| Abstract No.: | C-B3060 |
| Country: | Canada |
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| Title: | SYNAPTOTAGMIN 4 AND 7 ARE REQUIRED FOR SOMATODENDRITIC DOPAMINE RELEASE IN CULTURED DOPAMINE NEURONS |
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| Authors/Affiliations: | 1 J Alfredo Mendez*; 1 Marie-Josée Bourque; 1 Louis-Éric Trudeau;
1 Université de Montréal, QC, Canada
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| Content: | Objectives: In addition to axonal dopamine (DA) release in projection areas, DA neurons also release DA locally within the mesencephalon through their cell body and dendrites, a process called somatodendritic (STD) DA release. STD DA release is thought to regulate the excitability of DA neurons through STD D2 autoreceptor activation. Two mechanisms have been proposed to explain STD DA release: reversal of DA transport and release of DA through some form of exocytosis. A growing body of evidence suggests that under physiological conditions, the latter is more likely: STD DA release is activity dependent, sensitive to depletion of vesicular stores, requires calcium and moreover, disruption of SNARE proteins that are required for exocytosis severely decreases STD DA release. To further identify the molecular mechanism of STD DA release and to search for an explanation of the differential calcium-sensitivity of axonal and STD DA release, we decided to test the hypothesis that different synaptotagmin (Syt) isoforms are present in the axonal and STD compartment of DA neurons and mediate both forms of DA release.
Materials and methods: Release of DA was measured by radioassay using tritiated tyrosine in primary cultures of postnatal mesencephalic DA neurons isolated from TH-EGFP transgenic mice. Immunocytochemistry and confocal microscopy was used to localize Syt isoforms, while single-cell RT-PCR was used to profile individual DA neurons for the presence of Syt mRNA. Short interfering RNA (siRNA) were used to knockdown various Syt isoforms and evaluate their participation in both total and STD DA release. RESULTS: DA release in primary cultures was found to be activity-dependent. A substantial fraction of DA release persisted in 0.5 mM extracellular calcium, under conditions where axonal release is known to be abrogated, as we have previously reported in rat cultures. We attribute this residual release to the STD compartment. All DA neurons were found to contain Syt1 and Syt4 mRNA. However, Syt7 mRNA showed increased expression with time in culture. Immunocytochemistry showed that while Syt1 was found exclusively in the axonal compartment, Syt 4 was found only in the STD compartment and Syt7 was found in both compartments. Cultured neurons were transfected with siRNA directed against Syt1, 4, 7 or a combination of these. We found that compared to controls, Syt1 inhibition had no effect on STD release of DA, but strongly reduced total DA release, compatible with an exclusive role in axonal DA release. Knockdown of either Syt4 or Syt7 reduced STD DA release, although the later produced a larger effect. Inhibition of both isoforms at the same time induced an almost complete block of STD DA release.
Conclusions: The present results are compatible with the exocytotic nature of STD DA release. Furthermore, we suggest that while Syt1 plays a major role in axonal DA release, Syt4 and Syt7 play critical roles in STD DA release. We propose that the differential calcium sensitivity of axonal and STD DA release is due to the differential compartmentalized distribution of Syts in DA neurons. |
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