Gabriel, LukeWu, SijiaKearney, PatrickBellve, Karl D.Standley, CliveFogarty, Kevin E.Melikian, Haley E2022-08-232022-08-232013-11-062015-11-25J Neurosci. 2013 Nov 6;33(45):17836-46. doi: 10.1523/JNEUROSCI.3284-13.2013. <a href="http://dx.doi.org/10.1523/JNEUROSCI.3284-13.2013">Link to article on publisher's site</a>0270-6474 (Linking)10.1523/JNEUROSCI.3284-13.201324198373https://hdl.handle.net/20.500.14038/30533<p>Co-authors Luke Gabriel and Sijia Wu are doctoral students in the Neuroscience Program in the Morningside Graduate School of Biomedical Sciences (GSBS) at UMass Medical School.</p>Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.en-USCopyright © 2013 the authors. Copyright of all material published in <em>The Journal of Neuroscience</em> remains with the authors. The authors grant the Society for Neuroscience an exclusive license to publish their work for the first 6 months. After 6 months the work becomes available to the public to copy, distribute, or display under a <a href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International (CC BY 4.0) license</a>. Publisher PDF posted as allowed by the publisher's author rights policy at http://www.jneurosci.org/site/misc/ifa_policies.xhtml#copyright.http://creativecommons.org/licenses/by/4.0/Journal Articlehttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1811&amp;context=faculty_pubs&amp;unstamped=1https://escholarship.umassmed.edu/faculty_pubs/8097880385faculty_pubs/809