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Dopamine Synapse Is a Neuroligin-2–Mediated Contact Between Dopaminergic Presynaptic and Gabaergic Postsynaptic Structures

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Dopamine Synapse Is a Neuroligin-2–Mediated Contact Between Dopaminergic Presynaptic and Gabaergic Postsynaptic Structures Dopamine synapse is a neuroligin-2–mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures Motokazu Uchigashimaa, Toshihisa Ohtsukab, Kazuto Kobayashic, and Masahiko Watanabea,1 aDepartment of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan; bDepartment of Biochemistry, Graduate School of Medicine, University of Yamanashi, Chuo 409-3898, Japan; and cDepartment of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University, Fukushima 960-1295, Japan Edited by Robert C. Malenka, Stanford University School of Medicine, Stanford, CA, and approved February 26, 2016 (received for review July 17, 2015) Midbrain dopamine neurons project densely to the striatum and (MSNs), and exhibit ultrastructural features common to symmet- form so-called dopamine synapses on medium spiny neurons (MSNs), ric synapses (12–14). MSNs constitute around 90% of all striatal principal neurons in the striatum. Because dopamine receptors are neurons. They are divided equally into direct and indirect pathway widely expressed away from dopamine synapses, it remains unclear MSNs (d-MSN and i-MSN), differing in their connectivity with how dopamine synapses are involved in dopaminergic transmission. output nuclei of the basal ganglia and in the expression of dopa- Here we demonstrate that dopamine synapses are contacts formed mine receptors (i.e., D1R in d-MSNs and D2R in i-MSNs) (10). between dopaminergic presynaptic and GABAergic postsynaptic Considering the broad extrasynaptic expression of D RandDR structures. The presynaptic structure expressed tyrosine hydroxylase, 1 2 (15–17), the mode of dopaminergic transmission at dopamine vesicular monoamine transporter-2, and plasmalemmal dopamine synapses (i.e., whether it is mediated by wired transmission like transporter, which are essential for dopamine synthesis, vesicular filling, and recycling, but was below the detection threshold for that at conventional glutamatergic and GABAergic synapses) re- molecules involving GABA synthesis and vesicular filling or for GABA mains unclear. It is also unknown which types of molecules com- itself. In contrast, the postsynaptic structure of dopamine synapses prise pre- and postsynaptic membrane specializations at dopamine expressed GABAergic molecules, including postsynaptic adhesion synapses. NEUROSCIENCE molecule neuroligin-2, postsynaptic scaffolding molecule gephyrin, In the present study, we show that the presynaptic side of do- and GABAA receptor α1, without any specific clustering of dopamine pamine synapses is indeed dopaminergic, whereas the postsynaptic receptors. Of these, neuroligin-2 promoted presynaptic differentia- side is exclusively GABAergic, expressing GABAA receptor α1 tion in axons of midbrain dopamine neurons and striatal GABAergic (GABAARα1), gephyrin, and NL2. In vivo knockdown of NL2 in neurons in culture. After neuroligin-2 knockdown in the striatum, a the striatum reduced the density of dopamine synapses on MSN significant decrease of dopamine synapses coupled with a reciprocal dendrites, but reciprocally increased that of GABAergic synapses. increase of GABAergic synapses was observed on MSN dendrites. Our findings suggest that NL2 regulates striatal synapse formation This finding suggests that neuroligin-2 controls striatal synapse for- by giving competitive advantage to dopamine synapses over mation by giving competitive advantage to heterologous dopamine GABAergic synapses, and that the formation of dopamine syn- synapses over conventional GABAergic synapses. Considering that apses may serve to provide dopamine-sensing MSNs with struc- MSN dendrites are preferential targets of dopamine synapses and tural anchorage of dopamine release sites. express high levels of dopamine receptors, dopamine synapse for- mation may serve to increase the specificity and potency of dopami- nergic modulation of striatal outputs by anchoring dopamine release Significance sites to dopamine-sensing targets. Nigrostriatal dopaminergic projections form a number of do- dopamine synapse | neuroligin-2 | medium spiny neuron | striatum pamine synapses onto medium spiny neurons in the striatum, and have strong influence on emotion, motivation, voluntary hemical synapses comprise presynaptic machinery for transmit- movement, and cognition. Despite the functional importance, Cter release and postsynaptic machinery for receptor-mediated the molecular composition at dopamine synapses remains un- signal transduction in a neurochemically matched manner. They known. Here we demonstrate that dopamine synapses are are classified into Gray type-I