1. Postsynaptic Plasticity Triggered by Ca2+-Permeable AMPA Receptor Activation in Retinal Amacrine Cells 
Mean-Hwan Kim, Henrique von Gersdorff 
Neuron 
Volume 89, Issue 3, Pages (February 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
Diverse Morphological Synaptic Connections between Mb Cell Terminals and AC Dendrites
(A) Schematic drawing of presynaptic recording from a goldfish Mb terminal and postsynaptic recording from an AC soma. GCL, ganglion cell layer, IPL, inner plexiform layer, INL, inner nuclear layer.
(B) The Mb terminal and AC soma were filled with 100 μM Alexa 488, a fluorescent dye, after patch-clamp recording, and images were captured at two different focal planes (1 and 2). The green arrows indicate AC dendritic varicosities; the red arrow indicates a telodendria from a Mb terminal. The left green arrow in (1) shows a putative synaptic contact between the Mb terminal and the AC dendrite.
(C–F) Axotomized (C; axon cut from soma) and intact Mb cells (D–F) form putative synapses with AC dendrites. The whole-cell patch pipette was positioned on the Mb terminals, which contact both morphological ON type AC (C and D) and bi-stratified ON-OFF (E,F) types of ACs. Insets: the Mb terminal (100 μM Alexa 555, red) and AC (100 μM Alexa 488, green) were filled with fluorescence dyes, and putative synaptic contacts are shown in yellow. All these paired recordings (B–F) displayed EPSCs in the ACs when the Mb terminal was depolarized (see Figure 2).
Neuron  , DOI: ( /j.neuron )
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3. Figure 2 Paired Recordings between Mb Terminals and AC Somata
(A) Patch-clamp recordings of different Mb terminals showing Cm changes and simultaneous EPSC paired recordings from ACs. Only one example of the Mb cell Ca2+ current (ICa2+) is shown for clarity. Cm was monitored with a 2 kHz sine wave before and after the step depolarization. The evoked EPSCs (IEPSC) are averages from three to five traces, and the AC was held at −70 mV. Note the diverse types of double-peaked EPSC waveforms with fast and slow components.
(B) EPSCs were evoked when a Mb terminal was stepped from −60 mV to between −50 mV (red trace) and +10 mV (black trace) in 20 mV increments for 100 ms. The corresponding ΔCm jump and EPSC charge transfer (QEPSC) are highly correlated (r2 = 0.97; inset).
(C) Average response of the presynaptic ΔCm jump and postsynaptic QEPSC (r2 = 0.89 ± 0.04; data points from n = 3 or 4 pairs).
Neuron  , DOI: ( /j.neuron )
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4. Figure 3
EPSCs from a Subgroup of ACs Can Display Postsynaptic Potentiation
(A1) An example of a paired recording that shows no plasticity (∼70% of paired recordings). Presynaptic Cm jumps were evoked in the Mb terminal by a depolarizing pulse to 0 mV, which elicits a double-peaked EPSC in the AC. The Mb cell and the AC were held at −70 mV. The black trace shows the data at the beginning of the recording and the red trace toward the end of the recording. No change is observed in Cm jumps or the EPSC. Note the short synaptic delay of the fast component of the EPSC.
(A2) The EPSC shown in A1 was integrated and plotted as EPSC charge during the recording. The solid line shows a linear fit to the data.
(B1) An example of a paired recording that shows plasticity in the EPSC (∼30% of paired recordings). Note that the Cm jumps do not change during the recording (indicating that the amount of exocytosis from the Mb terminal was not changing), although the EPSCs increased in amplitude and charge.
(B2) The EPSC shown in (B1) was integrated and plotted as EPSC charge during the recording. The data were fit with a sigmoid curve. Note that the EPSC remained potentiated for about 10 min. The decrease in EPSC charge at ∼16 min was accompanied by a decrease in the corresponding ΔCm jump.
(C–E) The normalized Cm changes (Norm. ΔCm), EPSC charges (Norm. QEPSC), and synaptic gains (Norm. gain) are averaged from the initial 3 min and 3–6 min time intervals after starting the paired recordings. All the non-plasticity-showing (gray) and plasticity-showing (light red color) paired recordings are included in the average analysis (n = 36 paired recordings; two-tailed Student’s t test, ∗p < 0.05, ∗∗p < 0.01).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
ACs with High Internal Ca2+ Buffering Do Not Show EPSC Potentiation
(A1) An example of paired recordings with high Ca2+ buffering in the AC. The internal solution of the ACs contained 20 mM BAPTA. The black trace shows the ΔCm and EPSC of the paired recordings at the beginning of the recording, and the red trace shows the ΔCm and EPSC toward the end of the recording (8 min later). No EPSC potentiation was observed.
