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H. Hirasawa, R. A. Betensky, E. Raviola; Co-Localization of Dopamine and GABA in Retinal Dopaminergic Amacrine (DA) Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3861.
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Retinal DA cells release both dopamine and GABA by exocytosis over their entire surface. In this study, we investigated whether the two transmitters are contained within the same or separate cytoplasmic organelles.
(i) Mouse retinas were stained with antibodies to tyrosine hydroxylase, vesicular monoamine transporter-2 (VMAT2) and vesicular GABA transporter (VGAT). We measured the intensity of all pixels in confocal z-sections of DA cell bodies in both the green (VGAT) and red (VMAT2) images. Then, we fit logistic regression models, with adjustment for the association within pixel, to measure the strength of the association of VGAT and VMAT2 in any given pixel. (ii) We examined with the electron microscope synaptic endings of DA cells in frozen ultrathin sections treated with antibodies to VMAT2 and VGAT followed by Protein A-gold of different sizes. (iii) In isolated cell bodies, we simultaneously used amperometry to measure dopamine release and patch clamp to measure GABAA-mediated Cl- currents generated by the cells in response to release of their own GABA.
(i) In the confocal images of the DA cell bodies there was dependence between VGAT+ and VMAT2+ pixels (lnOR=1.09; 95% confidence interval: 0.82, 1.4). There was 2.74 times the likelihood for VGAT of being associated with a VMAT2+ pixel than with a VMAT2- pixel. (ii) With the electron microscope, dopaminergic endings contained a mixture of vesicles positive for VMAT2 alone, for VGAT alone and for both. (iii) With electrophysiology, we found that 16% of the amperometric events were temporally coincident with GABAergic events within a time window of ± 5ms. Cross-correlation analysis showed that coincidence of the two types of events was statistically significant.
Dopamine and GABA are co-localized in a proportion of the synaptic vesicles of DA cells. This result is consistent with the idea that the two transporters are distributed stochastically among vesicles, possibly in the course of the process of synaptic vesicle membrane recycling.
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