Ultrastructural and molecular analysis of cortico-striatal dopamine hub synapses

Overview & Synopsis

Dopamine is a neurotransmitter that modulates neuronal activity and governs essentialfunctions such as reward prediction, motivation, and motor control. In the striatum, dopaminetypically acts over a large volume through slow-acting metabotropic receptors. However,recent studies have demonstrated that dopamine can also operate in localized hotspotsmeasuring a few cubic micrometers. Additionally, dopamine may trigger excitatory synapsepotentiation when released in synchrony with glutamate. Despite these advances, molecularand ultrastructural studies have been limited by technical challenges.During my doctoral training, I developed cutting-edge techniques to explore the molecular andultrastructural features of dopaminergic terminals and their relationship to synapses in themouse striatum.In a first piece of work, we aimed at analyzing the composition of dopaminergic (DA) terminals.To that end, we labeled dopaminergic neurons with a green fluorescent reporter under thecontrol of the Dopamine Transporter promoter (DAT-Cre line). Following subcellularfractionation of the striatum, we isolated green fluorescent synaptosomes (resealed terminalsbound to synaptic partners) with fluorescence-activated synaptosome sorting (FASS).Immunolabeling of these isolated DA synaptosomes confirmed the presence of genuinedopaminergic markers apposed to dopamine receptors. Surprisingly, 30% of dopaminesynaptosomes were bound to cortico-striatal excitatory synapses containing the type 1vesicular glutamate transporter (GLU). We termed these connections cortico-striataldopamine hub synapses (DHS). On these samples, I used 6 markers of the GLU pre- andpost-synapse to scrutinize the effect of the association in DHS and I could identify that theassociation in DHS corelates with a molecular remodeling of the cortico-striatal synapses.Dopamine hub synapses may thus serve as a structural substrate for localized dopamineactivity in the striatum and could further potentiate glutamatergic signaling.Next, I established a cryo-correlative light and electron microscopy (cryo-CLEM) protocol onlabeled synaptosomes to determine the ultrastructure of dopamine terminals and DHS in threedimensions. Compared to cortico-striatal pre-synapses, DA synaptosomes were three timessmaller and contained ten times fewer synaptic vesicles (SVs). The size and shape of SVs inDA terminals were more heterogeneous, they were generally larger and sometimes elongated.While GLU synapses exhibited active zones (AZ) and postsynaptic densities, DA terminalslacked distinct vesicle clusters or clear synaptic organization. Only 35% of dopamine terminalscontained at least one tethered SV required for exocytosis. Compared to synaptosomeswithout tethered vesicle, SVs were more abundant and closer to the plasma membrane,suggestive of a higher release activity. However, primed SVs (tethered within 5 nm ofthe plasma membrane) were absent. Interestingly, GLU terminals in DHS had more primedSVs compared to regular GLU synapses, implying that the presence of DA terminalsreorganizes SVs in glutamatergic terminals. These results suggest that the interaction of DAterminals with synapses modifies the release properties of GLU pre-synapses by a localdopamine-dependent plasticity.Given that dopamine dysregulation is implicated in various diseases as addiction, thediscovery of this multipartite structure responsible for specific dopamine activity onglutamatergic inputs represent a new conceptual framework for future studies in the field.Modulating the physical interaction between DA and GLU synapses in vivo could provide anew method to influence dopamine signaling in the striatum.