Embedded Files
Subjects
Subjects
13 female C57BL/6J mice (C57) (Jackson Laboratory) were bred in-house at Miami University.
Surgery
Surgery
A custom-made AAV expressing shRNA for the mouse Gprasp1 gene (AAV8-GFP-U6-m-GPRASP1-shRNA from Vector Biolabs, Malvern, PA). Control mice received a scrambled version of the shRNA sequence (AAV8-GFP-U6-scrmb-shRNA). Both the experimental and control viruses were injected into the mPFC (AP: 1.8, ML: ±0.5, DV: -3.0).
Drinking in the Dark (DID)
Drinking in the Dark (DID)
Following 4 weeks of recovery, mice began DID. Access to two bottles, one containing reverse-osmosis (RO) drinking water, the other containing 15% ethanol (EtOH), was provided for two hours. Consumption of water and EtOH was measured by bottle weight, using two “dummy” cages to account for spillage. This procedure was repeated 5 days/week for three weeks, after which quinine was added to the EtOH incrementally (0, 10, 100, 200 μM) to assess aversion resistance. After 2 weeks, mice were tested for sensitivity to quinine in water.
Why use DID?
Why use DID?
The Drinking in the Dark (DID) paradigm models human binge drinking and typically induces an escalation of ethanol consumption.
Histology
Histology
Upon completion of drinking, mice will be sacrificed and intracardially perfused with saline and formalin. Brains will then be harvested, sectioned using a cryostat (40 μm), and mounted on slides. Slides will be coverslipped with Vectashield mounting medium counterstained with DAPI. Finally, they will be visualized with an Olympus AX70 fluorescent microscope to confirm the correct placement of viral knockdown in the mPFC.
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