An experimental test of the role of postsynaptic calcium levels in determining synaptic strength using perirhinal cortex of rat

Abstract

1. We have investigated the prediction of a relationship between the magnitude of activity-dependent increases in postsynaptic calcium and both the magnitude and direction of synaptic plastic change in the central nervous system. 2. Activity-dependent increases in calcium were buffered to differing degrees using a range of concentrations of EGTA and the effects on synaptic plasticity were assessed. Activity-dependent synaptic plasticity was induced during whole-cell recording in rat perirhinal cortex in vitro. In control conditions (0.5 mM EGTA) low frequency stimulation (LFS; 200 stimuli) delivered to neurones held at -40 or -70 mV induced long-term depression (LTD) or, at -10 mV, induced long-term potentiation (LTP). 3. The relationship between EGTA concentration (0.2 to 10 mM) and the magnitude of LTD was examined. This relationship described a U-shaped curve, as predicted by models of synaptic plasticity. This provides strong evidence that the magnitude of LTD is determined by the magnitude of the increase in intracellular calcium concentration. 4. LFS paired with depolarisation to -10 mV induced LTD, no change or LTP as activity-dependent postsynaptic calcium levels were allowed to increase progressively by the use of progressively lower concentrations of buffer (10 to 0.2 mM EGTA). 5. We investigated if the lack of plasticity that occurs at the transition between LTD and LTP is due to induction of both of these processes with zero net change, or is due to neither LTD nor LTP being induced. These experiments were possible as LTP but not LTD was blocked by the protein kinase inhibitor staurosporine while LTD but not LTP was blocked by the mGlu receptor antagonist MCPG. At the transition between LTD and LTP, blocking LTP mechanisms did not uncover LTD whilst blocking LTD mechanisms did not uncover LTP. This suggests that the transition between LTD and LTP is due to the lack of induction of both of these processes and also suggests that these two processes are induced independently of one another.

Figure 1. The magnitude of LTD is dependent on the activity-dependent increase in intracellular calcium

A, LTD is not induced by pairing LFS with depolarisation to -40 mV when the filling solution contains 0.2 mm EGTA n = 4. B, however, LTD is induced with 2 mm EGTA in the filling solution n = 4. C, increasing EGTA to 5 mm reduces but does not prevent the induction of LTD n = 4. In this and subsequent figures the period of LFS is indicated by the arrows. Synaptic traces are the average of 4 consecutive responses from the time points indicated. D, graph illustrating the magnitude of LTD induced in the presence of each of the 5 different concentrations of EGTA: a U-shaped relationship exists between [EGTA] and LTD when LTD is induced at either -40 mV (○) or -70 mV (•). *Significant difference from baseline (P < 0.05).

Figure 2. Altering [EGTA] appropriately can result in LTP, no change, or LTD

LFS delivered during depolarisation to -10 mV results in LTP in the presence of 0.5 mm EGTA n = 4 (A) but LTD in 10 mm EGTA n = 4 (B). C, changes in synaptic strength are completely prevented by the inclusion of 20 mm EGTA in the whole-cell solution n = 4. D, graph showing the effect of a range of different concentrations of EGTA on synaptic strength. *Significant difference from baseline (P < 0.05).

Figure 3. Possible interactions between LTD and LTP in determining the outcome of synaptic strength

A, the change in synaptic strength () may be a function of the induction and expression of both LTD and LTP. The induction and expression of LTD (○) occurs at low concentrations of calcium and then reaches a plateau level. As calcium levels increase, the induction of LTP (•) also occurs and then reaches a maximum plateau level. The change in synaptic strength therefore describes the middle curve () due to both LTD and LTP and the transition between LTD and LTP is due to a sum of these two opposite processes. B, an alternative explanation is that LTD (○) and LTP (•) do not co-exist and that the change in synaptic strength () is a function of the selective presence of either LTD or LTP. Thus LTD is induced by low calcium concentrations. As the calcium concentration increases, LTD induction is prevented. As the calcium levels increase further the induction of LTP occurs. Therefore the two processes rely on different calcium concentrations and occur essentially independently of one another, and the transition between LTD and LTP is due to the lack of both LTD and LTP.

Figure 4. MCPG, which blocks LTD but not LTP, has no effect on the position of the transition between LTP and LTD

A, LTP, induced by depolarisation to -10 mV in the presence of 0.5 mm EGTA, is not blocked by MCPG n = 3. B, LTD, which is normally induced by depolarisation to -10 mV in the presence of 10 mm EGTA, is blocked by MCPG n = 3. C, the transition between LTP and LTD, observed at -10 mV in the presence of 5 mm EGTA, is not affected by MCPG n = 3. D, graph showing the results from the above experiments plotted together with the plasticity curve obtained from Fig. 2.

Figure 5. Staurosporine, which blocks LTP but not LTD, has no effect on the transition between LTP and LTD

A, LTP, induced by depolarisation to -10 mV in the presence of 0.5 mm EGTA is blocked by staurosporine n = 3. B, LTD, induced by depolarisation to -10 mV in the presence of 10 mm EGTA is not blocked by staurosporine n = 3. C, the transition between LTP and LTD is unaffected by staurosporine n = 5. D, graph showing the results from the above experiments plotted together with the plasticity curve obtained from Fig. 2.