Adaptive alterations to cerebellar synapse function in the ataxic PMCA2 knockout mouse

The plasma membrane Ca2+ ATPase isoform 2, PMCA2, also called the calcium pump, functions to transport calcium ions out of the cytosol at the expense of ATP. Although widely expressed, PMCA2 is enriched within the cerebellum where it contributes to presynaptic calcium dynamics and short term synaptic facilitation. In Purkinje neurons (PNs) from the ataxic, PMCA2 knockout mouse, we provide evidence for a reduction in excitatory synaptic inputs and a coincident enhancement of inhibitory synaptic events.
Saggital cerebellar slices (270 um thick) were prepared from previously genotyped PMCA2-/- mice (p23-30) and litter mate WT controls. Whole cell patch clamp recordings from PNs were used to analyse both excitatory and inhibitory spontaneous synaptic currents (EPSCs and IPSCs), mGluR mediated slow inward currents in the presence of TBOA (20µM) in response to 100Hz parallel fibre stimulation and complex spike behavior in response to climbing fibre stimulation. Experiments were conducted in accordance with local ethical guidelines. Some cells were filled with biocytin for post hoc identification, two-photon confocal reconstruction and morphological analysis. 10uM carboxyeosin (CE) pharmacologically inhibited PMCA activity. Values are mean ± SEM.
In PMCA2-/- PNs the mean median mEPSC inter event interval (IEI) was increased from 441±88 ms to 766±126 ms (n=8, p<0.05, t-test) with no change in mEPSC amplitude (p=0.74, t-test). The amplitude of the mGluR mediated inward current evoked during 100Hz parallel fibre activation was reduced from 169±7 pA in WT to 26±0.4 pA in PMCA2-/- PNs (n=8, p<0.05, t-test). The number of spikelets within the complex spike were almost completely abolished in the PMCA2-/- cells to 0.6±0.2 compared with 3±0.4 in WT cells (n=6, p<0.05, t-test). Spine density was also significantly reduced from 1.1±0.03 spines per micron of dendrite length in WT PNs to 0.89± 0.03 spines per micron in PMCA2-/- PNs (n=60, p<0.05, ANOVA) and mean dendrite width was also reduced from 3.2±0.06 µm in WT to 2.5 0.1 µm in PMCA2-/- PNs (n=54, p<0.05, ANOVA). In contrast spontaneous IPSC frequency was enhanced in cells from PMCA2-/- cells; mean median IEI changed from 224±45 ms in WT to 95±15 ms in PMCA2-/- cells, and this was accompanied by an increased mean median amplitude from 122±6 pA in WT to 171±22 pA in PMCA2-/- PNs (n=10, p<0.05, t-test).