Extrasynaptic NMDA Receptors on Rod Pathway Amacrine Cells: Molecular Composition, Activation, and Signaling

This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular Extrasynaptic NMDA receptors on rod pathway amacrine cells: molecular composition, activation, and signaling Margaret L. Veruki1, Yifan Zhou1, Áurea Castilho1, Catherine W. Morgans2 and Espen Hartveit1 1University of Bergen, Department of Biomedicine, Bergen, Norway. 2Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR, USA. https://doi.org/10.1523/JNEUROSCI.2267-18.2018 Received: 10 August 2018 Revised: 29 October 2018 Accepted: 14 November 2018 Published: 20 November 2018 Author contributions: M.L.V., C.W.M., and E.H. designed research; M.L.V., Y.Z., x.C., and E.H. performed research; M.L.V., Y.Z., x.C., and E.H. analyzed data; M.L.V. and E.H. wrote the first draft of the paper; M.L.V., Y.Z., x.C., C.W.M., and E.H. edited the paper; M.L.V. and E.H. wrote the paper; E.H. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. Financial support from The Research Council of Norway, grant numbers 213776 and 261914 (M.L.V.) and 182743, 189662 and 214216 (E.H.) and the Faculty of Medicine at the University of Bergen is gratefully acknowledged. A research stay of C.W.M. at the University of Bergen was supported by a SPIRE visiting researcher grant (M.L.V.). We thank Torhild Sunde for excellent technical assistance. Confocal imaging was performed at the Molecular Imaging Center (MIC) and was supported by the Department of Biomedicine and the Faculty of Medicine at the University of Bergen. Corresponding authors: Margaret L. Veruki and Espen Hartveit, University of Bergen, Department of Biomedicine, Jonas Lies vei 91, N-5009 Bergen, Norway. [email protected]; [email protected] Cite as: J. Neurosci 2018; 10.1523/JNEUROSCI.2267-18.2018 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Extrasynaptic NMDA receptors on rod pathway amacrine cells: molecular composition, 2 activation, and signaling 3 4 Margaret L. Veruki1, Yifan Zhou1, Áurea Castilho1, Catherine W. Morgans2, Espen Hartveit1 5 1University of Bergen, Department of Biomedicine, Bergen, Norway. 6 2Department of Physiology and Pharmacology, Oregon Health and Science University, 7 Portland, OR, USA. 8 9 10 Corresponding authors: Margaret L. Veruki and Espen Hartveit, University of Bergen, 11 Department of Biomedicine, Jonas Lies vei 91, N-5009 Bergen, Norway. 12 [email protected]; [email protected] 13 14 Abbreviated title: NMDA receptors on AII and A17 amacrine cells 15 16 Number of pages: 65. Number of figures: 14. Number of tables: 1 17 Number of words: Abstract 250, Introduction 650, Discussion 1499. 18 Conflict of interest: The authors declare no competing financial interests. 19 20 Acknowledgments 21 Financial support from The Research Council of Norway, grant numbers 213776 and 261914 22 (M.L.V.) and 182743, 189662 and 214216 (E.H.) and the Faculty of Medicine at the University 23 of Bergen is gratefully acknowledged. A research stay of C.W.M. at the University of Bergen 24 was supported by a SPIRE visiting researcher grant (M.L.V.). We thank Torhild Sunde for 25 excellent technical assistance. Confocal imaging was performed at the Molecular Imaging 26 Center (MIC) and was supported by the Department of Biomedicine and the Faculty of 27 Medicine at the University of Bergen. 28 Abstract 29 In the rod pathway of the mammalian retina, axon terminals of glutamatergic rod bipolar 30 cells are presynaptic to AII and A17 amacrine cells in the inner plexiform layer. Recent 31 evidence suggests that both amacrines express NMDA receptors, raising questions 32 concerning molecular composition, localization, activation, and function of these receptors. 33 Using dual patch-clamp recording from synaptically connected rod bipolar and AII or A17 34 amacrine cells in retinal slices from female rats, we found no evidence that NMDA receptors 35 contribute to postsynaptic currents evoked in either amacrine. Instead, NMDA receptors on 36 both amacrine cells were activated by ambient glutamate and blocking glutamate uptake 37 increased their level of activation. NMDA receptor activation also increased the frequency of 38 GABAergic postsynaptic currents in rod bipolar cells, suggesting NMDA receptors can drive 39 release of GABA from A17 amacrines. A striking dichotomy was revealed by 40 pharmacological and immunolabeling experiments, which found GluN2B-containing 41 NMDA receptors on AII amacrines and GluN2A-containing NMDA receptors on A17 42 amacrines. Immunolabeling also revealed a clustered organization of NMDA receptors on 43 both amacrines and a close spatial association between GluN2B subunits and connexin 36 on 44 AII amacrines, suggesting that NMDA receptor modulation of gap junction coupling 45 between these cells involves the GluN2B subunit. Using multi-photon Ca2+ imaging, we 46 verified that activation of NMDA receptors evoked an increase of intracellular Ca2+ in 47 dendrites of both amacrines. Our results suggest that AII and A17 amacrines express 48 clustered, extrasynaptic NMDA receptors, with different and complementary subunits that 49 are likely to contribute differentially to signal processing and plasticity. 