(A2) The EPSCs from the cell in (A1) were integrated and plotted as EPSC charge during the recording. The solid line shows a linear fit to the data.
(B–D) The normalized Cm changes (Norm. ΔCm), EPSC charges (Norm. QEPSC), and synaptic gains (Norm. gain) were averaged from the initial 3 min and 3–6 min time intervals after starting the recording. Note that the normalized QEPSC and synaptic gain did not significantly change in both the initial 3 min and 3–6 min time intervals. Both individual (open circle) and average (closed circle with SEM) values are shown (n = 9 pairs).
Neuron  , DOI: ( /j.neuron )
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6. Figure 5
Repetitive Puff Application of 1 mM AMPA onto AC Dendrites Reveals an Ipuff Current Potentiation that Is Blocked by High BAPTA Concentrations in the AC
(A) AMPA puffs (1 mM) onto the IPL with the duration of 25–50 ms evoked inward currents in ACs held at a membrane potential of −70 mV. Repetitive AMPA-evoked currents gradually increased (2 mM EGTA internal solution). Gray (0 min; control) and black (2.3 min later) traces reveal the current potentiation.
(B) The normalized charge transfers of AMPA-evoked currents (Norm. Qpuff) are plotted and averaged for the initial 3 min and 3–6 min time interval after starting the recordings. The black trace shows the average values and includes all the non-plasticity-showing (gray, n = 16) and plasticity-showing (light red, n = 11) (two-tailed Student’s t test, ∗p < 0.05, ∗∗p < 0.01) AC recordings.
(C) The amount of Ipuff potentiation is significantly reduced with 10 mM BAPTA in the AC.
(D) Only two of eight recordings showed potentiation of Ipuff with 10 mM BAPTA in the internal solution.
(E) Ipuff potentiation is completely suppressed with a 20 mM BAPTA internal solution.
(F) The normalized charge transfers of Ipuff with a 20 mM BAPTA internal solution are not significantly changed at the initial 3 min and 3–6 min time intervals (n = 12; two-tailed Student’s t test).
Neuron  , DOI: ( /j.neuron )
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7. Figure 6 CP-AMPARs Mediate the Postsynaptic Plasticity
(A) After repetitive stimulation with 1 mM AMPA in the IPL, Ipuff potentiation was observed (compare trace 2 with trace 1). When 60 μM IEM 1460, a specific CP-AMPAR antagonist, is bath-applied (trace 3), the Ipuff is greatly reduced indicating, that it is composed largely of CP-AMPAR-mediated currents. Recovery was obtained in trace 4.
(B) The calculated charge transfers of the traces shown in (A) are plotted during the time course of the experiment (IEM was bath-applied in the time window of the shaded rectangular box).
(C and D) Repetitive iontophoretic stimulation with 1 M L-glutamate (L-Glu ionto) with NMDAR blockers (50 μM D-AP5 or 20 μM R-CPP) on AC dendrites can evoke current potentiation in some ACs. The same protocol as AMPA puff experiments were used here except for the L-Glu iontophoretic stimulation. Some ACs did not show Iionto potentiation (C, representative traces from n = 8), whereas some ACs showed a gradual Iionto potentiation (D, representative traces from n = 5). Internal solutions of the AC contained 2 mM EGTA. Possible activation of inhibitory currents and Ca2+ channels were pharmacologically blocked in all the experiments shown here (see Experimental Procedures).
(E) The evoked current charge (Qpuff) after treatment with CP-AMPAR blockers, 60 μM IEM (n = 6) or 1 μM philanthotoxin (PhTx; n = 3), were compared with evoked charges (Qpuff) right before drug treatments. Internal AC solutions contained 2 mM EGTA. We also note that 60 μM IEM significantly suppressed Qionto in both non-plasticity-showing ACs (n = 4) and plasticity-showing ACs (n = 5), and the degree of suppression between these two conditions was not significantly different (p = 0.52; n = 9).
(F) For non-plasticity-showing ACs, evoked Qpuff were compared before and after 5 min of treatment with 60 μM IEM. Internal solutions contained 10 mM BAPTA (n = 4) or 20 mM BAPTA (n = 10). There was no significant difference in the amount of IEM block.
