In the cerebellum, sensory information is conveyed to Purkinje cells (PC) via the granule cell/parallel fibre (PF) pathway. Plasticity at the PF-PC synapse is considered to be a mechanism of information storage in motor learning. The induction of synaptic plasticity in the cerebellum and elsewhere usually involves intracellular Ca2+ signals. Unusually, postsynaptic Ca2+ signalling in PF-PC spines does not involve ionotropic glutamatergic receptors because postsynaptic NMDA receptors are absent and the AMPA receptors are Ca2+-impermeable; postsynaptic voltage-gated Ca2+ channels therefore constitute the sole rapid Ca2+ signalling mechanism. Low-threshold activated T-type calcium channels are present at the synapse, although their contribution to PF-PC synaptic responses is unknown. Taking advantage of 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide, a selective T-type channel antagonist, we show in the mouse that inhibition of these channels reduces PF-PC excitatory postsynaptic currents and excitatory postsynaptic potentials by 15-20%. This contribution was preserved during sparse input and repetitive activity. We characterized the biophysical properties of native T-type channels in young animals and modelled their activation during simulated dendritic excitatory postsynaptic potential waveforms. The comparison of modelled and observed synaptic responses suggests that T-type channels only activate in spines that are strongly depolarized by their synaptic input, a process requiring a high spine neck resistance. This brief and local activation ensures that T-type channels rapidly deactivate, thereby limiting inactivation during repetitive synaptic activity. T-type channels are therefore ideally situated to provide synaptic Ca2+ entry at PF-PC spines.

Ca(V)3.1 T-type channels are abundant at the cerebellar synapse between parallel fibers and Purkinje cells where they contribute to synaptic depolarization. So far, no specific physiological function has been attributed to these channels neither as charge carriers nor more specifically as Ca2+ carriers. Here we analyze their incidence on synaptic plasticity, motor behavior, and cerebellar motor learning, comparing WT animals and mice where T-type channel function has been abolished either by gene deletion or by acute pharmacological blockade. At the cellular level, we show that Ca(V)3.1 channels are required for long-term potentiation at parallel fiber-Purkinje cell synapses. Moreover, basal simple spike discharge of the Purkinje cell in KO mice is modified. Acute or chronic T-type current blockade results in impaired motor performance in particular when a good body balance is required. Because motor behavior integrates reflexes and past memories of learned behavior, this suggests impaired learning. Indeed, subjecting the KO mice to a vestibulo-ocular reflex phase reversal test reveals impaired cerebellum-dependent motor learning. These data identify a role of low-voltage activated calcium channels in synaptic plasticity and establish a role for Ca(V)3.1 channels in cerebellar learning.

The limited choice and poor performance of red-emitting calcium (Ca2+) indicators have hampered microfluorometric measurements of the intracellular free Ca2+ concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca2+ indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH2 linker arm. The low-affinity variant (KD,Ca ∼30 μM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca2+ transients. While Calcium Ruby is a mitochondrial Ca2+probe, its conjugation, via the NH2 tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca2+ probe with an affinity matched to microdomain Ca2+ signals. As an example, we show depolarization-evoked Ca2+ signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca2+ measurements.