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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Genes Brain Behav. 2017 May 2;16(7):699–708. doi: 10.1111/gbb.12384

Figure 4. Mdk knockdown by RNAi in the ventral tegmental area (VTA) of male mice increases binge-like ethanol consumption.

Figure 4

C57BL/6J mice were stereotaxically injected in the VTA with a lentivirus expressing a short hairpin RNA (shRNA) targeting Mdk (shMdk) or a control shRNA (shScr) and green fluorescent protein (GFP). Mice were allowed to recover for three weeks and tested for ethanol consumption in the drinking in the dark protocol. (a) Schematic diagram showing the location of the 19 nucleotide targeting sequence on the Mdk transcript (transcript variant 1). The blue box indicates the open reading frame. The arrow points to a vertical black line indicating the location of the targeting sequence. Numbers indicate the nucleotide position on the transcript. (b) Demonstration of knockdown efficacy of shMdk in Neuro-2a cells transfected with the lentiviral plasmid encoding shMdk compared with cells transfected with shScr. RNA was isolated from cells 48 h after transfection and analyzed by quantitative real-time PCR (qPCR, n = 3) (c) Demonstration of knockdown efficacy of shMdk in mouse VTA three weeks after lentiviral infection. Infected VTA was dissected from mice 3 weeks after injection, RNA isolated and analyzed by qPCR. shScr, n = 9; shMdk, n = 11. (d) Representative image of lentiviral infection in the VTA. Immunohistochemistry with a GFP antibody was performed on brain sections containing the VTA. IPR, interpeduncular nucleus, rostral. Scale bar, 200 μM. (e) Ethanol consumption in the DID test by mice infected in the VTA with lentivirus expressing shMdk (open squares) or shScr (filled circles). Shown is the amount of ethanol consumed in g ethanol/kg body weight during the 2 h drinking sessions each day. There were significant main effects of shRNA and day. (f) Ethanol consumption in mice infected in the VTA with lentivirus expressing shMdk (white bar) or shScr (black bar) during the 4 h drinking session on day 4 in the DID test, expressed as g ethanol/kg body weight. shScr, n = 16; shMdk, n = 17. *P < 0.05 by two-way ANOVA. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by student’s t-test. Data are presented as the mean ± SEM.