Investigation of Dopamine D3 Receptor and Transporter Dual Mutant for Molecular Mechanism of Behavioral Sensitization and Psychosis

This 3-year grant proposal aims to investigate the cellular and molecular mechanism that lead to the development of behavioral sensitization. To achieve this purpose, we will first produce a novel animal model of psychosis/mania and behavioral sensitization, i.e. the application of dopamine D3 receptor and transporter double knock out mice, the DRD3-/-/DAT-/- mutants. Considering the nature of D3R and DAT KO individually develop the hyperkinetic activity, we expect the DRD3-/-/DAT-/- mutants will exhibit an even higher psychomotor activity, the spontaneous or fully sensitized mice. This animal model would allow us to investigate the mechanism of psychosis/mania, besides provide us a valuable platform to evaluate the potential anti-psychotic drug efficacy. Once DRD3-/-/DAT-/- mutants produced, at the first year we will characterize its behavioral and biochemical phenotypes, i.e. the spontaneous locomotor activity and stereotypy, learning and memory via Morris water maze test, the anxiety response via tail suspension and force-swimming tests and prepulse inhibition (an valid index for schizophrenia). In addition, to evaluate if this fully sensitized mutants carry excess dopamine efflux, we will use in vivo microdialysis to measure the amount of dopamine release from nucleus accumbens. Previous evidence strongly implicate that D1 dopamine receptor-dependent PKA/DARPP-32/Thr34/Cdk5, ERK1/2 and D2 dopamine receptor-dependent PI3K/Akt/GSK3β/β-catenin pathways are critically involved in the development of behavioral sensitization and psychosis. We will evaluate if these signal regulators explore a similar pattern as in methamphetamine- or ketamine-sensitized mice. A series of western immunoblotting, immunoprecipitation and kinase assays will be performed in this regards. In addition, we will also treat the DRD3+/-/DAT+/- heterozygous with chornic methamphetamine or ketamine followed by behavioral sensitization test to serve as drug-induced behavioral sensitization controls. If all the behavioral and biochemical results are expected, we will further test if in sensitized mice, the responsiveness of D1 and D2 receptors would behave similar to drug-induced behaviorally sensitized animals. For this purpose, in a accumbal brain slices preparation, we will treat the tissues with selective D1 or D2 agonist and evaluate, in a dose- and time-dependent manner, the response of particular signaling pathway. Since the LTP and LTD are characterized as neuroplasticity associated with behavioral sensitization and/or addiction, we will also analyze the binding activity of NMDA and AMPA, as well as their associated scaffolding proteins, PSD-95 and GRIP, respectively. By the end of 2nd grant year, we will adopt proteomic study taking advantage of in-house Proteomic Center. The pooled prefrontal cortex and nucleus accumbens from mutants and wild-type controls will subject to 2-D PAGE to resolve the proteins, followed by MALDI-TOF and/or LC-MS/MS analyses. The identified protein species will first be confirmed via either western immunoblot, EMSA or ChIP assays, then compared with signal regulators that resulted from previous experiments to formulate the possible protein complex or signal route that are responsible for the sensitized or psychotic behaviors. At the 3rd grant year, we will try to link the behavioral sensitization with possible alteration of neuregulin-ErbB system, both are candidate genes in the cause of schizophrenia, in the prefrontal cortex. Other than quantification identification, we will also evaluate, in incubated brain slices, extragenous recombinant NRG1 would evoke altered PI3K/Akt or ERK1/2 signaling. Finally, based on recent reports linked gliosis and apoptosis with behavioral sensitization, we will measure a series of apoptotic regulatory proteins and astrogliosis (marked by GFAP), in addition to treat mutants (or drug-induced sensitized mice) with selective caspase inhibitors or glial modulator, PPF followed by behavioral sensitization evaluation. Overall, we believe through the creation of novel animal model and these experimental attempts, we will gain valuable information to dissect further the molecular mechanism underlying the behavioral sensitization and psychosis/mania.

Project ID:PC9709-0936
External Project ID:NSC97-2320-B182-018-MY3