(G) Before starting 1 mM AMPA puffs on the AC dendrites, the slices were treated with 60 μM IEM, which was continuously present in all these recordings. These AMPA-puff-mediated currents did not exhibit any potentiation. Normalized charge transfers (Norm. Qpuff) were plotted (the red dotted line indicates 1). Internal solutions contained 2 mM EGTA.
(H) With 60 μM IEM present in the bathing solution, there was no significant change in Qpuff after 3 min of recordings, but a significant decrease was observed in the 3–6 min time interval (n = 11; two-tailed Student’s t test, ∗∗p < 0.01).
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
Potentiation of Ca2+ Influx through CP-AMPARs in AC Dendrites during Ipuff Plasticity
(A) An example of ON-type AC morphology visualized with a fluorescent dye (100 μM Alexa 488). The red arrows indicate the locus of dendritic varicosities, where 1 mM AMPA puffs were targeted.
(B–D) Simultaneous recording of dendritic Ca2+ rises and evoked currents. The AC dendrites were filled with 100 μM Oregon green BAPTA1 and stimulated with 1 mM AMPA puffs. Activation of Ca2+ channels on AC dendrites was blocked by 200 μM Cd2+. Example traces from a plasticity-showing AC (C) and a non-plasticity-showing AC (D). AMPA mediated currents and dendritic Ca2+ rises were simultaneously measured. Repetitive 1 mM AMPA puffs were applied to targeted AC dendrites in every 30 s. In the initial trace (0 min, gray, ΔF/F) of (C), three data points (1, 2, and 3) are displayed with original images from (B) (regions of interest on the dendrite). The black arrow in (C) indicates the increase of the peak Ca2+ rise amplitude compared with the initial Ca2+ rise (gray trace in C). Bath application of 60 μM IEM significantly blocked the AMPA-evoked currents and Ca2+ rises (red in C and D).
(E and F) Normalized charge transfers (Norm. Qpuff, E) and peak Ca2+ rises (Norm. peak of ΔF/F, F) are plotted (n = 12; the blue dotted line indicates 1).
(G) Both charge transfers (Qpuff) and dendritic Ca2+ rises (ΔF/F) were significantly potentiated, but the amounts of the potentiation between Qpuff and ΔF/F was not significant (p = 0.29; n = 12).
(H) The amount of block by 60 μM IEM was greater in the dendritic Ca2+ rises compared with the Qpuff charge transfers (n = 6; two-tailed Student’s t test, ∗p < 0.05, ∗∗p < 0.01).
Neuron  , DOI: ( /j.neuron )
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9. Figure 8 Ipuff Currents Are Blocked by IEM at Mouse AII-ACs
(A) AMPA puffs (1 mM) onto the IPL with the duration of 20 ms evoked inward currents in AII-ACs held at a membrane potential of −60 mV. After repetitive stimulation with 1 mM AMPA in the IPL, Ipuff potentiation was observed (compare trace 2 with trace 1). When IEM is bath-applied (trace 3), the Ipuff is greatly reduced, indicating that it is composed largely of CP-AMPAR-mediated currents. Recovery was obtained in trace 4. Note that inward action currents are present during activation of AMPAR (see D).
(B) The calculated charge transfers of the traces shown in (A) are plotted during the time course of the experiment (60 μM IEM was bath-applied in the time window of the blue rectangular box).
(C) The normalized charge transfers of AMPA-evoked currents (Norm. Qpuff) are plotted and averaged for the initial 3 min and 3–6 min time interval after starting the recordings. The black trace shows the average values and includes all the non-plasticity-showing (gray, n = 9) and plasticity-showing (light red, n = 4; two-tailed paired Student’s t test, ∗p < 0.05) mouse AII-AC recordings.
(D) Onset delay of both AMPA evoked current and action current generation is shortened by Ipuff plasticity. The action currents are due to unclamped action potential spikes in the AII-AC.
(E) The evoked current charge (Qpuff) after treatment with CP-AMPAR blocker, 60 μM IEM (n = 6 for goldfish, shown in Figure 6E; n = 7 for mouse) was compared with evoked charges (Qpuff) right before drug treatments (two-tailed Student’s t test, ∗∗p < 0.01).
(F and G) AMPA puff-mediated Ca2+ rises were shown in both proximal and distal dendrites of mouse AII-AC. The AII-AC dendrites were filled with 100 μM Oregon green BAPTA1 and stimulated with 1 mM AMPA puffs. Activation of L-type Ca2+ channels on AII-AC dendrites was blocked by 2 μM isradipine (n = 3).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions