This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Development/Plasticity/Repair Tonic activation of GluN2C/GluN2D-containing NMDA receptors by ambient glutamate facilitates cortical interneuron maturation Elizabeth Hanson1,2, Moritz Armbruster1, Lauren A. Lau1,2, Mary E. Sommer1, Zin-Juan Klaft1, Sharon A. Swanger3, Stephen F. Traynelis3, Stephen J. Moss1,4, Farzad Noubary5, Jayashree Chadchankar4 and Chris G. Dulla1 1Department of Neuroscience, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA 2Neuroscience Program, Tufts Sackler School of Biomedical Sciences, 136 Harrison Avenue, Boston, MA 02111, USA 3Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322, USA 4AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Tufts University School of Medicine, Boston, MA, 02111 5Department of Health Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA, USA. https://doi.org/10.1523/JNEUROSCI.1392-18.2019 Received: 31 May 2018 Revised: 29 January 2019 Accepted: 26 February 2019 Published: 7 March 2019 Author contributions: E.H., M.A., M.E.S., Z.-J.K., S.A.S., S.F.T., S.J.M., J.C., and C.G.D. designed research; E.H., M.A., L.L., M.E.S., Z.-J.K., S.A.S., J.C., and C.G.D. performed research; E.H., M.A., L.L., M.E.S., Z.- J.K., S.A.S., S.F.T., F.N., J.C., and C.G.D. analyzed data; E.H., M.A., S.F.T., and C.G.D. wrote the first draft of the paper; E.H., M.A., L.L., M.E.S., S.A.S., S.F.T., S.J.M., J.C., and C.G.D. edited the paper; E.H., M.A., L.L., M.E.S., Z.-J.K., S.A.S., S.F.T., S.J.M., J.C., and C.G.D. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by the Epilepsy Foundation (CD), National Institute of Neurological Disease and Stroke R01-NS076885 (CD), R56-NS076885 (CD), NS036554 (ST), Synapse Neurobiology Training Grant (EH, NINDS T32-NS061764, M. Jacob, P.I.), and Tufts Center for Neuroscience Research (P30 NS047243). We would like to thank Jennifer Shih, Thomas Papouin, Paul Rosenberg, Chinfei Chen, Christian Bjorbaek, Dan Cox, and John Lisman for their helpful comments. We would also like to thank Pedram Hamrah, Arisa Jamali, and Allen Parmelee for their help in establish a FACs protocol. Corresponding Author: Chris Dulla, [email protected], Tufts Sackler School of Biomedical Sciences, South Cove 203, 136 Harrison Avenue, Boston, MA 02111, 617-636-3418 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1392-18.2019 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 © 2019 the authors 1 Tonic activation of GluN2C/GluN2D-containing NMDA receptors by ambient glutamate 2 facilitates cortical interneuron maturation 3 4 Elizabeth Hanson1,2, Moritz Armbruster1, Lauren A. Lau1,2, Mary E. Sommer1, Zin-Juan Klaft1, 5 Sharon A. Swanger3, Stephen F. Traynelis3, Stephen J. Moss1,4, Farzad Noubary5, Jayashree 6 Chadchankar4, Chris G. Dulla1 7 8 9 1. Department of Neuroscience, Tufts University School of Medicine, 136 Harrison Avenue, 10 Boston, MA 02111, USA 11 2. Neuroscience Program, Tufts Sackler School of Biomedical Sciences, 136 Harrison 12 Avenue, Boston, MA 02111, USA 13 3. Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322, 14 USA 15 4. AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Tufts University 16 School of Medicine, Boston, MA, 02111 17 5. Department of Health Sciences, Bouvé College of Health Sciences, Northeastern 18 University, Boston, MA, USA. 19 20 21 Abbreviated Title: Ambient glutamate and interneuron maturation 22 23 Corresponding Author: 24 Chris Dulla 25 [email protected] 26 Tufts Sackler School of Biomedical Sciences 27 South Cove 203 28 136 Harrison Avenue, Boston, MA 02111 29 617-636-3418 30 31 Lead Contact: Chris Dulla 32 33 Number of pages: 41 34 Number of figures: 8 35 Number of words: Abstract, 249; Introduction, 646; Discussion, 1397 36 37 Conflict of interests: SFT is on the Scientific Advisory Board for Sage Therapeutics, and a co- 38 founder of NeurOp Inc. 39 40 Acknowledgements: This work was supported by the Epilepsy Foundation (CD), National 41 Institute of Neurological Disease and Stroke R01-NS076885 (CD), R56-NS076885 (CD), 42 NS036554 (ST), Synapse Neurobiology Training Grant (EH, NINDS T32-NS061764, M. Jacob, 43 P.I.), and Tufts Center for Neuroscience Research (P30 NS047243). We would like to thank 44 Jennifer Shih, Thomas Papouin, Paul Rosenberg, Chinfei Chen, Christian Bjorbaek, Dan Cox, 1 45 and John Lisman for their helpful comments. We would also like to thank Pedram Hamrah, Arisa 46 Jamali, and Allen Parmelee for their help in establish a FACs protocol. 