Article Figures & Data
Figures
- Figure 1.
Cav1.2 is expressed in the embryonic mPFC. A, Representative images of histologic sections from Nkx2.1/Ai14 E16.5 mouse brain (A1) immunostained for Cav1.2 (A2) and overlayed with images of DAPI counterstaining and Nkx2.1/tdTomato-fluorescent GINs (A3). Images were captured at 10× magnification on a spinning disk confocal microscope. B, Representative images at 40× magnification. These images are magnified images of the area demarcated by the white box in A1–A3. C, Representative images of no primary antibody negative control of Cav1.2 staining.
- Figure 2.
Prenatal ethanol exposure does not alter Cav1.2 expression. A, Representative images of Cav1.2 staining overlayed with DAPI and Nkx2.1 in the mPFC of control (A1) and ethanol-fed (A2) E16.5 mouse brain. B, Quantification of fluorescence intensity ratio of Cav1.2 to Nkx2.1 in the mPFC of control and ethanol-treated mice. Unpaired t test. For statistical details, see Results.
- Figure 3.
Nifedipine prevents ethanol-induced aberrant migration in organotypic slice cultures. A, Representative image of Nkx2.1/Ai14 embryonic mouse brain with five bins (100 μm wide each) above CSJ and one 200-μm bin below CSJ. B, Quantification of mean Nkx2.1+ cells per individual bins in control (vehicle; 3 litters, 3 females, 5 males), ethanol (EtOH; 3 litters, 6 females, 3 males), ethanol + nifedipine (EtOH + Nifed; 3 litters, 7 females, 3 males), and nifedipine (Nifed; 3 litters, 2 females, 4 males) treated organotypic slice cultures. C, Quantification of crossing index for control (vehicle), ethanol (EtOH), ethanol + nifedipine (EtOH + Nifed), and nifedipine (Nifed) treated organotypic slice cultures; * compared with control, # compared with EtOH + Nifed. *,#p < 0.05, **,##p < 0.01, ***,###p < 0.001, two-way ANOVA with Tukey’s post hoc test.
- Figure 4.
Maternal nifedipine treatment normalizes ethanol-induced enhancement of tangential migration in vivo. A, Experimental timeline. Graded blue box highlights the period of tangential migration, beginning at ∼E01.5 and tapering out by ∼P0. Binge-type ethanol exposure and nifedipine co-treatment begins on E13.5 and ends on E16.5 (red dashed lines). B, Fluorescent images of mPFC counterstained with DAPI in mice that received control, binge-type maternal ethanol consumption from E13.3 to E16.5 (EtOH), ethanol exposure in combination with maternal nifedipine treatment in liquid diet (0.15 mg/kg body weight; EtOH+Nifed), or nifedipine only treatment (Nfed). Scale bars: 100 μm. C, Quantification of Nkx2.1+ cells in all treatment groups; **p < 0.01 compared with control, one-way ANOVA with Tuckey’s post hoc test.
- Figure 5.
Ethanol acutely potentiates calcium current mediated by L-type calcium channels. A, Representative images of the recording setup. Hoffman modulation contrast image of an acute slice from E16.5 mouse brain with the patch clamp recording pipette (A1, right) pointed at a tdTomato-fluorescent Nkx2.1+ GIN to be recorded in the whole-cell voltage clamp configuration. A multibarrel drug pipette is navigated to the vicinity of the cell being recorded (A1, left). B, Calcium current mediated by LTCCs was isolated by performing 40-mV depolarizing voltage steps in the presence of TTX, TEA (black trace). This current was subtracted with current obtained from 40-mV depolarizing steps in the presence of TTX, TEA, and nifedipine (nifedipine, gray trace). L-type calcium current was measured again in the presence of 18 mm ethanol (EtOH, red trace). C, Quantification of peak amplitude of calcium current mediated by LTCC in the presence of TTX and TEA (control) and nifedipine (nifedipine); **p < 0.01 paired t test. D, Peak amplitude of calcium current mediated by LTCC in the before (control) and during acute 18 mm ethanol exposure (EtOH); *p < 0.05 unpaired t test.
- Figure 6.
Schematic diagram summarizing the effect of ethanol on migrating embryonic cortical GINs. A, The depolarizing action of GABA in embryonic cortical GINs is established by the predominant expression of NKCC1 chloride importer relative to that of the KCC2 chloride exporter, resulting in heightened intracellular chloride. GABA binding to its cognate GABAA receptor thus causes chloride efflux and membrane depolarization. B, Following ethanol exposure, chloride efflux from GABAA receptors is enhanced, shifting the membrane potential of embryonic GINs to more depolarized levels. This enhanced depolarization of the membrane potential promotes the activation of LTCCs. It is postulated that the increased depolarization may also activate NMDA receptors by releasing the voltage-dependent Mg++ block. The increase in the activity of calcium-permeable channels may, in turn, increase calcium influx, raising intracellular calcium, and further activate downstream calcium signaling mechanisms. The net result of this chloride-to-calcium interplay mobilizes changes in the actin-microtubule dynamics and promote migration of cortical GINs.
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