Synaptomic analysis of dopaminergic inputs reveal hub synapses in the mouse striatum

Abstract

Highlights:1. Fluorescence Activated Synaptosome Sorting allows for the establishment of a molecular synaptome of striatal dopaminergic projections. 2. Dopaminergic varicosities adhere to receptor filled post-synaptic membranes. 3. Dopaminergic projections build hub synapses with excitatory and inhibitory projections. 4. SynCAM 2 is a strong candidate for the adhesion and differentiation of dopamineeffector hub synapses. SUMMARYDopamine transmission is a monoaminergic system involved in reward processing and motor control. Volume transmission is thought to be the main mechanism by which monoamines modulate effector transmission though synaptic structures are scarcely described. Here, we applied a fluorescence activated synaptosome sorting workflow to dopaminergic projections to the striatum and explored cellular and molecular features of the dopaminergic synaptome. This demonstrated that dopaminergic varicosities adhere to post-synaptic membrane baring cognate receptors. We further identified a specific bond of varicosities to glutamatergic or GABAergic synapses in structures we named dopaminergic "hub synapses". Finally, we showed that the synaptic adhesion protein SynCAM 2 is strongly expressed at dopaminergic hub synapses. Our data strongly suggest that neuromodulation frequently operates from hubsynapses on local receptors, presumably in conjunction with extra-synaptic volume transmission. We provide a new framework for the molecular exploration of dopaminergic synapses and more generally on discrete synapse populations ex-vivo.

Dopaminergic neurons establish a distinctive axonal arbor with a majority of non‐synaptic terminals

“…One possibility is that NL‐2 plays a role in the establishment of GABA release sites by DA neurons 10,51 . The implication of other cell adhesion molecules such as SynCAM2 also needs to be examined as recent work has also revealed the presence of this protein in striatal synaptosomes containing DAergic terminals linked to postsynaptic domains 82 …”

“…10,51 The implication of other cell adhesion molecules such as SynCAM2 also needs to be examined as recent work has also revealed the presence of this protein in striatal synaptosomes containing DAergic terminals linked to postsynaptic domains. 82 In the context of the strong ability of NL-1 to promote synapse formation by DA neurons, a limited formation of synapses by DA neurons in vitro and in vivo could perhaps be due to limited levels of Nrxn-1 at individual terminals. Although we found that Nrxn-1α and Nrxn-1ß are robustly expressed by DA neurons, the fact that DA neurons in rodents are thought to establish upwards of 100 000 individual release sites along their axonal arbor 15 could give rise to limited levels of presynaptic transsynaptic proteins in these terminals.…”

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

“…Dissecting the molecular composition of specific connections remains a major challenge. Sorting of growth cones 69 and specific synapse types (Pfeffer et al 70 , this study), imaging-based approaches [71][72][73] , affinity purification 74 , and proximity biotinylation [75][76][77] each have their advantages and disadvantages 78 . Here, we combined biochemical enrichment, antibody labeling of a synapse type-enriched surface marker, and fluorescent sorting to isolate MF synapses.…”

Subcellular proteomics of dopamine neurons in the mouse brain reveals axonal enrichment of proteins encoded by Parkinson’s disease-linked genes

“…We suspect this is due to the high density of boutons en passant along dopaminergic axons, which would provide a strong representation of the entire axonal proteome upon resealing as synaptosomes. Our data thus highlight the utility of the synaptosome sorting technique developed by Herzog and colleagues as a complementary approach for study of the presynaptic proteome (Biesemann et al, 2014;Pfeffer et al, 2020). Future work will determine whether the strong correlation between axonal and synaptosomal proteomes is a unique feature of DA neurons.…”