Toward Precise Modeling of Dopamine Release Kinetics: Comparison and Validation of Kinetic Models Using Voltammetry.

Dopamine (DA) is a neurotransmitter present within the animal brain that is responsible for a wide range of physiologic functions, including motivation, reward, and movement control. Changes or dysfunction in the dynamics of DA release are thought to play a pivotal role in regulating various physiological and behavioral processes, as well as leading to neuropsychiatric diseases. Therefore, it is of fundamental interest to neuroscientists to understand and accurately model the kinetics that govern dopaminergic neurotransmission. In the past several decades, many mathematical models have been proposed to attempt to capture the biologic parameters that govern dopaminergic kinetics, with each model seeking to improve upon a previous model. In this review, each of these models are derived, and the ability of each model to properly fit two fast-scan cyclic voltammetry (FSCV) data sets will be demonstrated and discussed. The dopamine oxidation current in both FSCV data sets exhibits hang-up and overshoot behaviors, which have traditionally been difficult for mathematical models to capture. We show that more recent models are better able to model DA release that exhibits these behaviors but that no single model is clearly the best. Rather, models should be selected based on their mathematical properties to best fit the FSCV data one is trying to model. Developing such differential equation models to describe the kinetics of DA release from the synapse confers significant applications both for advancing scientific understanding of DA neurotransmission and for advancing clinical ability to treat neuropsychiatric diseases.

TargeTron inactivation of plasmid-regulated Chlamydia trachomatis CT084 results in a nonlytic phenotype.

Chlamydia trachomatis is an obligate intracellular bacterium that causes blinding trachoma and sexually transmitted disease. The chlamydial plasmid is a critical virulence factor in the pathogenesis of these diseases. Plasmid gene protein 4 (Pgp4) plays a major role in chlamydial virulence by regulating the expression of both chromosomal genes and Pgp3. Despite the importance of Pgp4 in mediating lytic exit from host cells the pathogenic mechanism by which it functions is unknown. CT084 is a highly conserved chromosomal gene with homology to phospholipase D. We showed CT084 expression is regulated by Pgp4 and expressed late in the chlamydial developmental cycle. To investigate the function of CT084 in chlamydial lytic exit from infected cells, we made a CT084 null strain (ct084::bla) by using Targetron. The ct084::bla strain grew normally in vitro compared to wild-type strain; however, the strain did not lyse infected cells and produced significantly less and smaller plaques. Collectively, our finding shows Pgp4-regulated CT084-mediated chlamydia lytic exit from infected host cells.

A Chlamydial Plasmid-Dependent Secretion System for the Delivery of Virulence Factors to the Host Cytosol.

Chlamydia are obligate intracellular Gram-negative bacteria distinguished by a unique developmental biology confined within a parasitophorous vacuole termed an inclusion. The chlamydial plasmid is a central virulence factor in the pathogenesis of infection. Plasmid gene protein 4 (Pgp4) regulates the expression of plasmid gene protein 3 (Pgp3) and chromosomal glycogen synthase (GlgA), virulence factors secreted from the inclusion to the host cytosol by an unknown mechanism. Here, we identified a plasmid-dependent secretion system for the cytosolic delivery of Pgp3 and GlgA. The secretion system consisted of a segregated population of globular structures originating from midcycle reticulate bodies. Globular structures contained the Pgp4-regulated proteins CT143, CT144, and CT050 in addition to Pgp3 and GlgA. Genetic replacement of Pgp4 with Pgp3 or GlgA negated the formation of globular structures, resulting in retention of Pgp3 and GlgA in chlamydial organisms. The generation of globular structures and secretion of virulence factors occurred independently of type 2 and type 3 secretion systems. Globular structures were enriched with lipopolysaccharide but lacked detectable major outer membrane protein and heat shock protein 60, implicating them as outer membrane vesicles. Thus, we have discovered a novel chlamydial plasmid-dependent secretion system that transports virulence factor cargo from the chlamydial inclusion to the host cytosol. IMPORTANCE The Chlamydia trachomatis plasmid regulates the expression and secretion of immune evasion virulence factors to the host cytosol by an unknown mechanism. In this study, we identified a novel plasmid gene protein 4 (Pgp4)-dependent secretion system. The system consists of globular structures distinct from typical chlamydial developmental forms that export Pgp3 and GlgA to the host cytosol. Globular structures emerged at mid-chlamydial growth cycle from distinct populations of reticulate bodies. The formation of globular structures occurred independently of known chlamydial secretion systems. These results identify a Pgp4-dependent secretory system required for exporting plasmid regulated virulence factors to the host cytosol.