To investigate the mechanism of DP, LTP was first induced in 18 cells by pairing afferent stimulation (baseline frequency) with a holding potential of 0 mV. This resulted in stable LTP (EPSC amplitude = 186 ± 16 % of baseline, n = 18).

In agreement with a previous study 18, cells fell into two groups with respect to changes (> 20%) in γ of AMPA receptor channels during LTP. In the majority of cases (11/18 cells), LTP was associated with an increase in γ (LTPγ; 246 ± 26% of baseline; range 136 – 363%). In the other 7 cells there was no increase in γ during LTP (100 ± 4 % of baseline; range 85 – 118 %), indicating that there was an increase in the functional number of channels activated (LTP_N). As noted previously 18, there were no differences between the two groups of neurons with respect to a variety of baseline parameters.

Figure 1 shows two examples from the group of cells that exhibited LTPγ, as indicated by the change in the current-variance plot obtained from non-SFA (Figure 1B). As previously reported 18, the increase in γ was not associated with any change in EPSC kinetics (Figure 1C) indicating that AMPA receptor channel kinetics were not affected 29. Failures analysis (Figure 1C) of this group of cells (Figure 2) revealed that LTP was associated with changes in success rate (1 – failure rate) in some cells (Figure 2B), and potency (mean EPSC amplitude excluding failures) in all cells (Figure 2C), as previously reported under these recording conditions 1828.

For this group of cells, DP, induced by pairing stimulation (baseline frequency) with a holding potential of -40 mV, always resulted in a reversal of the γ increase, as indicated by the current-variance plot (Figure 1B; Figure 2D). Similar to the preceding LTPγ, this form of DP (DPγ) was also associated with no change in EPSC kinetics (τ_rise: baseline = 1.6 ± 0.2 ms, LTPγ = 1.7 ± 0.3 ms, DPγ = 1.7 ± 0.2 ms, n = 11; τ_decay: baseline = 8.3 ± 0.6 ms, LTPγ = 8.2 ± 0.6 ms, DPγ = 8.9 ± 0.7, n = 11; Figure 1C). Failures analysis showed that DPγ was associated with a decrease in success rate in most cells (Figure 2B) and a full reversal of the potency increase (Figure 2C). These data show that the primary mechanism for DP is the reversal of any increase in γ caused by LTP. Indeed, the changes in γ were sufficient to account for the potency changes during both LTPγ and DPγ. (In most cells the alterations in γ actually exceeded the potency changes. This is most likely due to an underestimate of the potency change due to dendritic filtering, which affects measurements at the peak of EPSCs greater than during the tail, from where the non-SFA estimates are obtained; see 18).

Figure 3 shows an example from the group of cells that exhibited no change in γ during LTP (LTP_N), as indicated by the current-variance plot (Figure 3B). The change in EPSC amplitude for LTP_N neurons (Figure 4A) was similar to that for LTPγ neurons (Figure 2A). Failures analysis (Figure 3D) of this group of cells (Figure 4) revealed that LTP was associated with changes in success rate in some cells (Figure 4B), and potency in most cells (Figure 4C; P < 0.01), as previously reported under these recording conditions 1828.

In contrast to DPγ, DP in these cells was never associated with a change in γ (DP_N; Figure 3B, Figure 4D). There was also no change in EPSC kinetics with LTP or DP (τ_rise: baseline = 1.9 ± 0.4 ms, LTP_N = 2.0 ± 0.3 ms, DP_N = 1.8 ± 0.3 ms, n = 7; τ_decay: baseline = 9.7 ± 0.7 ms, LTP_N = 9.7 ± 0.6 ms, DP_N = 10.1 ± 0.9, n = 7; Figure 3C). Failures analysis of LTP_N and DP_N (Figure 3D, Figure 4) showed similar changes to the LTPγ group of cells in EPSC amplitude (Figure 4A), success rate (Figure 4B) and potency (Figure 4C). These data suggest that there is a second mechanism for DP, not involving a decrease in γ, which co-exists at CA1 synapses. Therefore, there are two mechanisms for the expression of DP that depend upon the expression mechanism of the previous potentiation.

It has been shown previously that DP at CA1 synapses may be blocked either by NMDA receptor antagonists 26 or by mGlu receptor antagonists 27 and that this may depend on previous history 30. In the present study we wished to focus on NMDA receptor-dependent synaptic plasticity and therefore used pairing protocols designed to activate NMDA receptors sufficiently to, firstly, induce NMDA receptor-dependent LTP and, secondly, to induce NMDA receptor-dependent DP. To verify that we were indeed investigating NMDA receptor-dependent DP we performed a series of experiments using the NMDA receptor antagonist D-AP5 interleaved with control experiments. Following the induction of LTP, D-AP5 (50 μM) was bath applied for 15 minutes before delivering the DP induction stimulus. Whilst DP was induced in the control experiments (25 ± 11% of baseline, n = 5; p < 0.05) it was blocked by D-AP5 (89 ± 21%, n = 4; Figure 5).

