Previous work has shown that modified whisker input, where all but a single-whisker (single-whisker experience; SWE) has been removed from one side of the mouse face, leads to the potentiation of synapses at layer 4-layer 2/3 excitatory inputs in the neocortical representation of the spared whisker. This potentiation can be accompanied by an increase in presence of CP-AMPARs, determined by their electrophysiological and pharmacological properties. The trafficking of CP-AMPARs is associated with age and input identity, where they are not implicated for plasticity induced at later developmental ages (when SWE begins at P13 or older ages) or at layer 2/3-layer 2/3 inputs (Wen and Barth, 2011
Assays to demonstrate the experience-dependent increase in excitatory synaptic strength have relied upon a method to isolate the post-synaptic response in a pathway-specific manner, using an ACSF where Sr++
. Under these conditions, neurotransmitter release is triggered by electrical stimulation to a specific input, but vesicle release is slowed such that the post-synaptic response to individual quanta can be evaluated (Goda and Stevens, 1994
; Xu-Friedman and Regehr, 1999
). In previous analyses (Clem and Barth, 2006
; Clem et al., 2008
; Wen and Barth, 2011
), the AMPAR-EPSC was pharmacologically isolated from NMDAR currents by the application of D-APV. However, during the course of our investigations, we discovered that when both NMDAR- and AMPAR-mediated currents were present, there was sometimes a voltage-dependent run-down in the amplitude of average Sr-EPSC for a given cell. This run-down was only present at layer 4-2/3 synapses from SWE-treated animals, suggesting that it might be related to the recent potentiation at these synapses.
Sr-depression is induced by a modest post-synaptic depolarization
We formalized a method to examine this synaptic lability, named Sr-depression, by comparing mean Sr-EPSC amplitude before and after post-synaptic depolarization. We use the term Sr-depression to indicate specifically that this depression was not what has typically been considered “short-term depression” in other studies, since its onset is immediate and it is stable for many minutes following pairing. Experiments were carried out in acute brain slices from SWE-treated animals at postnatal day 13 (P13), a time when experience-dependent plasticity is pronounced (Figures ; Wen and Barth, 2011
). Typically, Sr-EPSC amplitude from a given cell was calculated from the average of a 50–100 individual, well-isolated events (Figure ). The mean amplitude of these events was constant over the recording period when the post-synaptic cell is maintained at hyperpolarized holding potentials. Sr-EPSCs are primarily mediated by AMPARs, since application of NMDAR-antagonists does not change Sr-EPSC amplitude at hyperpolarized potentials when NMDARs exhibit a characteristic Mg-dependent voltage block.
Figure 1 Sr-depression can be elicited at layer 4-2/3 synapses from SWE-treated mice. (A) Schematic of an SWE animal (top) and recording configuration in the spared barrel column (bottom). (B) Fluorescence image of a slice that contains the spared barrel column, (more ...)
Depolarization of the post-synaptic cell (−20 mV, 5 min, 0.1 Hz) leads to a rapid, change in Sr-EPSC amplitude (Figure ), without any change in event frequency between the baseline and post-pairing period (Figure , before frequency 2.85 ± 0.35, vs. after 2.63 ± 0.21, n = 8 cells, p = 0.65 by paired t-test). Since stimulation strength is not altered during the experiment, and since individual Sr-EPSCs are thought to represent individual release events at distinct synaptic contacts, these results indicate that this depression is likely to be post-synaptic in origin.
Because statistical comparisons were carried out for a large number of events before and after pairing, this method was very sensitive to small changes in Sr-EPSC amplitude. In tissue from SWE animals, 16/20 (Figures and ) cells showed a significant reduction in Sr-EPSC amplitude, with a mean depression of ~20% (Figures and ; average of 16 cells 20 ± 2%; p value range for individual cells 0.025–0.00002 for baseline vs. post-pairing window by Mann–Whitney U-test). A cumulative distribution histogram for all cells from the spared barrel column showed a highly significant reduction in event amplitude in the post-pairing window (Figure ; Kolmogorov–Smirnov test p < 0.00001, n = 10 cells).
Figure 8 Sr-depression occurs irrespective of CP-AMPARs after 24 h SWE. (A) The RI of cells before the induction of Sr-depression is not correlated with the magnitude of depression in the same cells (n = 8). Cells presented here all exhibited significant Sr-depression (more ...)
Figure 7 CP-AMPARs are not required for Sr-depression. (A) Example scatter plot of a Sr-depression experiment in the presence of NASPM that still showed depression. Inset: average traces. Scale: 5 pA, 5 ms. (B) Within-cell comparison of Sr-EPSC amplitude between (more ...)
