Fig. 9

Increased MCH⁺ neurons closely related to radial glia cells and processes in NEP and mHYP. Photomicrographs illustrate how maternal ethanol administration (2 g/kg/day, E10–E15) compared to isocaloric Control group (n = 7/group) increases the number of MCH⁺ neurons in close relation to the radial glia cells and along their processes (measured by IF for BLBP⁺) in female E19 embryos, with the quantification and data described in detail in the “Methods” and “Results” section. a Top panel, negative controls (sense probe and no fluorescence antibody) and positive control (double-labeling of MCH DIG-ISH with BLBP IF) (× 10 regular fluorescence images). The sense probe control revealed no MCH-expressing neurons, and the no fluorescence antibody control revealed no BLBP immunofluorescent cells and processes. The positive control shows the MCH-expressing neurons (black) to be very dense in the LH, with some evident in the mHYP (indicated by white arrowheads) and a few in the NEP, and the BLBP-labeled cells (green) to be most concentrated in NEP while their processes (green) are densest in the mHYP (indicated by white arrows) as they project laterally toward the LH. Bottom panel, photomicrographs combining DIG-labeled in situ hybridization for MCH⁺ neurons and immunofluorescence histochemistry for BLBP⁺ radial glia (× 20 confocal images). In ethanol-exposed embryo compared to control, these images illustrate several MCH⁺ neurons (two indicated by white arrowheads) in NEP, not evident in Control, that lie lateral to the BLBP⁺ radial glia cells and the increased number and percentage of MCH⁺ neurons in mHYP that are positioned close to the radial glia processes as they project laterally toward LH (see text for data). b This close relationship between the MCH⁺ neurons and radial glia is more clearly illustrated in the enlarged image (from the area between dotted white lines in of ethanol-exposed embryo), showing one MCH⁺ neuron in the NEP and three MCH⁺ neurons (white arrowheads) along of a single BLBP⁺ process (white arrow) projecting laterally through the mHYP. c Further analyses using double-labeled immunofluorescence histochemistry confirmed this close relationship between embryonic MCH⁺ neurons (green) and radial glia (red) in the mHYP, with × 40 image showing six MCH⁺ neurons (white arrowheads) along a single BLBP⁺ process (white arrows) projecting toward the LH. BLBP, brain lipid-binding protein; mHYP, medial hypothalamus; MCH, melanin-concentrating hormone; NEP, neuroepithelium; V, third ventricle. Scale bars, 100 μm