To gain further insights into the underlying mechanisms of DP we compared changes in EPSC amplitude, success rate, potency and γ for the individual experiments (Figure 6). A decrease in success rate indicates a reduction in probability of transmitter release (Pr) and/or a reduction in the number of functional synapses (n). A decrease in potency indicates a reduction in quantal amplitude (postsynaptic response to the release of a single quantum of transmitter, q) or, if multiple synapses are activated, a reduction in Pr or n.

In both types of neuron (i.e., LTPγ and LTP_N), a small depression of less than 50 % (to 71 ± 7% of baseline; n = 4) was associated with no change in success rate (Figure 6A; success rate ratio = 1.00 ± 0.01) but an equivalent decrease in potency (Figure 6B; potency ratio = 0.70 ± 0.08), as is also observed for de novo LTD 17). This indicates that the depression in these cells was associated primarily with a decrease in q. For larger depressions (to 26 ± 4 % of baseline; n = 14) there was also a marked decrease in success rate (success rate ratio = 0.52 ± 0.08; P < 0.01 vs success rate ratio for the group with DP < 50%) as well as a decrease in potency (potency ratio = 0.54 ± 0.04). This suggests that in these cells there was an additional decrease in Pr or n. Therefore, for DPγ, the change in γ did not fully account for the amplitude change in every cell (Figure 6C) but did account for the potency change (Figure 6D).

What might be the mechanism underlying DP that is not associated with a decrease in γ (i.e., DP_N)? It is unlikely that this type of depression is due to a change in channel kinetics because there was no change in EPSC kinetics (see 1829). Therefore the mechanism is most likely a reduction in the number of activated AMPARs. This could be due to a presynaptic mechanism such as a reduction in release probability, the L-glutamate content of vesicles or the amount of L-glutamate discharged during fusion. Indeed there is evidence that some forms of LTD are expressed presynaptically 535. Postsynaptic mechanisms for a reduction in the number of activated AMPA receptors include a reduction in their P_open 36or in the physical number of receptors present in the postsynaptic membrane 37.

The present observations for DP_N are indistinguishable from those that we and others have reported recently for de novo LTD 1738. For example, we showed that similar effects were obtained using the postsynaptic injection of a peptide (pep2m) that disrupts the interaction between NSF and GluR2 39404142. The effects of pep2m and those of de novo LTD were mutually occlusive, indicating a convergence of mechanisms. These data argue strongly for a postsynaptic mechanism of expression. Furthermore, since pep2m causes the removal of AMPA receptors from the membrane surface, as determined immunocytochemically 3842, it is most likely that de novo LTD is due to the physical elimination of synaptic AMPA receptors. Other evidence for a postsynaptic mechanism for de novo LTD includes a reduction in the postsynaptic sensitivity to glutamate 4344, the dephosphorylation of serine 845 of the GluR1 subunit 4546 and a rapid internalisation of AMPA receptors 47 associated with LTD. Therefore, by analogy, we feel that the postsynaptic removal of AMPA receptors is also the most likely explanation for DP_N (Figure 7A). Accordingly, a reduction in AMPA receptor number would account for the changes in potency without changes in success rate observed with modest DP_N. The removal of an entire synaptic complement of AMPA receptors would explain the additional change in success rate seen with large depressions associated with DP_N in some cells.

A number of possible underlying mechanisms could account for the change in γ during LTPγ and DPγ. Non-SFA cannot distinguish between 1) a change from a single low conductance state to a single high conductance state, 2) changes in open times within a burst and, 3) changes in the proportion of time spent in different conductance states. Since AMPA receptors are known to have multiple conductance states 3233 we have postulated that the proportion of time spent in different conductance states is the modifiable parameter 18. Such a change is detectable with the type of analysis that we have used, which provides a weighted mean of the various sub-conductance states 48. Independent support for this hypothesis is provided by a study which shows that phosphorylation of GluR1 at serine 831 increases the proportion of time AMPA receptors spend in high conductance states, as determined by single channel recording 20. Indeed, phosphorylation of this residue occurs during LTP 19. Thus a possible mechanism of DPγ is the dephosphorylation of serine 831 45, perhaps involving calcineurin 49, resulting in a lower proportion of time AMPA receptors spend in the higher conductance states (Figure 7B).

