (F) and percentage of c-Fos positive neurons co-labeled with GAD67 (G) or GFP (H) in L2/3 or L5 of prelimbic cortex (n = 9, 9 for Ctrl and KD, respectively). Open symbols, individual data. All statistical data are represented as mean ± S.E.M.; ****, P<0.0001; unpaired t-test.

STP model in light of known Ca2+ binding kinetics of Syt7

(A-D) Left, Schematic of allosteric calcium binding to Syt7. The number of Ca2+ bound to Syt7 was denoted as # in ‘S#’ in the reaction scheme. kon = 7/μM/s, koff = 10/s. Middle, Simulated changes of k1 (black) in response to 5 Hz train of Ca2+ transients (light blue traces). The priming step of the simple refilling model was assumed to be catalyzed by full Ca2+-bound from of Syt7. Accordingly, Ca2+-dependent increase in k1 was calculated as K1,max multiplied by a fraction of full Ca2+-bound form of Syt7. We assumed that local [Ca2+]i(t) follows a Gaussian function: (1/σ √2π) exp[-(t-tp)2/ 2σ2], in which tp = 0.25 ms and σ = 0.085 ms. Right, Fits of the Syt7 model to the STP data. To fit this model to the STP data, K1,max was set to 300/s (A), 220/s (B), and 180/s (C). Cooperativity factor (b) was set to 0.35 (A), 0.2 (B), and 0.05 (C). k1 is set to be constant for Syt7 KD (D). Same frequency color codes were used as in Figure 1.