The membrane raft protein Flotillin-1 is essential in dopamine neurons for amphetamine-induced behavior in Drosophila

Abstract

The dopamine transporter (DAT) is the primary molecular target responsible for the rewarding properties of the psychostimulants amphetamine (AMPH) and cocaine. AMPH increases extracellular dopamine (DA) by promoting its nonexocytotic release via DAT-mediated efflux. Previous studies in heterologous cells have shown that phosphorylation of the amino terminus of DAT is required for AMPH-induced DA efflux but not for DA uptake. However, the identity of many of the modulatory proteins and the molecular mechanisms that coordinate efflux and the ensuing behavioral effects remain poorly defined. Here we establish a robust assay for AMPH-induced hyperlocomotion in Drosophila melanogaster larvae. Using a variety of genetic and pharmacological approaches we demonstrate that this behavioral response is dependent on DA and on DAT and its phosphorylation. We also show that methylphenidate (MPH), which competitively inhibits DA uptake but does not induce DAT-mediated DA efflux, also leads to DAT-dependent hyperlocomotion, but this response is independent of DAT phosphorylation. Moreover, we demonstrate that the membrane raft protein Flotillin1 is required for AMPH-induced but not MPH-induced hyperlocomotion. These results are the first evidence of a role for a raft protein in an AMPH-mediated behavior. Thus, using our assay we are able to translate molecular and cellular findings to a behavioral level and to differentiate in vivo the distinct mechanisms of two psychostimulants.

Drosophila melanogaster as a genetic model system to study neurotransmitter transporters

“…By disrupting the function of membrane rafts in DA cells, the authors elegantly showed that localization of dDAT to these sites is required for efflux but not for transport in vivo (Pizzo et al, 2013) consistent with in vitro studies (Cremona et al, 2011). These studies also demonstrated that the behavioral effects of amphetamines in the fly are mediated through DA neurons rather than another aminergic cell type (Pizzo et al, 2013; Pizzo et al, 2014).…”

“…Efflux represents an additional functional mode in which neurotransmitter moves out of rather than into the cell (Fleckenstein et al, 2007; Sulzer, 2011). The mechanism by which this occurs is a very active area of investigation, and includes work in Drosophila described in more detail below (Pizzo et al, 2013; Pizzo et al, 2012).…”

“…Treatment of adult mutant flies with reserpine also increases sleep (Nall and Sehgal, 2013). In contrast, amphetamines decrease sleep in flies (Andretic et al, 2005), presumably via release of DA (Pizzo et al, 2013). In each case, changes in sleep were distinguished from changes in locomotion using previously established criteria (Hendricks et al, 2000; Shaw et al, 2000).…”

“…Importantly, this type of in vivo structure-function analysis provides an opportunity to determine the effects of altered dVMAT trafficking on behavior. Similar studies on Drosophila dopamine transporter ( dDAT) point mutants have been used to determine the behavioral effects of N-terminal phosphorylation and an hDAT mutant linked to autism (Hamilton et al, 2013; Pizzo et al, 2013; Pizzo et al, 2014). This method of analysis is likely to useful for the study of a variety of other transporters (see Future Directions below).…”

“…Mutations in dDAT have also been used to study the mechanism of action of amphetamines (Pizzo et al, 2013; Pizzo et al, 2014). By disrupting the function of membrane rafts in DA cells, the authors elegantly showed that localization of dDAT to these sites is required for efflux but not for transport in vivo (Pizzo et al, 2013) consistent with in vitro studies (Cremona et al, 2011).…”