Other theoretical possibilities exist to explain DPγ. For example, the silencing of synapses close to the patch electrode leaving more distant synapses contributing a lower net γ due to electrotonic filtering, or a decrease in trial-to-trial asynchrony of transmitter release. We believe that these possibilities are unlikely because in every cell in which de novo LTD was induced there was never a change in γ 17. If such possibilities were likely, statistically one would expect similar changes to occur for both de novo LTD and DP. Moreover, we have also investigated these possibilities using our standard compartmental model 29. This shows that 1) the electrotonic filtering required to achieve an artefactual decrease in γ would have a pronounced effect on τ_decay which was never observed experimentally, and 2) pronounced changes in asynchrony necessary to cause an artefactual change in γ cause large deviations from the parabolic relationship in the current-variance plot (unpublished observations) and alterations in τ_rise 29, also never observed experimentally. Another possibility is that alterations in vesicle fusion pore dynamics leading to substantial changes in the peak and time-course of cleft glutamate are caused by synaptic plasticity 7. Such changes could differentially affect estimates of γ 50, however they would be associated with substantial changes in τ_rise and τ_decay 7, which were never observed experimentally.

Hippocampal slices (400 μm) were obtained from 12–15 day old rats and perfused with an extracellular solution containing (in mM): 124 NaCl, 3 KCl, 1.25 NaHPO₄, 26 NaHCO₃, 2 CaCl₂, 1 MgSO₄, 15 glucose, 2 ascorbic acid, 0.05 picrotoxin, saturated with 95% O₂ / 5% CO₂, at room temperature (23–25°C). Individual dendrites were visualised using infrared illumination and DIC optics and approached under visual control. Whole-cell dendritic recordings of synaptic currents were obtained at a holding potential of -70 mV using patch electrodes (6–10 MΩ) filled with a solution containing (in mM): 135 CsMeSO₄, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 5 QX-314, pH 7.25, 285 mOsm. Schaffer collateral-commissural fibers were stimulated at 0.5 Hz using a platinum monopolar or concentric bipolar electrode, which was positioned 20–40 μm from the dendrite parallel to the input pathway. The stimulus intensity was set to evoke some failures to enable a failures analysis to be performed and to ensure that the majority of EPSCs were, for any given trial, evoked by release from a single site. However, to elicit sufficient EPSCs to obtain a baseline estimate of γ before "washout" of LTP it was usually necessary to set the success rate fairly high (usually above 50%). As a result it is likely that multiple release sites contributed to the recordings. LTP was induced by pairing 40–60 stimuli (baseline frequency) with a holding potential of 0 mV. DP was induced following the induction of stable LTP by 100–200 stimuli (baseline frequency) at -40 mV holding potential. All recordings were made using an Axopatch 1B amplifier, signals were filtered at 5 kHz (8 pole Bessel filter), digitised at 10 kHz and stored on computer. EPSC amplitude and input resistance were analysed and displayed on-line using the 'LTP' program 51). Series resistance was estimated by measuring the peak amplitude of the fast whole-cell capacitance current in response to a -1 mV step applied to the cell during each sweep. The amplitude was estimated by fitting the capacitance transient with a double exponential (from 0.5 ms after the peak) and determining the current at the beginning of the step. Series resistance was stable throughout recordings (series resistance values [MΩ]: baseline = 42 ± 3, LTP = 40 ± 3, DP = 43 ± 3, n = 17).

Non-SFA was performed as described previously 18. Briefly, synaptic currents were aligned by their point of maximal rise, and averaged. The average response waveform was scaled to the peak, subtracted from individual responses and the variance of the decays calculated. The variance was plotted vs. the mean current amplitude and the single channel current was estimated by fitting the data to: σ² = iI - I²/N + b_l, where σ² is the variance, I is the mean current, N is the number of channels activated at the peak, i is the single channel current and b_l is the background variance. The single channel conductance (γ) is then γ = i/V, where V is the driving force (holding potential – assumed reversal potential of 0 mV). Response amplitude was measured in two ways. For failures, amplitude was estimated by measuring the difference between the average current over two time windows of equal length, one immediately before the stimulus artefact and the other centred on the peak of the mean EPSC. For estimation of the amplitude of successes the peak amplitude over a set time window was determined. Failures were identified visually, and potency was calculated as the mean EPSC amplitude excluding failures. For analysis of EPSC kinetics, rise time was estimated by the time-constant of a single exponential fit of the rising phase of the mean EPSC waveform. For decay, the time-constant of the single exponential fit to the decay phase was used. For display of individual EPSC traces in the figures, the stimulus artefact was digitally subtracted using an average of identified failures. All histograms are represented as smoothed line plots (SigmaPlot ver 5.0). Data are expressed as % of baseline (i.e., 100 % = no change). Statistical significance was assessed using the Student's t-test (one or two-tailed, paired or unpaired as appropriate; P < 0.05 as significant). All values are expressed as mean ± s.e.m.