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 (vertical broken line) was used for estimation of baseline pocc. Dashed parabolas indicate MPFA fits to variance-mean plot of WT synapses reproduced from Figure 3. (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) Recovery 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 3.

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 0.2 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 0.2 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 form 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.

Intrinsic membrane properties of mPFC neurons.

(A) Neuronal subtypes in mPFC layer 2/3. Cells were characterized based on their electrophysiological properties (n = 66, 64 for PC and FSIN, respectively). Representative voltage responses to current injection (-200 or +400 pA) of pyramidal cells (left) and fast-spiking interneurons (right). (B-D) Mean resting membrane potential (B), input resistance (C), and Sag ratio (D) measured in layer 2/3 PCs and FSINs. Gray symbols, individual data. (E) Current-spike relationship in each cell type. All statistical data are represented as mean ± S.E.M.; n.s. = not significant; ****, P<0.0001; unpaired t-test or two-way ANOVA.

Test for consistency of light stimulation in oChIEF-expressing L2/3 pyramidal cells.

(A) Representative images of an oChIEF-expressing neuron. Whole-cell recordings were performed from the fluorescently labeled neurons in layer 2/3 of the prelimbic cortex. Left, IR-DIC image (60×) of the pyramidal cell under whole-cell patch clamp. Right, fluorescence image of td-Tomato in the same cell excited at 530 nm wavelength. (B) Current-clamp recordings of action potentials evoked by 600 light pulse trains from an oChIEF-expressing L2/3 cell. For somatic stimulation, we used the typical light stimulation conditions used in STP experiments (5 ms in duration and 4 μm in radius; see Methods). Uniform action potentials were produced with high fidelity, albeit not without compromise towards the end of the train in the case of 40 Hz stimulation. Note that these results are similar to previously reported values from neocortical neurons (Lin et al., 2009; Yoon et al., 2020). (C) Same experiments as

(B) but in Syt7 KD pyramidal cells that express oChIEF in L2/3 of the mPFC.

Properties of EPSCs evoked by minimal stimulation.

(A) Optical minimal stimulation. A collimated DMD-coupled LED was used to confine the area of excitation to a small region near the soma. To stimulate a minimal number of excitatory axon fibers, the radius of illumination area was increased from 2 μm by 50% at each step, and selected the smallest illumination area that elicits EPSCs (Yoon et al., 2020) before starting each recording session. The illumination area was in the range of 3 to 8 μm in radius, when measured at the focal plane (typically 3–4 μm) of a 60× water immersion objective (blue circle in the photograph). (B-E) Properties of minimally stimulated baseline EPSCs (failure-excluded). Medians of frequency distributions are presented. Decay times below 1 ms were excluded from analysis. From left to right, representative traces (100 pulses), EPSC amplitude, rise time (20-80%), decay time (80-20%), and time to peak (from stimulus onset) from WT PC-PC (B, n = 81), WT PC-FSIN (C, n = 77), Syt7 KD PC-PC (D, n = 72), and Syt7 KD PC-FSIN (E, n = 78) synapses.

