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. 2020 Dec 9;108(5):919–936.e11. doi: 10.1016/j.neuron.2020.08.030

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© 2020 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Reduced PAP Presence after LTP Induction at CA3-CA1 Synapses

(A) Left: 2PE point-spread function (PSF) excites dye-filled PAPs (yellow, 3D EM fragment) within an ~1 μm focal plane (dotted lines; bottom). Right: fluorescence within ROI (F_ROI) scales with PAP VF, reaching ~100% VF inside the 5–7 μm wide soma (F_S).

(B) Astrocyte filled with AF 594 (single focal section; λ_x²^P = 800 nm); dashed cone, extracellular recording pipette. F_ROI and F_S, areas of VF readout; see Video S1 for extended dynamic range.

(C) Traces, s. radiatum fEPSPs, before (pre) and ~25 min after LTP induction (post); graph, relative fEPSP slope (mean ± SEM; arrow, induction onset); ^∗∗∗p < 0.001 (25–30 min post-induction: 151.0% ± 6.7% compared to baseline, n = 18).

(D) Relative change in PAP VF (%, mean ± 95% confidence interval [CI]) in control (green; n = 24 cells) and during LTP induction (arrow, onset; orange; n = 29); red line, best-fit exponential decay to steady state $V F (t) = V F_{s s} + (1 - V F_{s s}) \exp (- t / τ)$ ; VF_ss = 0.77 ± 0.04, τ = 14 ± 5 min.

(E) Relative change in PAP VF (%, mean ± SEM) plotted against initial PAP VF, in control (n = 8 cells) and ~25 min after LTP induction (orange; n = 13; ^∗p < 0.05, ^∗∗p < 0.01, compared to control, df = 19).

(F) Grey, PAP VF change (%, sample size n shown) in hypo-osmotic (220 mOsm/L) and hyper-osmotic (420 mOsm/L) solutions, as shown. Green and orange, PAP VF change 25–30 min after LTP induction in control (LTP, mean ± SEM: −25% ± 7%), in 50 μM APV (+APV, 3.1% ± 9.9%), with no HFS (−0.8% ± 7.3%), under Ca²⁺ clamp (Ca-clamp, 6.8% ± 9.5%), under Ca²⁺ clamp with 10 μM D-serine added (Ca-clamp⁺ D-ser, −24% ± 7%); ^∗∗p < 0.01; ^∗p < 0.05; dots, individual cells.

(G) Evaluating diffusion coupling inside astroglia using FRAP of dialyzed AF 594 (STAR Methods; single focal section; ~80 μm depth); arrow, example line scan position.

(H) Top: line scan as in (G) (baseline conditions; gray segment, shutter closed). Bottom: the corresponding fluorescence time course, before (Cntrl) and ~20 min after LTP induction (LTP); F₀, initial intensity; arrows, FRAP during shutter-on period (full recovery takes 39–40 s).

(I) Summary of FRAP tests (G and H); diagram, LTP induction may taper PAPs lowering diffusion coupling. Graph (mean ± SEM), FRAP rate relative to baseline (left ordinate): ~25 min after LTP induction (LTP, 62% ± 12%, n = 11; ^∗p < 0.05); in 50 μM APV (108% ± 32%, n = 7); no-HFS control (87% ± 15%, n = 7). Grey (right ordinate), change in extracellular diffusivity ~25 min post-induction (LTP-ECS; 104% ± 7%, n = 5; Figures S1F–S1H); dots, individual tests.