and type-II synapses by asym- neurochemically mismatched contacts formed between dopa- metric or symmetric membrane density, respectively, of the pre- minergic presynaptic and GABAergic postsynaptic structures. and postsynaptic structures (1). Most, if not all, asymmetric and Intriguingly, neuroligin-2 expressed at the GABAergic post- symmetric synapses are excitatory and inhibitory, respectively, as synaptic structure controls striatal synapse formation by giving competitive advantage to heterologous dopamine synapses they selectively express ionotropic glutamate or GABA/glycine over conventional GABAergic synapses. Therefore, our findings receptors together with their specific scaffolding proteins (2). suggest that neuroligin-mediated formation of such neurochemi- Neurochemical matching of chemical synapses is controlled by cally mismatched synapses could be a novel strategy to increase activity-dependent mechanisms (3, 4), and mediated by transmem- the target selectivity and potency of modulation by anchoring brane adhesion proteins and secreted molecules (5). The neuroligin release sites of neuromodulators to their receptive targets. (NL) family comprises postsynaptic adhesion molecules that form transsynaptic contacts with presynaptic neurexins (Nrxn) (6). Author contributions: M.U. and M.W. designed research; M.U. performed research; T.O. Of note, NL1 and NL2 are selectively expressed at glutamatergic and K.K. contributed new reagents/analytic tools; M.U. analyzed data; and M.U. and and GABAergic synapses, respectively (7, 8), and are required for M.W. wrote the paper. activity-dependent specification of the corresponding synapses (9). The authors declare no conflict of interest. Midbrain dopamine neurons project densely to the striatum This article is a PNAS Direct Submission. and are strongly involved in motor and cognitive functions (10, 1To whom correspondence should be addressed. Email: [email protected]. 11). Anatomically, dopamine synapses are frequently found on This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dendritic shafts and spines of GABAergic medium spiny neurons 1073/pnas.1514074113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1514074113 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Results microscopy for GABA and TH or VIAAT. According to previous Presynaptic Phenotypes at Dopamine Synapses. To characterize the studies (12, 13, 18), TH- or VIAAT-labeled terminals forming molecular–anatomical organization of the so-called dopamine symmetric synapses were judged to be dopaminergic or GABAergic, synapse, we studied the dorsolateral striatum in the adult mouse. respectively. Terminals that were unlabeled for either TH or We first examined the neurochemical properties of dopaminergic VIAAT and formed asymmetric synapses were judged to be terminals, which were identified by immunolabeling for plasma- glutamatergic, although a few asymmetric synapses are serotonergic lemmal dopamine transporter (DAT) or tyrosine hydroxylase (TH) (19). GABAergic terminals were intensely labeled for GABA (Fig. A B 1D, red), whereas dopaminergic (Fig. 1E, green) and glutamatergic (Fig. 1 and ). Triple immunofluorescence revealed that DAT- D labeled dopaminergic terminals overlapped extensively with TH (Fig. 1 , blue) terminals were scarcely labeled for GABA. Density and vesicular monoamine transporter-2 (VMAT2) (Fig. 1C). DAT- quantification revealed a significantly higher level of immunogold labeled dopaminergic terminals had no immunodetectable signals labeling for GABA in GABAergic terminals than in dopaminergic and glutamatergic terminals (Fig. 1F). Therefore, dopaminergic for glutamatergic terminal markers, vesicular glutamate trans- terminals contain very little, if any, GABA and GABAergic pro- porters VGluT1, VGluT2, and VGluT3, or for GABAergic ter- teins, except for GAT1. minal markers, 65/67-kDa glutamic acid decarboxylases (GAD) Presynaptic differentiation of dopaminergic terminals was tested or vesicular inhibitory amino acid transporter (VIAAT) (Fig. S1 A–E by expression of the active zone protein CAST and the presynaptic ). However, plasmalemmal GABA transporter GAT1 was adhesion molecule Nrxn. Immunofluorescence showed punctate + expressed in dopaminergic terminals, albeit at a much lower in- immunolabeling for CAST in DAT dopaminergic terminals (Fig. tensity than in VIAAT-labeled GABAergic terminals (Fig. S1 F H G S1 ). Immunoelectron microscopy revealed CAST expression and ). We then compared the GABA content in dopaminergic, beneath the presynaptic membrane of dopaminergic (Fig. 1G), GABAergic, and glutamatergic terminals using immunoelectron GABAergic (Fig. S1I), and glutamatergic (Fig. S1J)terminals.The density of immunogold labeling for CAST on the presynaptic membrane was comparable across the three types of terminals (Fig. 1H, open columns), and specificity
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