2 47 ABSTRACT 48 49 Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and 50 environmental cues to construct inhibitory circuits during early post-natal development. 51 Disruption of these events can cause long-term changes in cortical inhibition and may be 52 involved in neurological disorders associated with inhibitory circuit dysfunction. We 53 hypothesized that tonic glutamate signaling in the neonatal cortex contributes to and is 54 necessary for the maturation of cortical interneurons. To test this hypothesis, we utilized mice of 55 both sexes to quantify extracellular glutamate concentrations in the cortex during development, 56 measure ambient glutamate-mediated activation of developing cortical interneurons, and 57 manipulate tonic glutamate signaling using subtype-specific NMDA receptor antagonists in vitro 58 and in vivo. We report that ambient glutamate levels are high (≈100 nM) in the neonatal cortex 59 and decrease (to ≈50 nM) during the first weeks of life, coincident with increases in astrocytic 60 glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin (PV)- 61 positive interneurons in the cortex (identified using G42:GAD1-eGFP reporter mice) exhibit a 62 transient, tonic NMDA current at the end of the first post-natal week. GluN2C/GluN2D- 63 containing NMDA receptors mediate the majority of this current, and contribute to the resting 64 membrane potential and intrinsic properties of developing putative PV interneurons. 65 Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptors in vivo during the 66 period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron 67 morphological complexity and causes deficits in cortical inhibition later in life. These results 68 demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron 69 maturation via tonic activation of GluN2C/GluN2D-containing NMDA receptors. 70 71 72 3 73 SIGNIFICANCE STATEMENT 74 75 Inhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to 76 controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple 77 neurological disorders, including epilepsy. Establishing inhibitory cortical networks requires in 78 utero proliferation, differentiation, and migration of immature GABAergic interneurons, and 79 subsequent post-natal morphological maturation and circuit integration. Here, we demonstrate 80 that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive 81 GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this 82 activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks 83 exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of 84 developing cortical interneurons by ambient glutamate is critically important for establishing 85 normal cortical inhibition. 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 4 113 114 115 INTRODUCTION 116 117 Inhibitory GABAefrgic interneurons (INs) control cortical function by constraining local neuronal 118 activity and orchestrating circuit synchronization (Hensch et al., 1998; Buzsáki and Draguhn, 119 2004; Markram et al., 2004; Cardin et al., 2009). During embryonic development, cells destined 120 to become cortical INs are generated in the medial and caudal ganglionic eminences (MGE and 121 CGE) (Xu et al., 2004). These newborn INs migrate to the cortex, before radially migrating into 122 the cortical lamina (Angevine and Sidman, 1961; Berry and Rogers, 1965). Once in the cortex, a 123 combination of genetic programs and environmental cues drive interneuron localization, 124 morphological differentiation, and synaptic connectivity (Huang et al., 1999; Levitt et al., 2004; 125 Flames and Marín, 2005; BatistaǦBrito and Fishell, 2009; Bortone and Polleux, 2009). 126 GABAergic synaptic activity is established prior to glutamatergic, and GABA is excitatory in 127 early development due to developmentally specific expression of chloride transporters (Kaila et 128 al., 2014). This engenders GABAergic INs with the ability to drive spontaneous developmental 129 network activity (Kasyanov et al., 2004; Allene et al., 2008; Minlebaev et al., 2011), which is 130 critical to establishing synaptic properties, local microcircuits, and long-range neuronal 131 connections. The underlying developmental events that shape interneuron activity and 132