Enhanced synaptic inhibition in the cerebellar cortex of the ataxic PMCA2(-/-) knockout mouse.

Mice with genetic deletion of a calcium extrusion pump, the plasma membrane calcium ATPase isoform 2, PMCA2, exhibit overt cerebellar ataxia, but the cellular mechanisms are only partially understood. Here, we report an enhanced synaptic GABAergic inhibition within the molecular layer of cerebellar cortex slices from PMCA2 knockout (PMCA2(-/-)) mice, a finding that could contribute to the observed ataxia. Purkinje neurons from PMCA2(-/-) mice exhibited an increased frequency and amplitude of spontaneous inhibitory post-synaptic currents that was accompanied by an enhanced spontaneous firing frequency of molecular layer interneurons (both basket cells and stellate cells). The elevated inhibition was sufficient to reduce the frequency and regularity of spike firing by PMCA2(-/-) Purkinje neurons. Acute pharmacological inhibition of PMCA recapitulated some of these features in wild-type mice indicating that the changes were in part a direct result of PMCA2 removal. However, additional compensatory mechanisms within the PMCA2(-/-) mouse were also a major factor. Indeed, morphological studies revealed an abnormally large number of molecular layer interneurons (basket cells and stellate cells) and GABAergic synapses within the PMCA2(-/-) cerebellar cortex. We conclude that loss of PMCA2 adversely influences the function and organisation of Purkinje neuron synaptic inhibition as a major contributory mechanism to the ataxic phenotype of the PMCA2(-/-) mouse.

The contribution of the sodium-calcium exchanger (NCX) and plasma membrane Ca(2+) ATPase (PMCA) to cerebellar synapse function.

The cerebellum, a part of the brain critically involved in motor learning and sensory adaptation, expresses high levels of the sodium-calcium exchanger (NCX) and the plasma membrane calcium ATPase (PMCA). Both these transporters control calcium dynamics at a variety of synapses, and here, we draw upon the available literature to discuss how NCX and PMCA work together to shape pre-synaptic calcium dynamics at cerebellar synapses.

Functional contributions of the plasma membrane calcium ATPase and the sodium-calcium exchanger at mouse parallel fibre to Purkinje neuron synapses.

We investigated how two calcium clearance mechanisms, the sodium-calcium exchanger-NCX, and the plasma membrane calcium ATPase-PMCA2, function at the facilitating cerebellar parallel fibre to Purkinje neuron (PF-PN) synapse. Forward mode NCX helped recover PF presynaptic calcium elevations when the PFs received a double stimulation and the calcium load was sufficiently high. A smaller presynaptic calcium load evoked by a single PF stimulation failed to recruit NCX in wild-type mice but did so when PMCA2 was absent in PFs from PMCA2 knockout mice. Simulated calcium dynamics using a simple single-compartment model reported qualitatively similar effects. Functionally, reduced NCX activity in the absence of PMCA also prolonged the recovery of facilitation at the PF-PN synapse, beyond that seen by reduced NCX activity alone. We conclude that PMCA and NCX work in parallel to accurately shape residual presynaptic calcium recovery dynamics and fine-tune facilitation at this important cerebellar synapse.

Transient reversal of the sodium/calcium exchanger boosts presynaptic calcium and synaptic transmission at a cerebellar synapse.

The sodium/calcium exchanger (NCX) is a widespread transporter that exchanges sodium and calcium ions across excitable membranes. Normally, NCX mainly operates in its "forward" mode, harnessing the electrochemical gradient of sodium ions to expel calcium. During membrane depolarization or elevated internal sodium levels, NCX can instead switch the direction of net flux to expel sodium and allow calcium entry. Such "reverse"-mode NCX operation is frequently implicated during pathological or artificially extended periods of depolarization, not during normal activity. We have used fast calcium imaging, mathematical simulation, and whole cell electrophysiology to study the role of NCX at the parallel fiber-to-Purkinje neuron synapse in the mouse cerebellum. We show that nontraditional, reverse-mode NCX activity boosts the amplitude and duration of parallel fiber calcium transients during short bursts of high-frequency action potentials typical of their behavior in vivo. Simulations, supported by experimental manipulations, showed that accumulation of intracellular sodium drove NCX into reverse mode. This mechanism fueled additional calcium influx into the parallel fibers that promoted synaptic transmission to Purkinje neurons for up to 400 ms after the burst. Thus we provide the first functional demonstration of transient and fast NCX-mediated calcium entry at a major central synapse. This unexpected contribution from reverse-mode NCX appears critical for shaping presynaptic calcium dynamics and transiently boosting synaptic transmission, and is likely to optimize the accuracy of cerebellar information transfer.

Assessment of the contribution of the plasma membrane calcium ATPase, PMCA, calcium transporter to synapse function using patch clamp electrophysiology and fast calcium imaging.

The plasma membrane calcium ATPase, or PMCA, functions to extrude calcium out of cells as a key component necessary for adequate calcium homeostasis in all cells. However, calcium is particularly important at synapses between neurons, where communication relies on the controlled rise and fall in presynaptic calcium that precedes the release of neurotransmitter. Here we show how to infer the real-time contribution of PMCA-mediated calcium extrusion to this presynaptic calcium dynamic and how this influences the properties of the synapse. To do this we have taken advantage of a well-studied synapse in the cerebellum. We use electrophysiology to assess the timing of short-term facilitation at this synapse in the presence and absence of PMCA2 using PMCA2 knockout mice and pharmacology and fast calcium imaging to measure the presynaptic calcium dynamics. These approaches are all highly applicable to other synapses and can help determine the contribution of PMCA, and other transporters or exchangers, to the calcium dynamics that underpin reliable synaptic transmission.