Validation of STP of optically evoked EPSCs

(A) The high pv at PC-PC synapses may result from direct photo-stimulation of axon boutons. To test if optically evoked EPSCs depend on APs, the effect of 1 µM tetrodotoxin (TTX) was tested. Applying 1 μM tetrodotoxin (TTX) and additionally 0.1 mM 4-aminopyridine, a D-type K channel blocker abolished optically evoked EPSCs (n = 8). Even when repolarization was hindered by subsequent addition of 100 µM 4-aminopyridine (4-AP), a potassium channel blocker, EPSCs were not rescued. These findings suggest that the light-evoked responses are dependent on APs of presynaptic axon fibers rather than photo-induced depolarization of presynaptic boutons (Little and Carter, 2013). (B) Comparison of STP evoked by optical stimulation with that by dual patch techniques. To test validity of STP of photostimulation-evoked EPSCs, we compared STP of unitary EPSCs evoked by dual whole-cell patch at PC-FSIN synapses. Both of EPSC1 and STP at 40 Hz recorded by using paired whole-cell recordings were not significantly different from those evoked by photo-stimulation, supporting the validity of our optogenetic methods. (Ba) Representative EPSC traces evoked by 20-pulse 40 Hz trains stimulation at pyramidal cell to fast spiking interneuron (PC-FSIN) synapses. A train of APs in a presynaptic PC (top), evoked EPSCs in a FSIN by dual whole-cell patch clamp (middle) or by optical stimulation (bottom) under same conditions. (Bb) Mean values for baseline-normalized amplitudes of EPSCs evoked by 40 Hz train at PC-FSIN synapses. Opto, optical stimulation (n = 10); Dual, dual patch techniques (n = 5). STP evoked by the two techniques were not significantly different (two-way repeated measures ANOVA, F (1, 13) < 0.01, P = 0.955). (Bc) Mean values for the first EPSC in different stimulation methods (P = 0.5724, unpaired t-test). Open circles, individual data. All statistical data are represented as mean ± S.E.M.

Test for AMPAR desensitization and saturation

(A) Representative traces of EPSCs evoked by paired-pulse photostmulation at 20 ms ISI (left) and mean values for paired pulse ratio (PPR, right) before and after applying 50 μM cyclothiazide (CTZ, n = 8), an AMPA receptor (AMPAR) desensitization inhibitor. CTZ induced no significant change in PPR arguing against the possibility that strong PPD is caused by AMPAR desensitization. Gray symbols, individual data. (B) It has been shown that multi-vesicular release occurs at recurrent excitatory synapses in neocortical L2/3 (Holler et al., 2021). Whereas a single vesicle release is insufficient to saturate postsynaptic AMPARs, an increase in the number of vesicular release during facilitation may saturate AMPAR resulting in underestimation of facilitation. To test this possibility, facilitation at 20 Hz was measured in the presence of 0.5 mM kynurenic acid (Kyn), a fast competitive antagonist (Diamond and Jahr, 1997; Wadiche and Jahr, 2001). Fast blockade of AMPA receptors did not have significant effects on short-term facilitation at excitatory synapses in the mPFC L2/3, suggesting that AMPAR saturation may not be significant during facilitation. (Ba) Representative EPSC traces (left) and average of baseline-normalized EPSCs (right) evoked by 40 Hz 20-pulse trains in control and in the presence of 0.5 mM kynurenic acid (Kyn). Although facilitation was slightly increased by Kyn, it was not statistically significant (n = 12; two-way repeated measures ANOVA, F (1, 22) = 0.575, P = 0.456). The slightly higher facilitation may result from reduced blocking efficiency of Kyn upon increased glutamate concentration in the synaptic cleft (Tong and Jahr, 1994; Meyer et al., 2001). (Bb) Mean values for the baseline EPSC (left) and the steady-state EPSC (right) before and after applying 0.5 mM kynurenate as shown in Ba. The baseline EPSC was reduced by Kyn to 43.5% of control. Gray symbols, individual data. All statistical data are represented as mean ± S.E.M.; n.s. = not significant; ***, P<0.001; paired t-test.

Effects of EGTA-AM on synaptic transmission

(A) Representative traces (top) and time courses of normalized amplitudes of the first EPSCs (bottom) during paired-pulse photostimulation at 20 ms ISI in control and in the presence of 50 μM EGTA-AM (n = 8). The green horizontal bar indicates application of 50 μM EGTA-AM.

(B) Mean values for the first EPSC (left) and PPR (right) before and after EGTA-AM application. Gray symbols, individual data. All statistical data are represented as mean ± S.E.M.; n.s. = not significant; *, P<0.05; paired t-test.

