Prediction and validation of a mechanism to control the threshold for inhibitory synaptic plasticity

Yuichi Kitagawa^1,2, Tomoo Hirano^1,2 & Shin-ya Kawaguchi^1,2

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Synopsis

Neuronal activity-dependent synaptic plasticity, that is, sustained alteration of information transmission efficacy between neurons, is a cellular basis for learning and memory. Most forms of synaptic plasticity are induced by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and regulated by complex downstream molecular networks consisting of numerous molecules such as protein kinases and phosphatases. Computational models of the signaling cascades regulating synaptic plasticity have suggested that the [Ca²⁺]_i increase-triggered positive-feedback loops inherent in the molecular network have important functions in the establishment of synaptic plasticity (Bhalla and Iyengar, 1999; Kuroda et al, 2001; Pettigrew et al, 2005). However, the mechanism that determines the [Ca²⁺]_i threshold for inducing synaptic plasticity remains elusive.

At the GABAergic synapses on a cerebellar Purkinje neuron, postsynaptic depolarization induces long-term potentiation of GABA_A receptor (GABA_AR)-mediated inhibitory synaptic transmission (called rebound potentiation, RP) through a postsynaptic [Ca²⁺]_i increase and the subsequent activation of Ca²⁺/calmodulin-dependent protein kinase CaMKII (Kano et al, 1992). On the other hand, activation of presynaptic interneurons during postsynaptic depolarization suppresses RP through activation of postsynaptic metabotropic GABA_B receptors (GABA_BRs) (Kawaguchi and Hirano, 2000). The suppressive effect of GABA_BR is mediated by downregulation of PKA through inhibition of adenylyl cyclase. Reduction of PKA activity facilitates a protein phosphatase pathway consisting of calcineurin, DARPP-32, and PP-1, which counteracts CaMKII activity and suppresses RP induction (Kawaguchi and Hirano, 2002). However, it is enigmatic how the overall signaling pathways inferred from combining fragmentary experimental results operate to determine whether RP is induced or not in response to various patterns of inputs. To address this issue, we developed a kinetic simulation model of the molecular network and systematically analyzed the behavior of the signaling cascades regulating RP. Our simulation reproduced the RP induction and its suppression in response to different combinations of inputs, and suggested critical roles of two positive-feedback loops, CaMKII autophosphorylation at Thr286/287 and CaMKII-mediated downregulation of PDE1.

Using the simulation model, we attempted to address how the threshold for RP induction is controlled. There are two feedforward inhibitory pathways to CaMKII activity in the signaling network: one through calcineurin and the other through PDE1. To examine the involvement of these pathways in the [Ca²⁺]_i threshold for RP induction, we investigated how large an increase in [Ca²⁺]_i was required to persistently activate CaMKII by systematically altering the amplitude of the transient [Ca²⁺]_i increase (from 0.1 to 1 M for 10 s) with 0.05-M steps in the simulation model. To establish a sustained CaMKII activation, a [Ca²⁺]_i increase larger than 0.6 M was required (Figure 6A). Interestingly, when the concentration of PDE1 was reduced by 80% in the model, the increase of [Ca²⁺]_i to 0.35 M triggered sustained CaMKII activation (Figure 6B). The reduction of PDE1 caused leftward shift of the concentration–response curve for CaMKII activation by the transient [Ca²⁺]_i increase (Figure 6E). As the amount of PDE1 increased, the amplitude of [Ca²⁺]_i increase required for sustained CaMKII activation became larger (Figure 6F). Thus, the amplitude of [Ca²⁺]_i increase required for the sustained CaMKII activation was dynamically controlled by PDE1, and PDE1 reduction markedly lowered the [Ca²⁺]_i threshold. In contrast, when the concentration of Ca²⁺-independent PDE4 was altered, the [Ca²⁺]_i threshold was only slightly affected (Figure 6C, E, and F). Thus, Ca²⁺/CaM-dependent PDE1, but not Ca²⁺/CaM-independent PDE4, raises the [Ca²⁺]_i threshold for long-term activation of CaMKII. On the other hand, the calcineurin-mediated feedforward inhibition had little effect on the [Ca²⁺]_i threshold for persistent CaMKII activation. (Figure 6D–F). Taken together, these results show that, of the two feedforward inhibitory pathways, the PDE1 pathway rather than the calcineurin pathway predominantly controls the [Ca²⁺]_i threshold for sustained CaMKII activation.