In control animals, 0/7 cells showed a change in Sr-EPSC amplitude after pairing, (p value range for individual cells 0.19–0.89 for baseline vs. post-pairing window; Figures ). Depolarization did not change event frequency in the post-pairing window (Figure ). An absence of synaptic depression was confirmed by analysis of a cumulative distribution histogram of Sr-EPSCs from control cells, where depolarization failed to trigger any shift in event distribution (Figure , Kolmogorov–Smirnov test p = 0.715, n = 7 cells).
Figure 2 Layer 4-2/3 synapses from control animals did not exhibit Sr-depression. (A) Schematics of a control animal (top) and electrode configuration (bottom). (B) Example scatter plot of individual Sr-EPSC amplitudes from a control cell using the same pairing (more ...)
Requirements for Sr-depression
Many forms of synaptic depression, including depolarization-induced plasticity at layer 4-2/3 synapses in the spared barrel column (Clem et al., 2008
), require NMDAR-activation. To determine whether Sr-depression requires NMDARs, we examined whether bath application of the NMDAR-antagonist D-APV was sufficient to block depression in cells from SWE-treated animals. In the presence of D-APV, 0/5 cells showed a significant reduction in Sr-EPSC amplitude after pairing (Figures , p
value range 0.11–0.65, baseline vs. post-pairing window by Mann–Whitney U
-test). Analysis of a cumulative distribution histogram of Sr-EPSC events from all cells before and after pairing in Sr++
showed a small, but still significant reduction in amplitude (Figure ; Kolmogorov–Smirnov test p
= 0.021 for baseline vs. post-pairing window; note comparison from Figure where p
Figure 3 Sr-depression in SWE-treated animals requires NMDAR activation. (A) Within-cell comparison between pre- (filled) and post-pairing (open) windows in the presence of D-APV. (B) Cumulative distribution histogram of Sr-EPSC amplitude before (solid line) and (more ...)
We also found that a more modest depolarization to −40 mV was sufficient to block Sr-depression in most cells (Figure ; 4/6 did not show significant depression, within-cell p value range 0.14–0.73 for baseline vs. post-pairing window by Mann–Whitney U-test), consistent with a role for NMDAR-activation which remains partially blocked at this holding potential. As above, the cumulative distribution of Sr-EPSC event amplitudes showed a small shift following pairing at −40 mV (Figure ; Kolmogorov–Smirnov test p = 0.01 for baseline vs. post-pairing window). Thus, Sr-depression requires post-synaptic depolarization and NMDAR activation. Based on the small but statistically significant reduction in Sr-EPSC amplitudes shown in the cumulative distribution histograms, there may be additional pathways that are involved in this depression.
The role of CP-AMPARs in Sr-depression
Previous work from our lab has shown that CP-AMPARs are trafficked to layer 4-2/3 synapses after SWE. Because other investigations have found that these receptors can be highly labile at the synapse, we hypothesized that the rapid depression observed might be due to the NMDAR-dependent removal of CP-AMPARs. Previous Sr-depression experiments were carried out in tissue from P13 animals, since CP-AMPARs have been detected after SWE at this age (Wen and Barth, 2011
). Consistent with our previous findings, we observed significant rectification of pharmacologically isolated AMPAR-EPSCs after SWE at this age (Figures ; Control RI 0.57 ± 0.06 n
= 14 cells vs. SWE RI 0.42 ± 0.04 n
= 10 cells, Mann–Whitney U
= 0.03). To calculate the RI, EPSC amplitude was recorded at −70, 0, and +40 mV (see Methods). Cells with an RI smaller than 0.57 were classified as rectifying, and those with an RI equal to or larger than 0.57 were classified as non-rectifying.
Figure 4 CP-AMPARs are present at layer 4-2/3 excitatory synapses after 24 h SWE at P13. (A) Example AMPA-EPSC traces recorded at −70, 0, and +40 mV from a control animal (left, black) and a SWE-treated animal (right, green). Scale: 20 pA, 10 ms. (B) RI (more ...)
As a second method to quantify the contribution of CP-AMPARs at layer 4-2/3 inputs, we used PhTx or a synthetic analog of the CP-AMPAR antagonist Joro spider toxin, NASPM; (Koike et al., 1997
) to block CP-AMPARs (Figure ). Although layer 4-2/3 inputs from SWE animals show significant rectification compared to age-matched controls, the PhTx/NASPM-sensitive current was not significantly different between the two groups (Figure , reduction in EPSC amplitude for control 0.11 ± 0.09 n
= 7 vs. SWE 0.24 ± 0.12 n
= 7, p
= 0.32 by Mann–Whitney U
-test), likely due to large variability in magnitude of NASPM-sensitive current across cells in both control and SWE conditions. This is in contrast to previously published results showing minimal Joro spider toxin block at layer 4-2/3 synapses in control animals (Clem and Barth, 2006
), which were not focused on the specific developmental age (P13) tested here. It is notable that a subset of cells in control animals showed strong rectification (5/14 cells show RI less than 0.55) and NASPM blockade (3/7 control cells showed >15% block), despite the fact that we could not induce Sr-depression in cells from this group.
