Deactivation of L-type Ca current by inhibition controls LTP at excitatory synapses in the cerebellar nuclei

Abstract

Long-term potentiation (LTP) of mossy fiber EPSCs in the cerebellar nuclei is controlled by synaptic inhibition from Purkinje neurons. EPSCs are potentiated by a sequence of excitation, inhibition, and disinhibition, raising the question of how these stimuli interact to induce plasticity. Here, we find that synaptic excitation, inhibition, and disinhibition couple to different calcium-dependent signaling pathways. In LTP induction protocols, constitutively active calcineurin can replace synaptic excitation, and constitutively active alpha-CaMKII can replace calcium influx associated with resumption of spiking upon disinhibition. Additionally, nimodipine can replace hyperpolarization, indicating that inhibition of firing decreases Ca influx through L-type Ca channels, providing a necessary signal for LTP. Together, these data suggest that potentiation develops after a calcineurin priming signal combines with an alpha-CaMKII triggering signal if and only if L-type Ca current is reduced. Thus, hyperpolarization induced by synaptic inhibition actively controls excitatory synaptic plasticity in the cerebellar nuclei.

Figure 1

Calcineurin substitutes for synaptic excitation in the mossy fiber LTP protocol. (A) EPSC amplitudes before and after hyperpolarizing steps (“hyp only”; t = 0; upper inset, left) and the standard induction protocol (“standard”; t = 15 min; upper inset, right). Right lower insets: example EPSCs before and after the two induction protocols. Scale bar, 10 ms. (B) EPSC amplitudes in the presence of active calcineurin (CaN*) before and after the hyperpolarizing step conditioning protocol (applied at t = 0; upper inset, left) and the standard induction protocol (applied at t = 25 min; upper inset, right). Right lower insets show example EPSCs before and after the two protocols. (C) Mean EPSC amplitudes before and after hyperpolarizing steps in neurons infused with CaN* (closed circles; n = 11) or in control conditions (open triangles; n = 8). Error bars represent s.e.m. (D) Mean EPSC amplitudes before and after 3-sec hyperpolarizing steps applied either with synaptic stimulation (open triangles; n = 6) or in the presence of CaN* (closed circles; n=6).

Figure 2

CaN* combines specifically with disinhibition and requires CaMKII to induce LTP. (A) Upper panel: Examples of alternative induction protocols combined with CaN* infusion. Lower panel: Mean EPSC amplitudes before and after the protocols shown above; tonic hyperpolarization (circles; n = 6), spontaneous firing (squares; n = 7), synaptic excitation (down triangle; n = 8). (B) EPSC amplitudes in neuron infused with CaN* and CaMKII inhibitory peptide (fragment 290–309, 25 μM) before and after a hyperpolarizing step conditioning protocol at t = 0. Inset conventions as in Figure 1. (C) Mean EPSC amplitudes before and after hyperpolarizing step only protocol in neurons infused with CaN* and CaMKII inhibitors (inhibitory peptide, n=9; KN-62, n=1).

Figure 3

Constitutively active CaMKII* combines with synaptic excitation and hyperpolarization to trigger LTP. (A) EPSC amplitudes in the presence of heat inactivated CaMKII (HI-CaMKII) before and after trains of EPSPs. (B) EPSC amplitudes in the presence of active CaMKII (CaMKII*) before and after trains of EPSPs delivered to a spontaneously firing neuron. (C) EPSC amplitudes before and after excitatory synaptic trains delivered to a neuron held at −65 mV (inset). (D) Mean EPSC amplitudes before and protocols illustrated in A–C: neurons infused with either HI-CaMKII or no enzyme (open triangles; n = 6 in each condition; pooled); CaMKII* with spontaneous firing (circles; n = 10); or CaMKII* in voltage clamped neurons (closed triangles; n = 6).

Figure 4

EPSC amplitudes run up with CaN* and CaMKII* infused together in neurons held at −70 mV. (A) EPSC amplitudes at −70 mV in a neuron infused with both CaN* and CaMKII*. The standard conditioning protocol was delivered at t = 27 min (upper inset). (B) EPSC amplitudes in a neuron infused only with CaMKII*. (C) Mean EPSC amplitudes in neurons held at −70 mV and infused with both CaN* and CaMKII* (circles; n = 17) or either enzyme alone (triangles; n = 11).

Figure 5

Calcium blocks CaN*/CaMKII*-induced EPSC run up. (A) EPSC amplitudes in neurons infused with both CaN* and CaMKII* and held at −40 mV (open circles) and at −70 mV (closed circles). EPSC amplitudes were normalized to the first 10 measurements at each voltage. Scale: 200 pA, 10 ms. (B) EPSCs recorded at −40 mV in neurons infused with 10 mM BAPTA, CaN* and CaMKII*. (C) EPSCs at −40 mV in neurons infused with CaN* and CaMKII* during bath application of 10 μM nimodipine. (D) Mean EPSC amplitudes at −40 mV for neurons infused with both CaN* and CaMKII* in control solutions (open triangles; n = 9), with BAPTA (circles; n = 7) or in nimodipine (closed triangles; n = 6).

Figure 6

CaN* and spontaneous firing induce LTP without hyperpolarization in the presence of the L-type Ca channel blocker nimodipine. (A) EPSCs before and after conditioning protocol of CaN* and spontaneous firing in the presence of nimodipine (10 μM). (B) Mean EPSC amplitudes before and after conditioning protocols with nimodipine present (open circles; n = 5) or absent (triangles; data from Figure 2). (C) Diagram summarizing the interaction of Ca-dependent pathways that regulate LTP. Ca influx through NMDA receptors activates calcineurin. Activation of α-CaMKII by Ca influx through voltage-dependent Ca channels (VDCC) triggers LTP if the suppressive effect of L-type Ca current is reduced by hyperpolarization-driven closure of these channels.