Figure 6

Simulation showing that PDE1 determines the [Ca²⁺]_i threshold for induction of sustained CaMKII activation. (A–D) Time courses of the amount of active CaMKII before and after various amplitudes of [Ca²⁺]_i increase for 10 s without (A) or with 80% reduction of the amount of PDE1 (B), PDE4 (C), or calcineurin (CaN, D). (E) The amount of active CaMKII 30 min after the [Ca²⁺]_i increase in each condition was plotted against the amplitude of [Ca²⁺]_i increase. (F) The amplitude of the [Ca²⁺]_i increase required for the sustained CaMKII activation (>1 M at 30 min) was plotted against the amount of each enzyme (value relative to the default value). (G) Robustness of the predominant role of PDE1 in the Ca²⁺ threshold regulation. Cumulative distributions of the Ca²⁺ thresholds for induction of sustained CaMKII activation. The 843 different parameter sets were examined with or without an 80% decrease of PDE1, PDE4, or CaN.

Full figure and legend (418K)Source data for Figure 6E (21K)Source data for Figure 6F (19K)Source data for Figure 6G (77K)Figures & Tables index

We also experimentally evaluated the prediction that PDE1 has a predominant function in the [Ca²⁺]_i threshold for RP induction. RP was monitored by measuring the amplitude of current response to GABA iontophoretically applied to the proximal dendrites of a cultured Purkinje neuron under voltage-clamp conditions. As shown in Figure 8A, strong conditioning stimulation consisting of 5 depolarization pulses for 500 ms potentiated the amplitude of current response to GABA for >30 min, and this potentiation was impaired by inhibition of either CaMKII or PKA. Thus, RP is induced by a large [Ca²⁺]_i increase depending on CaMKII and PKA activities. Conditioning stimulation consisting of shorter pulses failed to induce RP (Figure 8B). Thus, RP was induced in an all-or-none manner dependent on the large [Ca²⁺]_i increase.

Figure 8

PDE1 predominantly regulates the threshold for RP induction. (A) Time courses of amplitude of GABA currents and representative current traces before and after the conditioning depolarization (0 mV for 500 ms, five times at 0.5 Hz) in the absence (control), or presence of AIP-II or KT5720. n=5 for each. (B–G) Time courses of amplitude of GABA currents before and after conditioning stimulation consisting of five depolarization pulses with duration of 20, 40, 60, 100, or 500 ms in the absence (B, control) or presence of 8-MM-IBMX (C), vinpocetine (D), rolipram (E), FK506 (F), or cyclosporine A (G). n=3 for each. Representative current traces before and 30 min after the conditioning stimulation composed of five depolarization pulses with duration of 20, 40, or 500 ms are also shown in each panel. Scale bars indicate 200 pA and 1 s. (H) The relationship between the strength of conditioning stimulation (duration of each depolarization pulse) and the extent of GABA response potentiation at 30 min. PDE1 inhibition by either 8-MM-IBMX or vinpocetine significantly lowered the threshold for RP induction compared with the control (P<0.001, two-way ANOVA, and Dunnet T3 test).

Full figure and legend (778K)Figures & Tables index

The dependency of RP induction on the [Ca²⁺]_i increase was clearly reduced by inhibition of PDE1. In the presence of a specific PDE1 inhibitor, 8-MM-IBMX, a weak [Ca²⁺]_i increase caused by a conditioning stimulation consisting of 40-ms depolarization pulses was sufficient to induce RP (Figure 8C). Another PDE1 inhibitor, vinpocetine, also lowered the threshold for RP induction (Figure 8D). In contrast, inhibition of PDE4 by a specific inhibitor, rolipram, did not affect the threshold (Figure 8E). Furthermore, the calcineurin-mediated feedforward inhibitory pathway was not involved in the control of the threshold for RP induction. Calcineurin inhibition by either FK506 or cyclosporin A did not affect the threshold (Figure 8F and G). These findings taken together, as summarized in Figure 8H, indicate that inhibition of PDE1, but not that of PDE4 or calcineurin, significantly lowered the threshold for RP induction. Thus, combined application of computational simulation and cell biological experiments revealed a critical role of the feedforward inhibition of CaMKII by PDE1 in regulating the threshold for inhibitory synaptic plasticity in the cerebellum.

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