CP-AMPAR blockade results in a small decrease in Sr-EPSC amplitude
To determine whether we could detect a contribution of CP-AMPARs in Sr-EPSC amplitude, we determined the effect of NASPM on layer 4-evoked Sr-EPSCs from SWE-treated animals. We predicted that if there were a small number of CP-AMPARs at individual layer 4-2/3 synapses, NASPM blockade should reduce the mean amplitude of these events. A comparison across cells from SWE-treated animals showed that mean Sr-EPSC amplitude was 11.62 ± 0.5 pA (n = 19), compared to the amplitude of Sr-EPSCs in NASPM at 10.65 ± 0.34 pA (n = 17), a difference that was not significant (Figure , p = 0.099 by Mann–Whitney U-test). Sr-EPSC amplitude before and after NASPM application within the same cell was compared for a smaller group of neurons. NASPM did not consistently decrease event amplitude (Figures ; mean EPSC amplitude before drug application, 11.94 ± 0.4 pA vs. after 10.95 ± 0.9 pA, n = 4, p = 0.43 by paired t-test).
Figure 5 NASPM as a tool to block CP-AMPARs. (A) Cross-cell comparison of Sr-EPSC amplitude between SWE-treated animals (SWE) and SWE-treated animals in the presence of NASPM (SWE+NASPM). (B) Example experiments of a cell that showed no reduction in Sr-EPSC amplitude (more ...)
Because CP-AMPARs, specifically those that are homomeric for GluA1, have been shown to have moderately faster decay kinetics than GluA1-GluA2 heteromers or GluA2 homomers (Oh and Derkach, 2005
), we also examined whether NASPM would slow the decay constant of the Sr-EPSC. There was no significant reduction in decay kinetics in the presence of NAPSM when compared across all cells (P13 SWE Sr-EPSC 3.27 ± 0.09 ms, n
= 17 vs. in NAPSM 3.19 ± 0.08 ms, n
= 15, p
= 0.31 by Mann–Whitney U
-test) and also when compared before and after drug application in the same cell (before 3.12 ± 0.16 ms vs. after 2.94 ± 0.19 ms, n
= 4, p
= 0.19 by Mann–Whitney U
-test). Although this difference might become significant with a larger sample size, the lack of a pronounced effect suggests that decay kinetics of the Sr-EPSC is not a reliable indicator for CP-AMPARs.
If there were some synapses that contained primarily CP-AMPARs, NASPM application might reduce the apparent frequency of layer 4-triggered Sr-EPSCs without influencing the mean amplitude of events. Such a scenario might explain why the multi-quantal EPSC amplitude might be reduced by antagonist application, but the single-quantal response would not be markedly affected. However, a comparison of event frequency before and after NASPM application showed that event frequency was significantly increased (Figure , baseline 3.96 ± 0.21 Hz vs. post-drug 4.73 ± 0.40 Hz, n = 4 cells), even when the mean amplitude of the Sr-EPSC was significantly reduced (Figure , bottom panel). This increase in frequency suggests that NASPM might have some presynaptic targets that normally suppress presynaptic neurotransmitter release. Thus, we ascribe the lack of statistical significance for NASPM-blockade of multiquantal EPSCs between control and SWE layer 4-2/3 synapses (Figure ) to large cell-to-cell, not simply synapse-to-synapse, heterogeneity in the distribution of CP-AMPARs.
NASPM effectively blocks CP-AMPARs
A critical assumption behind using NASPM to block CP-AMPARs is that this compound is sufficient to fully block these receptors under our recording conditions. To verify that this was indeed the case, the RI was determined before and after drug application in the same cell. If NAPSM is sufficient to eliminate the contribution of rectifying AMPARs, the RI should become linear after drug application. This is indeed what was observed in pharmacologically isolated AMPAR-EPSCs (Figure ). Cells were divided into two groups (rectifying vs. non-rectifying), based upon their RI values. Cells with a rectifying I–V showed a 23 ± 8% (n = 5) block in peak AMPAR-EPSC amplitude, compared to cells with a linear I–V with a 3.5 ± 5% (n = 5) block. As expected, blockade of CP-AMPARs made the I–V more linear (Figures , pre RI 0.4 ± 0.07 vs. post RI 0.59 ± 0.03, n = 5, p < 0.05 by paired t-test). Taken together, these data indicate that NASPM application is sufficient to eliminate rectification, most likely through the selective blockade of CP-AMPARs.
Figure 6 NASPM application blocks rectifying AMPARs. (A) Fraction AMPA-EPSC amplitude blocked by NASPM wash-in at +40 mV in rectifying cells (red) and non-rectifying cells (black). Dashed lines: horizontal, fraction NASPM = 0; vertical, RI = 0.57. Mean RI and (more ...)
