We, therefore, measured depolarization-evoked [3H]D-aspartate release in primary CGN cultures from the Tg(PG14) mice. Release was significantly lower in PG14 than in wild-type cells (Figure 3A). Single-cell calcium imaging found impaired calcium influx in response to depolarization (Figures 3B and 3C), and whole-cell patch-clamp recordings showed reduced calcium current densities in PG14 CGNs (Figures 3D and 3E). There were no apparent differences between wild-type and PG14 neurons in VGCC activation selleck compound and inactivation kinetics (Figure 3D), and in the voltage dependence of activation (Figure 3F), suggesting a reduction
in the number of functional channels rather than changes to their biophysical properties. Evoked excitatory postsynaptic currents (EPSCs) selleck chemicals llc recorded in cultured PG14 CGN by dual whole-cell patch clamp were significantly smaller than in wild-type cells, supporting the view that reduced calcium influx in the mutant neurons impaired
glutamate release (Figures 3G and 3H). The decrease in EPSC amplitude in PG14 neurons was not due to reduced postsynaptic sensitivity to glutamate, as suggested by the increased amplitude (wild-type = 12.07 ± 0.89 pA; PG14 = 16.51 ± 0.88 pA; mean ± SEM, n = 14 for wild-type and n = 13 for PG14; p < 0.01 by Mann-Whitney U test), and not frequency of miniature events (wild-type = 0.34 ± 0.05 Hz; PG14 = 0.28 ± 0.03 Hz, mean ± SEM; not significant by Mann-Whitney U test). The decrease in EPSC amplitude was rather due to reduced presynaptic calcium currents, as revealed by the increase in facilitation in a protocol of short-term plasticity (Figure 3I), which is sensitive to the amount of calcium entry (Zucker and Regehr, 2002). These results, which are in line with
previous reports for mutations of calcium Digestive enzyme channels affecting excitatory synaptic transmission (Liu and Friel, 2008, Ly et al., 2008 and Qian and Noebels, 2000), indicated abnormal VGCC function and impaired glutamatergic neurotransmission in CGN of Tg(PG14) mice. We used two complementary approaches to demonstrate that the VGCC defect was due to mutant PrP expression. First, we tested whether silencing PG14 PrP expression by lentivector-mediated RNAi restored the depolarization-induced calcium rise in mutant CGNs. CGNs from Tg(PG14) mice were transduced with a control lentivirus carrying enhanced green fluorescent protein (EGFP) cDNA (LV-E), or two different lentiviruses encoding EGFP and anti-PrP shRNAs (LV-MW1 and LV-MW2) that efficiently knock down PrP expression (Figure S4) (White et al., 2008), and the intracellular calcium rise in response to depolarization was measured in transduced neurons identified by EGFP fluorescence.