Design and validation of genetically encoded probes for the analysis of neuronal catecholamine and ATP co-transmission

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Sympathetic co-transmission
Neuronal differentiation
Vesicle trafficking
Vesicle co-localization
Vesicle exocytosis
PH-sensitive fluorescent protein

BACKGROUND: Sympathetic nerves co-release several neurotransmitters, including adenosine-5'-triphosphate (ATP) and norepinephrine (NE). Our studies are aimed at understanding how these nerves provide automatic regulation of blood vessel diameter and therefore blood pressure. Relatively little is known at the molecular level about how these nerves control the release of multiple neurotransmitters. Using immunofluorescence microscopy, we recently showed that clusters of vesicles containing ATP and NE are segregated within sympathetic nerve terminals. METHODS: To assess the mechanisms of ATP and NE release, we developed genetically encoded reporters of the vesicular monoamine transporter VMAT2 (SLC18A2) and the vesicular nucleotide transporter VNUT (SLC17A9) tagged with pH-sensitive fluorescent proteins to monitor the release of NE and ATP containing vesicles with molecular specificity and high spatial resolution. RESULTS: First, we characterized the dopaminergic Neuro-2a (N2a) cell line as a model to study catecholamine and ATP co-release. N2a cells express VMAT2 and VNUT, and we found that their expression is upregulated upon differentiation, induced by retinoic acid (RA) and serum deprivation. We optimized retinoic acid and serum concentrations to drive neurite outgrowth while minimizing cell death. Following differentiation, cells exhibited release of VMAT2-pHuji, evoked by field stimulation and the calcium Ionophore 4-Bromo-A23187. Second, we tested whether ATP and NE localize to separate vesicles in N2a cells. Nearest-neighbour colocalization analysis showed that VMAT2 and VNUT are located in common varicosities but in separate vesicles. VNUT and VMAT2 appear to traffic independently, and they appear to be localized into vesicles with pH <6.0 and >7.0, respectively. CONCLUSIONS: Our results corroborate reports that NE and ATP are stored in separate vesicles but segregated into separate pools within the varicosity. The N2a cell line is a promising model to further identify fundamental aspects of differential trafficking and release of VMAT2 and VNUT containing vesicles, while VMAT-pHuji and VNUT-pHluorin permit simultaneous detection of catecholaminergic and purinergic vesicle release.

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Damon Poburko
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.