50 Significance statement 51 Glutamate is the most important excitatory neurotransmitter in the central nervous system, 52 but not all glutamate receptors transmit fast excitatory signals at synapses. NMDA-type 53 glutamate receptors act as voltage- and ligand-gated ion channels, with functional properties 54 determined by their specific subunit composition. These receptors can be found at both 55 synaptic and extrasynaptic sites on neurons, but the role of extrasynaptic NMDA receptors is 56 unclear. Here, we demonstrate that retinal AII and A17 amacrine cells, postsynaptic partners 57 at rod bipolar dyad synapses, express extrasynaptic (but not synaptic) NMDA receptors, 58 with different and complementary GluN2 subunits. The localization of GluN2A-containing 59 receptors to A17s and GluN2B-containing receptors to AIIs suggests a mechanism for 60 differential modulation of excitability and signaling in this retinal microcircuit. 3 3 61 Introduction 62 When neurotransmitter molecules are released from a neuron, they can bind to and activate 63 heterogeneous populations of ligand-gated ion channels, typically formed by different 64 combinations of receptor subunits. This receptor heterogeneity can be expressed within a 65 single postsynaptic density, at different synapses received by a single neuron, and at 66 synapses made by the same neuron with different postsynaptic targets (Shepherd and 67 Grillner, 2018). Within a receptor family, individual receptors can differ in agonist affinity, 68 single-channel conductance, kinetics, selectivity and permeability for specific ions, and 69 influence on downstream signaling pathways, thus, the subunit composition of a given 70 receptor channel is of critical importance for the specific functional and signaling properties 71 (Traynelis et al., 2010; Smart and Paoletti, 2012). Considerable attention has therefore been 72 devoted to investigate how expression and regulation of receptors and receptor subunits are 73 adapted to and contribute to the specific signal processing taking place in different 74 microcircuits and networks. 75 The microcircuit formed in the mammalian retina by the rod bipolar cell and its dyad 76 postsynaptic partners, the AII and A17 amacrine cells, is structurally and functionally well 77 defined, plays a crucial role in visual processing (reviewed by Demb and Singer, 2012), and 78 has been identified as a target for molecular pathologies in early diabetes mellitus (Castilho 79 et al., 2015; Moore-Dotson et al., 2016). The cellular elements of this microcircuit are easily 80 identifiable in slice preparations, and, as such, the microcircuit provides a unique model to 81 investigate neurotransmitter interactions. Glutamate is released from axon terminals of rod 82 bipolar cells at specialized active zones called ribbons, with each ribbon typically 83 presynaptic to one AII process and one A17 process (Strettoi et al., 1990). AII amacrines 84 provide outputs to ON- and OFF-cone bipolar cells, via electrical and chemical (glycinergic) 85 synapses, respectively (Strettoi et al., 1992, 1994), whereas A17 amacrines provide 86 GABAergic feedback to the axon terminals of rod bipolar cells from which they receive input 87 (reciprocal synapses; reviewed by Diamond and Grimes, 2014). Glutamate from rod bipolar 4 4 88 cells activates non-NMDA-type glutamate receptors on both AII and A17 amacrines 89 (Hartveit, 1999; Veruki et al., 2003; Singer and Diamond, 2003; Chávez et al., 2006) and it was 90 originally assumed that these cells did not express NMDA receptors (Boos et al., 1993; 91 Menger and Wässle, 2000). There is increasing evidence, however, that both AII and A17 92 amacrines express functional NMDA receptors (Hartveit and Veruki, 1997; Zhou and 93 Dacheux, 2004; Kothmann et al., 2012; Zhou et al., 2016), raising questions concerning the 94 molecular identity, localization, and function of these receptors. NMDA receptors, 95 comprised of obligatory GluN1 subunits and either GluN2 or, less commonly,

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