A role for protein phosphatases 1, 2A, and 2B in cerebellar long-term potentiation

Abstract

Cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses can undergo postsynaptically expressed long-term depression (LTD) or long-term potentiation (LTP). PF-LTD induction requires the coactivity of the PF and CF (climbing fiber) inputs to PCs and a concomitant calcium transient and activation of protein kinase C (PKC). PF-LTP can be induced by PF activity alone and requires a lower calcium transient for its induction than PF-LTD. The cellular events triggering PF-LTP induction are not well characterized. At other types of synapses (e.g., in the hippocampus), bidirectional synaptic plasticity is under control of a kinase/phosphatase switch, with PKC and CaMKII (calcium/calmodulin-dependent kinase II) activity promoting LTP induction and phosphatase activity promoting LTD induction. Here, we have tested for the involvement of protein phosphatase 1 (PP1), PP2A, and PP2B (calcineurin) in cerebellar LTP induction using whole-cell patch-clamp recordings in rat cerebellar slices. LTP induction was blocked in the presence of the PP1/2A inhibitors okadaic acid and microcystin LR, the PP1 inhibitory peptide inhibitor-2, the PP2A inhibitor fostriecin, and the PP2B inhibitor cyclosporin A. LTP induction was not impaired by the PKC inhibitor chelerythrine. Conversely, LTD induction was not blocked by microcystin LR but instead was reduced when active PP2B was injected into PCs. These data indicate that a kinase/phosphatase switch controls bidirectional cerebellar plasticity, but in a manner "inverse" to the dependencies found at other types of synapses. Therefore, cerebellar LTP constitutes the only form of LTP described so far that depends on phosphatase rather than kinase activity.

Figure 1.

Postsynaptic PF-LTP is PKC independent. A, PF-LTP can be induced by PF stimulation at 1 Hz for 5 min (n = 11). Each data point represents the average of three successive test responses evoked at 0.05 Hz. The top traces show EPSCs before and after LTP induction. B, Paired-pulse facilitation ratio (EPSC2/1) obtained from a subset of the LTP cells shown in A (n = 7). C, Neither the PKC inhibitor chelerythrine (10 μm; n = 7) nor the general kinase inhibitor H-7 (50 μm; n = 7) blocks LTP induction. D, In the absence of tetanization, neither chelerythrine (n = 5) nor H-7 (n = 6) affects EPSC amplitudes. The bars in C and D indicate the presence of the drugs in the bath. Error bars are mean ± SEM.

Figure 2.

PP1/2A inhibitors block PF-LTP induction. A, Bath application of the PP1/2A inhibitor okadaic acid (1 μm) blocks LTP induction (n = 7). B, In the absence of tetanization, okadaic acid does not affect EPSC amplitudes (n = 5). C, When the PP1/2A inhibitor microcystin LR (10 μm) is added to the pipette saline, LTP induction is impaired (n = 7). D, The same pipette saline does not cause the depression observed in C in the absence of tetanization (n = 10). Error bars are mean ± SEM.

Figure 3.

PP1, PP2A, and PP2B are required for PF-LTP induction. A, I-2, a PP1 inhibitory protein, blocks PF-LTP induction when added to the pipette saline (100 nm; n = 7). B, The PP2A inhibitor fostriecin, which is also added to the pipette saline (50 nm), blocks PF-LTP induction (n = 7). C, Bath application of the PP2B-selective inhibitor cyclosporin A (100 μm) blocks PF-LTP induction as well (n = 8). D, In control experiments, neither I-2 (n = 4), nor fostriecin (n = 9), nor cyclosporin A (n = 4) affects EPSC amplitudes. The bars in C and D indicate the presence of cyclosporin A in the bath. Error bars are mean ± SEM.

Figure 4.

PF-LTD is not phosphatase dependent. A, PF-LTD can be induced after paired PF + CF stimulation at 1 Hz for 5 min (n = 8). The top traces show EPSCs before and after LTD induction. B, PF-LTD is not impaired when microcystin LR (10 μm) is added to the pipette saline (n = 9). PF-LTD was blocked in the presence of chelerythrine (10 μm; n = 5) and H-7 (50 μm; n = 6), respectively. C, When active calcineurin is added to the pipette saline (5 μg/ml), paired PF + CF stimulation no longer causes a depression (n = 8), whereas PF-LTP induction is unaffected (n = 8). D, In the absence of tetanization, adding calcineurin does not affect EPSC amplitudes (n = 7). Error bars are mean ± SEM.