Differences in synaptic transmission and plasticity between PFC and S1 synapses.

(A) Schematic representation of the mature mouse brain (top) and the positions at which coronal sections were made from PFC (orange; middle) or S1 (blue; bottom). Recordings were made in acute slices from L5PN-L5PN pairs or from L2/3-L5PN connections. (B) EPSCs (bottom) recorded from postsynaptic L5PNs after evoking action currents in the presynaptic L5PNs (top, inset: 3fold magnification) in PFC (left) or S1 (right), individual recordings in gray, averages in black). (C-F) Summary of basic synaptic properties: EPSC amplitudes (C), failure rates (D), synaptic delays (E) and their SD values (F) in PFC and S1 (medians ± IQRs, means as dashed lines, whiskers represent 10th and 90th percentiles, dots indicate outliers, numbers of cell pairs in brackets; ***P<0.001, MWU). Data from S1 are from ref. (Bornschein et al., 2019a). (G) EPSCs recorded from pairs of L5PNs at 50 Hz intervals in PFC (top) and S1 (bottom), presynaptic stimulations are omitted, averages from at least 10 individual recordings). (H, I) Summary of PPRs (H; ***P<0.001, MWU) and success rates (I) following the 1st (1_x) and 2nd stimulation (x_1; ***P<0.001, P=0.987, WSR). S1 data are from ref. (Bornschein et al., 2019a). (J) As in (G) but for L2/3-L5 synapses (bottom, averages from at least 15 individual recordings, stimulation artefacts were blanked for clarity, arrowheads denote the time points of extracellular stimulation). (K) Boxplots summarizing PPR data from lateral PFC (PFC), medial PFC (mPFC), older PFC (P90-100) and in S1 (***P<0.001, MWU; n.s., not significant, P=0.067, ANOVA on ranks). (L) Comparison of SDs of the synaptic delays at L2/3-L5 synapses between PFC and S1 (***P<0.001, MWU).

Area-specific differences in EGTA sensitivity.

(A) Averaged, baseline-normalized EPSC amplitudes (means ± SEMs, 2-min bins) recorded from L5PN-L5PN pairs in PFC with normal pipette solution (control, orange) and after re-patching the presynaptic neuron with a pipette solution containing 10 mM EGTA (blue). Insets: Average EPSCs from one pair during baseline (-10 to 0 min) and test period (20 to 30 min) for control and EGTA (averages normalized to baseline). (B) Boxplot comparing the normalized EPSC amplitudes prior and following the application of EGTA with control recordings in L5PN pairs in PFC (*P=0.041, MWU). Note the significant EGTA effect. (C) As in (A) but for L2/3-L5PN connections in PFC. Following baseline recordings (10 min), slices were perfused for 30 min (solid line) with ACSF containing either 10 μM EGTA-AM (blue), 10 μM BAPTA-AM (red), or only the solvent DMSO/Pluronic (control, orange), and thereafter rinsed for 10 min with ACSF (test period). Insets: Averaged EPSCs during baseline and test period. (D) Summary of chelator-AM effects in PFC, mPFC and older PFC (P90-100; *P=0.016, **P=0.007, 0.008, 0.008, MWU; P=0.404, ANOVA on ranks). Note the EGTA sensitivity of release in all PFC recordings. (E) As in (C) but for L2/3-L5PN connections in S1 (control in light blue). (F) Summary of chelator-AM effects in S1 (P=0.792, **P=0.008, MWU). Note the absence of a significant EGTA effect in S1.

Quantal synaptic parameters in PFC and S1.

(A) MPFA of EPSC amplitudes recorded at the indicated [Ca2+]e from a pair of L5PNs in PFC. Top: EPSCs (gray, average in black) recorded in 1, 2 and 5 mM [Ca2+]e. Middle: Plot of EPSC amplitudes over time. Bottom: Corresponding mean-variance plot fitted with a parabola estimating the quantal parameters of release. pN is for 2 mM [Ca2+]e. (B) As in (A), but for a L2/3-L5PN connection in PFC. Recordings were made in the presence of 0.25 mM Kyn and 50 µM APV. (C) As in (B), but in S1. (D) Summary of quantal release parameters in L5PN pairs from PFC and S1 and in S1 with 1-2 mM γDGG (cf. (1); P=0.121, *P=0.048, ANOVA on ranks; P=0.054, MWU PFC vs. S1). (E) Left: Example of evoked qEPSCs (top) and the corresponding amplitude histogram (bottom, gray range was used for calculating the qEPSC amplitude). Right: Summary of qEPSC amplitudes in PFC and S1 (**P=0.004, MWU). (F) Summary of pN values in L2/3-L5PN connections from PFC and S1 (P=0.222, MWU).

Presynaptic Ca2+ transients are similar in PFC and S1.

(A) Two-photon images of L5PNs in PFC (left) and S1 (right) filled with Fluo-5F and Alexa 594 (scale bar 50 µm). Insets: Magnifications showing presynaptic boutons from which Ca2+ transients are recorded (scale bar 5 µm). (B) G/R signals (top) evoked by single APs in individual cells (gray, average of 5-13 boutons each, grand average in black) and their conversion to Ca2+ increases (Δ[Ca2+]i, bottom). (C-F) Summary of G/R signals (C; P=0.112, MWU), Δ[Ca2+]i (D; P=0.361), basal Δ[Ca2+]i (E; P=0.597) and decay time constants τ1 and τ2 (F; P=0.532, 0.962) in PFC and S1. Note, that there is no significant difference in presynaptic Ca2+ transients between PFC and S1.

Model predicts microdomain coupling at PFC synapses.

(A) Fit of the model (blue line) to the measured average Ca2+ signal (A; gray line) of L5PN boutons in PFC. The fit was obtained with the indicated number of open VGCCs. (B) pN values were simulated with the Syt1 priming model (Bornschein et al., 2019a; Bornschein et al., 2025) for control conditions and in the presence of EGTA with different VGCC numbers yielded different coupling distances (CD) in the model. The simulated pN were subtracted from and normalized to the experimental values, i.e. a value close to zero indicates best agreement between simulation and experiment. This was obtained with 9 open VGCCs at a CD of 51 nm (see C). (C) Simulated pN for 9 open VGCCs under control conditions at increasing CDs (blue line). The experimental pN value (median ± IQR, dashed line, gray range, Figure 3D) was reproduced at a CD of 51 nm (48-55 nm). (D) Illustration of the ring model used in B and C with a CD of 51 nm between 9 VGCCs (orange) and the release sensor of a vesicle (blue). (E) Examples of averaged normalized paired pulse recordings at 50 Hz at L2/3-L5PN synapses before (black) and after (orange) application of 5 µM Cd2+ in PFC. (F) Bar graph quantifying normalized PPRs after Cd2+ application in PFC and S1 (**P=0.005, P=0.667, paired t-Test). Note that Cd2+ increased the PPR in PFC.