Calcium is an intracellular mediator of the climbing fiber in induction of cerebellar long-term depression.

Abstract

In cerebellar Purkinje cells, conjunctive stimulation of parallel fibers and the climbing fiber causes longterm depression of parallel fiber-Purkinje cell transmission. It has been postulated that calcium is an intracellular mediator of the climbing fiber to induce this synaptic modification. To directly test the hypothesis, a calcium-chelating agent, EGTA, was intracellularly injected into Purkinle cells. In these injected cells, conjunctive stimulation failed to induce depression. Instead, it caused potentiation similar to that observed after repetitive stimulation of parallel fibers alone.A unique and characteristic feature of the cerebellar cortical network structure is that the final output neuron, the Purkinje cell (PC), receives two distinct types of excitatory afferents: numerous parallel fibers (PFs), which are axons of cerebellar granule cells; and a single but powerful climbing fiber (CF), which derives from the inferior olive (1, 2). Synapses supplied by PFs to PCs exhibit prominent activity-dependent modifiability. A long-term depression (LTD) is induced by conjunctive stimulation of PFs with a CF (3-5), whereas a potentiation is produced by repetitive stimulation of PFs alone (5). This modifiability of PF-PC synapses would explain the role of the cerebellum in motor learning (1,6).Involvement of calcium in induction of LTD has been postulated (1,(3)(4)(5) from the findings that the CF response is associated with a large amount of calcium influx to PCs (7-9) and that strong postsynaptic inhibition of PC dendrites, which presumably depresses the calcium influx, prevented the induction of LTD (4). However, there has been no direct evidence for this hypothesis. Here, I report that when EGTA was injected into PCs, conjunctive CF-PF activation no longer produced LTD but, rather, elicited a potentiation. This observation suggests that calcium chelation by EGTA cancels the action ofCF impulses in inducing LTD, consequently leaving the same potentiating effect as with PF stimulation alone (5). The present results provide direct evidence for a role of calcium in induction of LTD. MATERIALS AND METHODSParasagittal slices (each 300-330 ,um thick) were prepared from guinea pig cerebellar vermis and maintained in vitro as described (5). Slices were prepared using a Microslicer (Dosaka EM, Kyoto) and preincubated for >1 hr in a standard medium containing 124 mM NaCl, 5 mM KCl, 1.24 mM KH2PO4, 1.3 mM MgSO4, 1.24 mM CaC12, 26 mM NaHCO3, and 10 mM glucose and equilibrated with a 95% 02/5% CO2 gas mixture. The temperature was kept constant at 360C.Intracellular recordings were made from PC somata or proximal dendrites. PCs were identified by their characteristic CF responses and/or antidromic spike responses (2). A stimulating electrode was placed at the superficial molecular layer for stimulating PFs with positive-negative current pulses. Another stimulating electrode was inserted into white matter for stimulating CUs. Since stimulation of the white matter often elicited inhibitory postsynaptic potentials...

Calbindin in Cerebellar Purkinje Cells Is a Critical Determinant of the Precision of Motor Coordination

“…The protocol used to induce LTD was comparable with methods previously applied to other mutants (Aiba et al, 1994;Conquet et al, 1994;Kashiwabuchi et al, 1995;De Zeeuw et al, 1998b). Whereas the calcium-binding protein calbindin is highly expressed in Purkinje cells and acts here as a powerful Ca 2ϩ buffer (Fierro and Llano, 1996;Airaksinen et al, 1997;Maeda et al, 1999), and although Ca 2ϩ signaling is essentially needed for the induction process (Sakurai, 1990;Konnerth et al, 1992), LTD is not affected in the Purkinje cell-specific calbindin null mutants. This preservation of LTD is most likely a consequence of the fact that mGluR-mediated Ca 2ϩ signaling, known to be critical for LTD induction (Daniel et al, 1998;Inoue et al, 1998;Wang et al, 2000;Ito, 2001), seems to be normal in the mutant mice.…”

“…IP 3 causes Ca 2ϩ release from intracellular stores (Mikoshiba et al, 1994;Finch and Augustine, 1998;Takechi et al, 1998), and synaptically induced dendritic Ca 2ϩ signaling has been known for a long time to be a critical step in LTD induction (Sakurai, 1990;Konnerth et al, 1992). On the basis of earlier evidence, it seemed that not merely the lack of synaptically mediated Ca 2ϩ signaling in Purkinje cells, but also a change in the temporal dynamics of postsynaptic Ca 2ϩ transients after general genetic deletion of the calcium-binding protein calbindin D-28k (calbindin), might cause impairment of motor coordination (Airaksinen et al, 1997).…”

Type-1 metabotropic glutamate receptor in cerebellar Purkinje cells: a key molecule responsible for long-term depression, endocannabinoid signalling and synapse elimination

“…These observations confirmed the validity of the Marr-Albus-Ito's synaptic plasticity assumption. Subsequently, Sakurai demonstrated LTD in cerebellar slice preparations from rat (Sakurai 1987) and that LTD requires an elevation of intracellular Ca 2C level in the Purkinje cell (Sakurai 1990). Hirano demonstrated LTD in cultured Purkinje cells (Hirano 1990).…”

Local dendritic Ca2+ signaling induces cerebellar long-term depression.

“…This increase in Ca 2+ concentration occurs throughout much (or all) of the dendritic field of the Purkinje cell. Second, a CF-induced rise in Ca 2+ concentration is necessary for LTD because induction of LTD is prevented by injection of Ca 2+ chelators into Purkinje cells (Sakurai 1990;Konnerth et al 1992). Finally, an increase in postsynaptic Ca 2+ concentration is sufficient to produce LTD because direct elevation of Ca 2+ concentration--either by depolarization-induced activation of voltage-gated Ca 2+ channels (Crepel and Jaillard 1991;Linden et al 1991;Kon-nerth et al 1992) or by photolysis of "caged Ca 2+'' compounds (Kasono and Hirano 1994;Lev-Ram et al 1995)--can substitute for activation of the CF synapse.…”