PMC

In utero electroporation of murine hippocampus. A, Diagram depicting the method for labeling specific areas of the hippocampal anlage. B, C, Two days after electroporation of CA1–CA3 and DG on E14.5, widespread RFP (B) and RFP mRNA (C) are present in the ventricular zone, SVZ, and initial differentiating fields of CA1–CA3 regions. DG precursors were also transfected, and the dentate migratory stream (DMS) was observed medially. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride. D, Expression of RFP in pyramidal neurons of CA1–CA3 and DG hippocampal subfields of E14.5→P15 animals. E, F, Electroporation in a restricted 45° plane resulted in extensive CA1 gene transfer after 1 month (E). F, Electroporation between 15 and 45° resulted in CA3+CA1 gene transfer. E, Inset, The RFP-mRNA at P30 after electroporation on E14.5.

Comparison of functional properties between transfected and nontransfected excitatory pyramidal neurons. Throughout this figure, red and green traces depict data from RFP⁺ and RFP⁻ hippocampal pyramidal neurons, respectively, in mice after electroporation of pCAG-RFP as described in Materials and Methods. Black traces depict data from hippocampal pyramidal neurons from mice that had undergone the surgical procedure but without electroporation pulses. A, Passive membrane responses to small subthreshold hyperpolarizing and depolarizing current injections (A1) from which the input resistances (A2) and membrane time constants (A3) were calculated. B, Suprathreshold depolarizing current injections (B1) were used to elicit action potential discharge from which the number of spikes (B2; elicited with the range of current injections) and accommodation ratio (B3; at the 320 pA injection) were calculated. C, Sample traces and ensemble average of mEPSCs from which amplitude, frequency, and decay values were calculated. See supplemental Table 1 (available at www.jneurosci.org as supplemental material) for a summary of the comparison of all parameters measured.

Electroporation on E14.5 resulted in selective transfection of pyramidal neurons. Different interneuronal markers were used to characterize RFP⁺ cells in the pyramidal layers. A, B, RFP⁺ neurons did not colocalize with neither calretinin (A) or parvalbumin (B). C, Single examples of action potential discharge of RFP⁺ (top) and RFP⁻ (middle and bottom) hippocampal CA1 neurons in electroporated brain. The traces illustrate the last 20 s epoch of a 60 s suprathreshold depolarizing current injection (+600 pA) from which the firing frequency was calculated (see values adjacent to traces). All RFP⁺ neurons (14 of 14 cells tested) displayed low-frequency discharge, whereas RFP⁻ neurons displayed either low (<4 Hz; 13 of 15 cells tested) or high-frequency discharge (>40 Hz; 2 of 15 cells tested). D, Morphology of an RFP-positive neuron located in the hippocampal CA1 region from which whole-cell current-clamp recording was performed. Scale bar, 100 μm. Note that because of dialysis and exchange of cell cytoplasm with the pipette solution during recording, varying amounts of RFP fluorescence are lost during whole-cell recording, and in many cases, it is not detected after post hoc analysis. SO, Stratum oriens; SR, stratum radiatum; SP, stratum pyramidale; SL-M, stratum lacunosam-moleculare.