Dopamine receptors (D1) will be tagged with green fluorescent protein (GFP), and inserted into the sindbis virus. This virus will be injected into organotypic cultures that include the mesolimbic pathway, the projections of the ventral tegmental area (VTA) to the nucleus accumbens (NAcc), a pathway believed to be important in the reinforcing properties of drugs of abuse, including alcohol. Specifically, D1 receptors will be injected into the NAcc. With this configuration D1 receptors will be located post-synaptically as in vivo. These explants will be exposed to ethanol in two ways: acutely and chronically. For acute ethanol exposure, multi-photon microscopy will be used to find neurons that are expressing our GFP tagged dopamine receptors while aCSF is perfused over the explant, then ethanol will be added to the perfusate. A difference in membrane expressed GFP tagged dopamine receptors should be seen after acute ethanol exposure. With chronic exposure the explants will be incubated in medium that contains ethanol. These explants may show a sensitization effect with less tagged receptors expressed on the membrane, compared to control. Additionally pretreatment with reserpine will be used to deplete dopamine, then acute ethanol exposure will be conducted to determine if ethanol is working only through dopamine release to cause internalization of receptors. ? ?
| Diaz, Laurea M; Maiya, Rajani; Sullivan, Matthew A et al. (2004) Sindbis viral-mediated expression of eGFP-dopamine D1 receptors in situ with real-time two-photon microscopic detection. J Neurosci Methods 139:25-31 |
