In our desire to better understand the roles of central adrenergic signaling and their relation to neuropsychiatric conditions such as depression, we propose to create a system in mice for the inducible inactivation of genetically defined neurons. While generally applicable, this system will be applied to the study of adrenergic and dopaminergic neurons. Such a technique will provide a potent method for determining the functions of these neurons in vivo, either during development or in the adult. We propose to inactivate neurotransmitter release through proteolytic cleavage of one of the proteins (SNAREs) essential for synaptic vesicle fusion with the plasma membrane. We will achieve this by developing transgenic lines of mice that can express the proteolytically active light chain of either a C. tetani (TeNT) or C. botulinum (BoNT/E) neurotoxin. Expression of the mouse codon-optimized neurotoxin transgenes will be regulated by the use of an inducible promoter sensitive to the presence of a transactivation factor in combination with a small molecule inducer drug. Cell-specific expression will be achieved by targeting insertion of the codon-optimized transactivator gene and a mammalian internal ribosome entry site to the 3'-untranslated region of a gene that defines the neurons of interest. This approach should permit true expression of the transactivator without disrupting expression of the endogenous gene. Neurons would be inactivated following administration of the inducer drug. The strengths of this system are likely to be the specificity of the neurons inactivated (due to the targeting scheme), the completeness of inactivation (due to the potency of the neurotoxin), and the stability of the temporally controlled inactivation (due to the half-life of the neurotoxin's effects). Our proposed technique will be applicable to any set of neurons that can be genetically defined. We will test this system in dopamine (DA) neurons because their inactivation is predicted to result in the Parkinsonian phenotypes of hypomotility and hypophagia. Block of DA release will be assessed by microdialysis in vivo and cyclic voltammetry in vitro. The system will also be applied to the study of adrenergic neurons, using similar techniques to document inducible and reversible inactivation of neurotransmitter release. The approach will permit the study of animals before and after inactivation of these neurons, providing a valuable internal control. This technique will complement other techniques used to study the same neurons, such as their genetic ablation or the genetic elimination of a single neurotransmitter from those neurons. Use of this technique will provide a model for diseases such as Parkinson's or depression in which a neuronal population is hypothesized to become dysfunctional. In general, the development of this technique should provide a powerful tool for the dissection of how the brain operates.