Presynaptic calcium kinetics at boutons of layer 2/3 pyramidal cells

(A-C) A mixture of Fluo-5F (150, 250 or 500 μM) and Alexa Flour 555 was loaded on their axonal boutons of L2/3-PCs through whole-cell patch techniques for dual excitation ratiometric measurements of the cytosolic [Ca2+] at axonal boutons. (A) The long-branched stretch of axon from L2/3 PCs visualized through maximum fluorescence intensity projection using confocal laser scanning of AF555 perfused via a patch clamp pipette. Scale bar, 10 μm. (B) Top, the selected bouton for recordings, among the three, is displayed with the corresponding scanning line. Scale bar, 5 μm. Bottom, Line-scan images for fluorescence of Fluo-5F and AF555. Scale bars, 50 ms and 2 μm. (C) Baseline-normalized fluorescence changes evoked by an AP. Fluorescence intensities were spatially averaged over the bouton shown in (B), background-subtracted and normalized to the baseline value (dF/F0). Inset, Ca2+ transient estimated from dF/F0. Scale bar, 50 ms and 100 nM. (D, E) The kinetic parameters of an AP-CaT were determined from a plot for the inverse of AP-evoked [Ca2+] increment (1/Δ[Ca2+]) vs. calcium binding ratio (κB; Eq. 4 in Material and Methods). (D) Reciprocal plot of the AP-induced [Ca2+] changes (1/Δ[Ca2+]) vs. corresponding calcium binding constants (κB) according to Eq. 2 (n = 14). A linear fit to the plot gave the amplitude of AP-CaT (ACa = 1.16 μM) from the y-intercept, representing the amplitude of free [Ca2+] increase in the absence exogenous Ca2+ buffer. The calcium binding ratio of endogenous static Ca2+ buffers (κS) was estimated as 96 from the x-intercept of the linear regression based on Eq. 2 in Material and Methods. Linear regression line, P = 0.035. (E) Plot of [Ca2+] decay time constant versus κB (n = 16). The time constant for [Ca2+] decay from mono-exponential fit was plotted vs. κB, and a linear regression based on Eq. 3 yielded the κS value of 77 from the x-intercept. The y-intercept was 43 ms, an estimate for a dye-independent value for Ca2+ decay time constant (τc) (Eq. 3 in Material and Methods). Linear regression line, P = 0.011. (F) Resting [Ca2+] measured from basal fluorescence ratio using Eq. 1 in Material and Methods as a function of [Fluo-5F] in patch pipettes. The horizontal line indicates 50 nM. The mean [Ca2+]rest at three different concentrations of Ca2+ indicator dye were not different and measured as 50 nM, suggesting that the dye did not influence [Ca2+]rest. (G-I) Strong PPD and the subsequent STF may be caused by Ca2+ channel inactivation and Ca2+-dependent facilitation (CDF) of calcium influx into presynaptic boutons, respectively. To test these possibilities, we measured presynaptic Ca2+ increments evoked by each of successive APs applied in a train at 40 Hz. (G) Fluorescence signals from a selected bouton with a scanning line. Scale bars, 3 μm (top), 100 ms and 2 μm (bottom). (H) Normalized fluorescence (dF/F0) responses to a train of APs at 40 Hz. (I) Total Ca2+ increments (Δ[Ca2+]T; free plus bound form) are plotted against the number of APs in the train (n = 13). Δ[Ca2+]T were calculated based on Eq. 2 with κS set to 90. No significant change was observed for the estimates of Δ[Ca2+]T at each of first 4 APs during 40 Hz train, indicating that contributions of Ca2+ channel inactivation and CDF to STP are unlikely. Mean ± S.E.M., One-way ANOVA; n.s. = not significant.

Quantal size estimated from Sr2+-induced asynchronous release.

(A) Representative traces (100 pulses) from PC-PC synapses. Black bars indicate the detection window (100 ms) for release events in sham stimulation (left bar) and photostimulation (right bar). (B) Representative distribution of asynchronous EPSCs at PC-PC synapses. Left, after photostimulation. Middle, before stimulation. Right, difference between left and middle panels. The difference distribution was fitted with a Gaussian function (m = 24.93, σ = 6.09). (C) Comparisons of estimates for quantal size (q) between WT and Syt7 KD synapses. From left to right: PC-PC (n = 16, 13 for WT and KD, respectively), PC-FSIN (n = 16, 15). Gray symbols, individual data. All data are represented as mean ± S.E.M., unpaired t-test; n.s. = not significant.

