Venue: CARF
Paul Lapios
Perrais’ team (IINS)
Thesis supervisor: David Perrais
Title
Ultrastructural and molecular analysis of cortico-striatal dopamine hub synapses
Abstract
Dopamine is a neurotransmitter that modulates neuronal activity and governs essential functions such as reward prediction, motivation, and motor control. In the striatum, dopamine typically acts over a large volume through slow-acting metabotropic receptors. However, recent studies have demonstrated that dopamine can also operate in localized hotspots measuring a few cubic micrometers. Additionally, dopamine may trigger excitatory synapse potentiation when released in synchrony with glutamate. Despite these advances, molecular and ultrastructural studies have been limited by technical challenges.
During my doctoral training, I developed cutting-edge techniques to explore the molecular and ultrastructural features of dopaminergic terminals and their relationship to synapses in the mouse 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 the control of the Dopamine Transporter promoter (DAT-cre line). Following subcellular fractionation of the striatum, we isolated green fluorescent synaptosomes (resealed terminals bound to synaptic partners) with fluorescence-activated synaptosome sorting (FASS). Immunolabeling of these isolated DA synaptosomes confirmed the presence of genuine dopaminergic markers apposed to dopamine receptors. Surprisingly, 30% of dopamine synaptosomes were bound to cortico-striatal excitatory synapses expressing the type 1 vesicular glutamate transporter (GLU). We termed these connections cortico-striatal dopamine hub synapses (DHS). On these samples, I used 6 markers of the GLU pre- and post-synapse to scrutinize the effect of the association in DHS. I could identify that the association in DHS corelates with a molecular remodeling of the cortico-striatal synapses (1). Dopamine hub synapses may thus serve as a structural substrate for localized dopamine activity in the striatum and could further potentiate glutamatergic signaling.
Next, I established a cryo-correlative light and electron microscopy (cryo-CLEM) protocol on labeled synaptosomes to determine the ultrastructure of dopamine terminals and DHS in three dimensions. Compared to cortico-striatal pre-synapses, DA synaptosomes were three times smaller and contained ten times fewer synaptic vesicles (SVs). The size and shape of SVs in DA terminals were more heterogeneous; they were generally larger and sometimes elongated. While GLU synapses exhibited active zones (AZ) and postsynaptic densities, DA terminals lacked distinct vesicle clusters or clear synaptic organization. Only 35% of dopamine terminals contained at least one tethered SV required for exocytosis. In these terminals, SVs were more abundant and closer to the plasma membrane, suggestive of a higher release activity. However, primed SVs (tethered within 5 nm of the plasma membrane) were absent. Interestingly, GLU terminals in DHS had more primed SVs compared to regular GLU synapses, implying that the presence of DA terminals reorganizes SVs in glutamatergic terminals. These results suggest that the interaction of DA terminals with synapses modifies the release properties of GLU pre-synapses by a local dopamine-dependent plasticity (2).
Given that dopamine dysregulation is implicated in addiction and Parkinson’s disease (for which effective treatments are limited), the discovery of this multipartite structure responsible for specific dopamine activity on glutamatergic 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 a new method to influence dopamine signaling in the striatum.
Keywords: Synapse, Dopamine, Synaptic plasticity, Striatum, Cryo-CLEM
Publications
(1) Paget-Blanc V et al., (2022) A synaptomic analysis reveals dopamine hub synapses in the mouse striatum. Nat Commun 13:3102
(2) Paul Lapios, Robin Anger, Vincent Paget-Blanc, Vladan Lučić, Rémi Fronzes, Etienne Herzog#, and David Perrais# (2024) In preparation
Jury
- Aude Panatier (University of Bordeaux)
- Ira Milosevic (University of Oxford)
- Benoit Zuber (University of Bern)
- Jean-Antoine Girault (Sorbonne University)