Abstract
The dopamine transporter (DAT) is a cell membrane protein whose main function is to reuptake the dopamine (DA) released in the synaptic cleft back into the dopaminergic neurons. Previous studies suggested that the activity of DAT is regulated by allosteric proteins such as Syntaxin-1A and is altered by drugs of abuse such as amphetamine (Amph). Because Caenorhabditis elegans expresses both DAT (DAT-1) and Syntaxin-1A (UNC-64), we used this model system to investigate the functional and behavioral effects caused by lack of expression of unc-64 in cultured dopaminergic neurons and in living animals. Using an inheritable RNA silencing technique, we were able to knockdown unc-64 specifically in the dopaminergic neurons. This cell-specific knockdown approach avoids the pleiotropic phenotypes caused by knockout mutations of unc-64 and ensures the transmission of dopaminergic specific unc-64 silencing to the progeny. We found that, similarly to dat-1 knockouts and dat-1 silenced lines, animals with reduced unc-64 expression in the dopaminergic neurons did not respond to Amph treatment when tested for locomotor behaviors. Our in vitro data demonstrated that in neuronal cultures derived from animals silenced for unc-64, the DA uptake was reduced by 30% when compared to controls, and this reduction was similar to that measured in neurons isolated from animals silenced for dat-1 (40%). Moreover, reduced expression of unc-64 in the dopaminergic neurons significantly reduced the DA release elicited by Amph. Because in C. elegans DAT-1 is the only protein capable to reuptake DA, these data show that reduced expression of unc-64 in the dopaminergic neurons decreases the capability of DAT in re-accumulating synaptic DA. Moreover, these results demonstrate that decreased expression of unc-64 in the dopaminergic neurons abrogates the locomotor behavior induced by Amph. Taken together these data suggest that Syntaxin-1A plays an important role in both functional and behavioral effects caused by Amph.
Highlights
The dopamine transporter (DAT) is a plasma membrane protein which reuptakes the DA released in the synaptic cleft back into the neurons
We found that the pdat-1::unc-64 sas animals show a slight but statistically significant reduction in body bends when tested in Abbreviations: C. elegans, Caenorhabditis elegans; BSR, basal slowing response; DAT, dopamine transporter; DA, dopamine; Amph, amphetamine; GFP, green fluorescent protein; sas, sense and antisense; RNAi, RNA interference; SWIP, swimming-induced paralysis
To dissect the role played by unc-64 in dopaminergic neurons, in otherwise wild type animals, we created transgenic animals in which a dopaminergic specific promoter drives the expression of part of the gene, in the sense and antisense directions (RNAi sas) (Esposito et al, 2007; Gallotta et al, 2016)
Summary
The DAT is a plasma membrane protein which reuptakes the DA released in the synaptic cleft back into the neurons. The neuronal specific isoform can bind to and regulate different plasma membrane proteins including ion channels (Naren et al, 1998; Arien et al, 2003; Condliffe et al, 2004; Tsuk et al, 2004) and neurotransmitter transporters (Deken et al, 2000; Geerlings et al, 2001; Haase et al, 2001; Horton and Quick, 2001; Quick, 2003, 2006; Sung et al, 2003; Wang et al, 2003; Fan et al, 2006). Few studies have been performed to assess whether the interaction and/or lack of interaction between Syntaxin-1A and DAT may cause behavioral outcomes in living animals (Carvelli et al, 2008; Cartier et al, 2015)
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