[Abstract Title]. - Society for Neuroscience

Topic: B.06.b. Vesicle recycling and biogenesis
Support: NIH Grant NS053050
Title: Multiphoton monoamine imaging reveals two modes of release by Raphe Nucleus
serotonin neurons
Authors: *L. A. COLGAN 1 , I. PUTZIER 2 , E. S. LEVITAN 2 ;
1 Neurobiol, 2 Pharmacol., Univ. Pittsburgh, Pittsburgh, PA
Abstract: Currently, neurotransmission imaging techniques rely on markers of cycling
membranes or vesicular proteins instead of direct detection of neurotransmitter. Here, a novel
approach, Multiphoton Monoamine Imaging (MMI), is established to investigate monoamine
dynamics in cultured cells and neurons in brain slices. Specifically, a fluorescent serotonin
analog was visualized by multiphoton microscopy, allowing monoamine storage, release, and
repackaging to be monitored. The analog was taken-up by PC12 cells expressing the serotonin
transporter (SERT) and uptake was blocked by fluoxetine, a SERT inhibitor. Intracellularly, the
analog was concentrated into growth cones where it colocalized with secretory vesicles. This
localization was disrupted by reserpine, an inhibitor of the vesicular monoamine transporter
(VMAT), suggesting packaging of the analog into secretory vesicles by VMAT. Furthermore,
stimulation with high potassium led to reserpine sensitive release, with kinetics and magnitude
comparable to peptidergic release. MMI was also feasible with Raphe neurons in rat brain slice.
Incubation of slices with analog gave a fluoxetine sensitive signal in the somatodendritic region
of neurons, which decreased upon stimulation with the glutamate receptor agonist AMPA.
Further examination revealed two phases of release. Initial release required calcium and was
blocked by reserpine, indicating exocytosis of vesicles. This release also decreased the cytosolic
pool of analog, suggesting that recycling vesicles were rapidly refilled. This was followed by a
slower, calcium and reserpine insensitive phase of release. Interestingly, this slow release was
not blocked by fluoxetine, suggesting a mechanism that is independent of calcium, VMAT, and
SERT.
Disclosures: L.A. Colgan , None; I. Putzier, None; E.S. Levitan, None.
Poster
236. Neurotransmitter Release: Docking and Fusion
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 236.25/D25
Topic: B.06.a. Docking and fusion