Fig. 2

Neonatal alcohol effects on microglial opioid receptors and TLR4 pathway proteins in mediobasal hypothalamus in vivo. Immunohistochemical characterization of double-labeled IBA-1 and MOR/DOR-positive microglia in neonatal pups. Nuclear stain DAPI (a, e), DOR (b), MOR (f), and IBA-1(c, g) immunolabeled cells. Representative merged photographs demonstrate colocalization of DOR/IBA-1 (d) and MOR/IBA-1 (h). Scale bars are 200 μm/each and arrows depict the same cell. i–x showing protein quantification in isolated microglia from ad libitum (AD), pair-fed (PF), alcohol-fed (AF), alcohol-fed and naltrindole-treated (AF + NTD), and alcohol-fed and naltrexone-treated (AF + NTX) neonatal rat pups at PND 6 by flow cytometry. Histograms show representative staining of isolated microglia for each protein compared to unstained or unlabeled control cells. Bar graphs show mean or median fluorescence intensity of staining of IBA-1 (i, l), MOR (k, l), DOR (m, n), TLR4 (o, p), p-38 MAPK (q, r), p-JNK (s, t), p-AKT (u, v), and NF-κB (w, x) from microglia isolated from each treatment group. Data are represented as mean ± SEM (n = 7–11). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug treatment groups are shown by lines with p values on the top of bar graphs