Figure 3.

LTP-Associated PAP Withdrawal Depends on NKCC1 and Cofilin

(A) Top left: astrocyte fragment (5 μm z stack); circles, uncaging spots (400 μM NPE-IP₃; AF 594 channel, λ_x^2P = 840 nm). Other panels: Ca²⁺ response (200 μM Fluo-4; false colors) to IP₃ spot-uncaging (at t = 0; five 5 ms pulses at 5 Hz; λ_u^2P = 720 nm); time lapse shown; circle, ROI for Ca²⁺.

(B) Time course of intracellular Ca²⁺ signal (ΔF/G) in ROI shown in (A); one-cell example; red arrow (gray segment), IP₃ uncaging.

(C) Relative change in PAP VF (%, mean ± SEM) 25 min after: spot-uncaging of intracellular IP₃ (−1.4% ± 4.1%, n = 10), application of DHPG (300 μM, 3.5% ± 3.9%, n = 10), CB1 receptor agonist WIN55 (1 μM, 4.1% ± 0.4%, n = 3), or GABA receptor agonist muscimol (20 μM, 0.4% ± 1.7%, n = 6).

(D) Relative change in PAP VF (%, mean ± SEM; top) ~25 min after LTP induction, and the corresponding LTP level (%, mean ± SEM; bottom, sample size shown): in the presence of 0.5–0.7 U/mL chondroitinase ABC (ChABC, −14% ± 3%), control ChABC-c (−11% ± 7%), 10 μg/mL EphA4-Fc (−17% ± 3%), 10 μg/mL Fc control (−20% ± 2%), wild-type C57BI6 mice (−17% ± 3%), AQP4^−/− knockout mice (−18% ± 2%), 20 μM intracellular bumetanide (Bmtnd, −4% ± 4.5%), 50 μM intracellular bumetanide + 100 μM extracellular TGN-020 (Bmtnd⁺, −5.5% ± 2.7%), DMSO control 0.2% external + 0.05% internal (−19% ± 2%); blue text, data from mice; gray shadow, 95% CI for PAP VF change after LTP induction in control conditions; ^∗p < 0.02 (df = 12 for AQP4⁻/ versus Bmtnd, df = 9 for Bmtnd versus DMSO), ^∗∗∗p < 0.005 (df = 13 for AQP4⁻/ versus Bmtnd⁺, df = 10 for Bmtnd+ versus DMSO; t test or Mann-Whitney independent sample tests).

(E) PAP VF change (%, mean ± SEM) during LTP induction (arrow) in key tests shown in (D), as indicated, and summary for other experiments (Rest).

(F) Relative fEPSP slope (mean ± SEM) during LTP induction at CA3-CA1 synapses in control (n = 10) and with S3 peptide inside astroglia (200 μM , n = 6), as shown (Figures S3B and S3C).

(G) Occlusion experiment: PAP VF change (%, mean ± SEM, sample size shown): ~25 min after LTP induction in control (LTP no S3; −23% ± 3%), no LTP induction, whole-cell loaded S3 (S3 no LTP; −29% ± 6%); same but recorded in gap-junction connected astrocytes devoid of S3 (S3-GJ cells; −0.4% ± 2.4%); and ~25 min after LTP induction with S3 (S3 and LTP; −27% ± 3%); ^∗∗∗p < 0.001 (df = 13 for “LTP no S3” versus “S3 GJ Cells,” df = 12 for the rest).

(H) Time course of PAP VF (%, mean ± SEM) in the occlusion experiments shown in (G); notations as in (G).