Analysis of double failure rate supports high pv at excitatory synapses

(Aa) Schematic of simple refilling model. (Ab) An exponential fit to the PPR plot as a function of ISIs reproduced from Figure 1G. Fitted function, PPR = 1.1 [1 − exp(− t/43.5 ms)]. (Ac) Representative traces for four combinations of success and failure of 1st and 2nd EPSCs evoked by paired pulses with ISI of 20 ms at PC-PC synapses, showing successes in both pulses (1_1), failures in both pulses (0_0), and mixed responses (1_0; 0_1). On the right side of each trace, corresponding observation probability is shown in percentages (mean ± S.E.M. n = 57). (B-C) Plots of the calculated P(F1, F2) values (B) and their differences (C) from the observed value (6.2%) under the simple refilling model are displayed on the plane of k1 versus pv.

Little effects of Syt7 KD on intrinsic properties of neurons

Whole-cell recordings were performed from the fluorescently labeled pyramidal neurons in the prelimbic L2/3 of WT (n = 30) or Syt7 KD (n = 16) rats. (A) Resting membrane potential. (B) Input resistance. (C) Sag ratio. Filled triangles, individual data. (D) Current-spike frequency relationship in WT and KD cells. All statistical data are represented as mean ± S.E.M., unpaired t-test or two-way ANOVA; n.s. = not significant.

Presynaptic expression of Syt7 rescues facilitation at L2/3 excitatory synapses

(A, D) Representative amplitudes of EPSC trains evoked by 600-pulse 10 Hz trains (flanked by 2 baseline periods comprised of 40 pulses each) at PC-PC (A) and PC-FSIN (D) synapses, in which presynaptic WT (left, gray triangles) pyramidal cells and those transfected with plasmid encoding shRNA against Syt7 (sh-Syt7, KD, middle, black triangles) or sh-Syt7 plus Syt7 insensitive to shRNA (rescue, right, red triangles). (B, E) STP of EPSCs evoked by 600-pulse trains at 10 Hz at L2/3 PC-PC (B; n = 9, 10, 7; WT, Syt7 KD, Rescue, respectively) and PC-FSIN (E; n = 12, 10, 8) synapses. Each data point represents an average from a moving window of 20 pulses, normalized to the baseline EPSC. (C, F) Mean values of 41-60th pulses from B and E. Mean ± S.E.M.; n.s. = not significant; **, P<0.01; ***, P<0.001; ****, P<0.0001; one-way ANOVA with Tukey’s post-hoc test.

Activity-dependent increase in the synaptic weight ratio at PC-PC to at PC-FSIN connections (Jee/Jie) is abolished by Syt7 KD

(A, C) Average amplitudes of baseline-normalized EPSCs during 20-pulse trains at frequencies from 5 to 40 Hz at PC-PC (left) and PC-FSIN (right) synapses in WT (A) and Syt7 KD (C). Data are reproduced from Figure 1 (A) and Figure 5 (C). (B, D) Relative synaptic weights of excitatory input to PCs over that onto FSINs (Jee/Jie) in WT (B) and Syt7 KD (D). Note that excitatory influence (Jee/Jie) to the recurrent network increases as the firing number and frequency increase in WT but not in Syt7 KD.

Locomotor activity and anxiety levels in Syt7 KD rats using OFT and EPM

(A, C) Representative tracking trace of the elevated plus maze (A) and open field test (C). (B, D) Mean values for total distance moved (left) and time spent (right) in each arm (B) or in periphery zone (D) in control (n = 13) or Syt7 KD rats (n = 9). Mean ± S.E.M.; Unpaired t-test or two-way ANOVA; n.s. = not significant.