Sr-depression does not require CP-AMPARs
If synaptic lability at recently potentiated synapses is mediated by the removal of CP-AMPARs, we should expect that when CP-AMPARs are blocked, Sr-depression can no longer occur. To test this hypothesis, NASPM was bath applied to slices from SWE-treated animals, and Sr-depression was induced by post-synaptic depolarization. Because this antagonist is an open-channel blocker, care was taken to bath apply the drug with afferent stimulation for at least 15 min prior to pairing. In half the cells (4/9), a significant depolarization-induced reduction in Sr-EPSC amplitude was observed (Figures ), indicating that in these cells reduced current through CP-AMPARs was not required for depression. The magnitude of depression in cells that showed a significant depolarization-induced change in Sr-EPSC amplitude was identical to that which we characterized earlier, ~20% (Figures and , 16 ± 3%, n = 4). The cumulative distribution of Sr-EPSC amplitude was also shifted after the pairing protocol in NASPM (Figure , p < 0.001 by Kolmogorov–Smirnov test). Overall, these findings are incompatible with an obligatory role for CP-AMPARs, either for induction or expression, in Sr-depression.
We also evaluated the decay kinetics of the post-pairing Sr-EPSC. If fast-decay CP-AMPARs are removed by this pairing protocol, it was reasonable to hypothesize that there might be an increase in the decay time constant. However, the lack of significant change after NAPSM application suggested we might not be able to detect a small change in decay kinetics. A comparison of the baseline and post-pairing decay time constant of the Sr-EPSC revealed that the decay time constant was not slower after Sr-depression (3.86 ± 0.09 vs. 3.27 ± 0.16 for baseline vs. post-pairing window, n = 8 cells, p = 0.52 by Mann–Whitney U-test). These data are inconsistent with the removal of fast-decay CP-AMPARs during Sr-depression.
Rectification is not correlated with Sr-depression
If CP-AMPARs are important for Sr-depression, either for its induction, or because they are selectively removed, then cells with more rectifying AMPAR-EPSCs should show greater depression. This was not the case (Figure ). The amount of Sr-depression was uncorrelated with the cell's RI, when RI measurements in a Ca++ based ACSF were made before Sr++ wash-in and depolarization (Figure , p = 0.53; rectifying cells, RI 0.45 ± 0.03, magnitude of depression 21 ± 4% vs. non-rectifying cells, RI 0.70 ± 0.05, magnitude of depression 23 ± 3%, n = 4 cells each). Cells that showed a linear I–V (Figure , RI 0.59) or a rectifying I–V (Figure , RI 0.53) showed similar depression.
In a subset of cells, the RI was determined in a Ca++ based ACSF both before and after Sr-depression. In these cells, we noted that the RI became more linear (Figure , pre RI 0.45 ± 0.03 vs. post 0.66 ± 0.02, n = 4, p < 0.05 by paired t-test), suggesting that when present, CP-AMPARs might be removed during depression. Thus, CP-AMPARs can be removed during, but their presence is not required for, Sr-depression.
Sr-depression is absent at older developmental ages
SWE-induced increased in the strength of layer 4-2/3 synapses can be observed at least until P14, although the contribution of CP-AMPARs to SWE-induced potentiation appears minimal at this time. The RI is identical for SWE at P14 compared to control cells (control RI 0.64 ± 0.59 n
= 11 vs. SWE 0.59 ± 0.13 n
= 4, see also Wen and Barth, 2011
), and NASPM showed a small effect on reducing the amplitude of the multiquantal EPSC (Figure , control −8 ± 12% n
= 6 vs. SWE −14 ± 11%, n
= 4). At this age, Sr-depression in the spared whisker barrel column could not be induced in any cell (Figure , 0/6 cells, p
value range 0.49–0.70 baseline vs. post-pairing window by paired t
Figure 9 Sr-depression was not observed at later developmental ages. (A) Fraction NASPM/PhTx sensitive current in P14 control and SWE-treated animals. (B) Comparison of Sr-EPSC amplitude of the pre-pairing window between P13 and P14 SWE-treated animals. (C) Within-cell (more ...)
The cumulative distribution of Sr-EPSC amplitudes were not different between pre- and post-pairing window (Figure , p
= 0.936). The amplitude of SWE-induced synaptic strengthening was comparable between the two ages [Figure , P13 11.62 ± 0.48 pA, n
= 19 cells vs. P14 11.24 ± 0.49 pA, n
= 8 cells, p
= 0.94 by Mann–Whitney U
-test; see also (Wen and Barth, 2011
)], suggesting that pre-pairing response amplitude was not a factor in the induction of Sr-depression. These data suggest the presence of a critical period for Sr-depression which concludes at the end of the second postnatal week.