The goal of this proposal is to advance our ability to inducibly manipulate gene expression within subsets of neurons. Four neurotransmitter systems have been chosen for this proposal based on their having been implicated in numerous neurophysiologic and behavioral processes, as well as disorders of those processes. The four are the adrenergic (norepinephrine and epinephrine), dopaminergic, serotonergic and orexinergic systems. Because of the unique ability to create gene-targeted mice among mammals, mice will be the model organism employed. One goal is to inducibly activate, inactivate or modify endogenous genes that have been endowed with unique loxP sites that are recognized by bacteriophage P1 Cre recombinase (Cre). The main approach for achieving this will be the neurotransmitter-specific expression of the tamoxifen-inducible Cre fusion protein, CreERT2, that is fused to a modified estrogen receptor ligand-binding domain (ERT2). Because there may be occasions when using tamoxifen should be avoided due to effects on endogenous estrogen receptor signaling, a second approach for achieving inducible Cre activity will also be pursued. For this approach, the tetracycline-inducible transactivator (tTA2) and reverse tTA (rtTA2S-M2) will be expressed specifically in each neurotransmitter system. When crossed with previously characterized tetO-Cre mice, the transactivator (TA) mice will induce the neurotransmitter-specific expression of Cre either in the absence (tTA) or in the presence (rtTA) of antibiotic. In addition, the TA mice will permit the induction of any other Ptet-cDNA transgene in a neurotransmitter-specific fashion. To obtain neurotransmitter-specific expression, genes that uniquely define these neurotransmitter systems will be utilized: for the adrenergic system - dopamine 2- hydroxylase, for the dopaminergic system - dopamine transporter, for the serotonergic system - tryptophan hydroxylase 2, and for the orexinergic system - orexin. CreERT2 and the TAs will be targeted to the 3'- untranslated region of each gene via homologous recombination in embryonic stem cells. To achieve bicistronic expression, CreERT2 and the TAs will be preceded by an internal ribosome entry site. Reporter mice for Cre activity will be used to correlate temporal and spatial expression patterns of Cre with those for the targeted endogenous gene. Finally, the utility of the CreERT2 and TA mice will be demonstrated by crossing them with mice harboring a floxed tyrosine hydroxylase gene for inducible transmitter depletion.
The study of these four neurotransmitter systems via inducible changes in gene expression that are specific to each system is highly relevant to the understanding of fundamental neurophysiological and behavioral processes such as sleep, arousal and attention, motivation and reward, and learning and memory, as well as disorders related to these processes that include narcolepsy, drug abuse, anxiety, depression and post- traumatic stress disorder. It is expected that the proposed mouse models will permit significant insights into the etiology, identification and treatment of these disorders.