Whole-cell patch-clamp

recordings showed that the input resistance and membrane capacitance of the EGFP-positive Purkinje cells from mice that underwent IUE at E11.5 www.selleckchem.com/products/dabrafenib-gsk2118436.html were similar to those of wild-type Purkinje cells (Table 1). In addition, there were no significant differences in either the PF– or CF–EPSC kinetics (Table 1). The PF– and CF–EPSCs in the EGFP-positive Purkinje cells showed the typical paired-pulse facilitation and paired-pulse depression, respectively, that were observed in wild-type Purkinje cells (Fig. 2B and Table 1). By the end of the third postnatal week in mice, most wild-type Purkinje cells lose their redundant CFs and become innervated by a single CF. EGFP-positive Purkinje cells electroporated at E11.5 were similarly innervated by a single CF, as shown by their single threshold for excitation (Fig. 2C). Furthermore, the input–output

relationships of the PF–EPSC were not significantly different between the electroporated EGFP-positive and wild-type Selleck ABT-888 Purkinje cells (Fig. 2D), indicating that the PF inputs to Purkinje cells were also intact. Finally, the conjunctive stimulation of PFs and the depolarization of Purkinje cells induced LTD similarly in both wild-type and electroporated Purkinje cells (Fig. 2E; 67 ± 5% at t = 25–30 min, n = 7 from four wild-type mice; 69 ± 6% at t = 25–30 min, n = 7 from four electroporated Purkinje cells; Mann–Whitney U-test, P = 0.947). Together, these results indicate that IUE

did not alter the basic membrane properties, EPSC parameters, or short-term or long-term synaptic plasticity of the transfected Purkinje cells. To examine whether cell-type-specific and inducible promoters were compatible with the IUE method for Purkinje cells, we employed an inducible Cre/loxP system (Matsuda & Cepko, 2007). The Purkinje-specific L7 promoter (Oberdick et al., 1990; Smeyne et al., 1991; Tomomura et al., 2001) was used to express the conditionally active form of Cre recombinase ERT2CreERT2, in which the ligand-binding domain of the estrogen receptor PLEKHB2 was mutated; the Cre recombinase is activated in response to 4OHT (Matsuda & Cepko, 2007). By coexpressing pCALNL-DsRed2, which contains the CAG promoter and a stop signal flanked by loxP sequences, the reporter gene DsRed2 was designed to be expressed in a 4OHT/Cre- and L7-dependent manner (Fig. 3A). To unconditionally label all the electroporated cells, pCAG-EGFP was co-electroporated with the pL7-ERT2CreERT2 and pCALNL-DsRed2. After IUE at E11.5, the mice received an intraperitoneal injection of 4OHT or vehicle at P6 and were fixed at P14 (Fig. 3A). As expected, only mice that received 4OHT displayed DsRed2 signals in the cerebellum (Fig. 3B). Confocal microscopy further confirmed that the DsRed2 signals were observed only in a subset of EGFP-positive Purkinje cells (Fig. 3C).