Figure 5.

Effects of PDBU and 4-AP on the amplitudes of [Ca²⁺] transients recorded from CA1 PC boutons targeting PV+ or mGluR1α+ INs

(A) Two-photon (2P) image stack of a CA1 PC (Cell #3) basal dendritic tree and axonal arbor filled with 20 μM Alexa Fluor 594 (white), 200 μM Fluo5F, and biocytin. Boxed area indicates the part of the imaged axonal arbor and is shown at higher magnification in (B).

(B) High magnification 2P image of the scanned axon collateral. Numbers indicate scanned boutons.

(C) Averaged [Ca²⁺] transients evoked by 5 APs at 40 Hz in local axon collaterals of a CA1 PC (Cell #1, trace is average of 17 boutons, black). Each bouton was scanned at the beginning of the imaging period (Baseline, black) and 30 min later (CTRL, gray) without perfusing any drug.

(D–F) Same as (C) for Cells #2–4 but, after a control imaging period (Baseline), 1 μM PDBU (D), 5 μM 4-AP (E), or both (F) were applied (average trace from 9, 12, and 21 boutons, respectively).

(G) Peak amplitudes of [Ca²⁺] transients for the first AP do not change in CTRL and in PDBU but increase significantly following the application of 4-AP and 4-AP + PDBU (WSR test, p = 0.10, 0.35, 0.003, 0.036, respectively). Data points represent individual cells (CTRL: n = 11; PDBU: n = 7; 4-AP: n = 12; 4-AP + PDBU: n = 6). Red dots indicate the cells shown in (C–F).

(H) KW test with post hoc Dunn’s test demonstrates that 4-AP (p = 0.0009) and 4-AP + PDBU (p = 0.0003) have significant effects on the peak amplitudes of [Ca²⁺] transients compared to control, while PDBU does not change the transients significantly (p = 1). Data points represent individual cells (CTRL: n = 11; PDBU: n = 7; 4-AP: n = 12; 4-AP + PDBU: n = 6). Red dots indicate the cells shown in (C–F).

(I) Maximum intensity projection of a confocal image z stack showing the imaged boutons after fixation and the visualization of biocytin. Each imaged bouton can be unequivocally identified.

(J and K) Some of the imaged boutons are in direct contact with mGluR1α+ (J, #5, 6, 10) or PV+ (K, #1, 3) dendrites.

(L) Averaged [Ca²⁺] transients evoked by 5 APs at 40 Hz in boutons #5, 6, and 10 targeting mGluR1α+ (cyan CTRL, dark cyan 4-AP) and in boutons #1 and 3 targeting PV+ dendrites (CTRL, light orange, 4-AP, dark orange). [Ca²⁺] transients are smaller in boutons targeting mGluR1α+ dendrites.

(M) Peak amplitudes of averaged [Ca²⁺] transients in response to the first AP in boutons with identified postsynaptic partners (cyan mGluR1α+, orange PV+) in CTRL and in the presence of drugs (CTRL: p = 0.87 and 0.50, n = 6 and 5 cells; PDBU: p = 0.42, n = 4 cells only for mGluR1α+; 4-AP: p = 0.0012 and 0.06, n = 12 and 11 cells; 4-AP + PDBU: p = 0.031 and 0.032, n = 5 and 4 cells for mGluR1α+ and PV+ INs, respectively, paired t test). Red and blue dots indicate data from Cell #3.

Horizontal lines in the boxplots: 25th, 50th, and 75th percentiles, rectangle: mean, whiskers: SD.