bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. NMDAR-mediated transcriptional control of gene expression during the development of medial ganglionic eminence-derived interneurons a b c a a Vivek Mahadevan , Apratim Mitra , Yajun Zhang , Xiaoqing Yuan , Areg Peltekian , Ramesh a b d c a Chittajallu , Caroline Esnault , Dragan Maric , Christopher Rhodes , Kenneth A. Pelkey , Ryan b c a,∗ Dale , Timothy J. Petros , Chris J. McBain aSection on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), Bethesda, 20892, MD, USA bBioinformatics and Scientific Programming Core, NICHD, Bethesda, 20892, MD, USA cUnit on Cellular and Molecular Neurodevelopment, NICHD, Bethesda, 20892, MD, USA dFlow and Imaging Cytometry Core Facility, National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, 20852, MD, USA ABSTRACT Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+ and Neurogli- aform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical and physiological properties. However, the molecular mechanisms regulating their diversity remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates subtype-specific transcriptional regulation of gene ex- pression in MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific conditional Grin1 loss results in a systemic downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders, particularly schizophre- nia. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders. Keywords: Interneurons, Medial ganglionic eminence, PV, SST, Neurogliaform, NMDA receptor, Transcriptional regulation, Neurodevelopmental disorders, Schizophrenia, NMDA-hypofunction, Hippocampus, Frontal Cortex, Mouse model, scRNAseq 1 1. INTRODUCTION entire forebrain accounting for approximately 6 60% of all cortical interneurons [1, 2]. In ad- 7 2 Medial ganglionic eminence (MGE)-derived dition, approximately half of all hippocampal 8 3 forebrain GABAergic interneurons comprise the neurogliaform-type cells (NGFCs), the so called 9 4 parvalbumin-containing (PV) and somatostatin- Ivy cells, originate from the MGE [3, 4]. In- 10 5 containing (SST) subpopulations throughout the terestingly, though only rarely found in rodent 11 neocortex such MGE-derived NGFCs are sig- 12 ∗Corresponding author. nificantly more populous in primate neocortex, 13 Email addresses: [email protected] (Vivek Mahadevan), [email protected] (Ryan Dale), including humans [5]. While PV neurons exert ro- 14 [email protected] (Timothy J. Petros), bust somatic, and proximal dendritic inhibition, 15 [email protected] (Chris J. McBain) Mahadevan et al., 2021 1/49 NMDAR-mediated control of gene expression in MGE-derived interneurons bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 16 the SST and NGFCs mediate domain-specific resembles global Grin1 -mutants [32] in their con- 63 17 dendritic inhibition on their downstream pyrami- stellation of schizophrenia-like behavioral aber- 64 18 dal neuron targets [6]. Collectively these classes rations. This indicates that Grin1 dysfunction 65 19 of interneurons shape diverse aspects of corti- across multiple interneuron-subtypes precipitates 66 20 cal and hippocampal circuit maturation during schizophrenia-like abnormalities [33]. In addition, 67 21 development, and critically regulate information this interneuron-specific NMDAR-hypofunction 68 22 processing in mature circuits by maintaining ap- model is sensitive to developmental age, since 69 23 propriate excitation-inhibition (E-I) balance [7]. adult-onset Grin1 loss does not result in the same 70 24 Recent evidence indicates a critical role for activ- phenotypes [29]. Despite the importance of devel- 71 25 ity, particularly through ionotropic glutamate opmental NMDAR function in interneurons, and 72 26 receptors (iGluRs), in driving the morpho- its relevance to human neurodevelopmental disor- 73 27 physiological maturation of MGE-derived in- ders, a comprehensive interrogation of the impact 74 28 terneurons [8–12]. Unlike mature interneurons of developmental NMDAR ablation in interneu- 75 29 where iGluRs differentially contribute towards rons, particularly across MGE-derived interneu- 76 30 synaptic transmission, immature and migrating rons, is lacking. 77 31 interneurons express different glutamate receptor It is clear that during the developmental win- 78 32 subunits including the NMDA-type iGluR (NM- dow between embryonic day (ED) 13.5 and post- 79 33 DAR) and AMPA/Kainate-type iGluR (AM- natal day (PD) ~10 [34], a combination of innate 80 34 PAR/KAR) [13–15] prior to the expression of genetic programs, external environment, and neu- 81 35 any functional synapses. This becomes particu- ronal activity shapes interneuron subtype spec- 82 36 larly important as the developing brain contains ification leading to remarkable diversity [2, 21, 83 37 higher ambient glutamate levels than the adult 35, 36] The NMDAR signaling complex comprises 84 38 brain [16]. Collectively, higher ambient gluta- an essential node for regulating gene expression 85 39 mate, developmental expression of iGluRs and via excitation-transcription (E-T) coupling in ma- 86 40 recruitment of glutamatergic signaling is con- ture circuits [37–39]. Moreover, different NMDAR 87 41 sidered to be trophic [8, 17, 18] and thought to subunits are widely expressed in the developing 88 42 engage mechanisms to regulate various aspects of brain [40] where they provide a critical source of 89 2+ 43 interneuron development including morphological Ca -entry via trophic glutamate signaling prior 90 44 and electrical maturation to promote appropriate to synaptogenesis [15, 19, 41]. However, it is not 91 2+ 45 circuit integration [9, 11, 14, 16, 19–22]. clear whether the NMDAR-mediated Ca cas- 92 46 Interneuron-specific impairments are increas- cades in nascent and developing MGE-derived in- 93 47 ingly considered central to the etiology of multi- terneurons engage transcriptional programs nec- 94 48 ple neurodevelopmental and circuit disorders [23]. essary for MGE-derived interneuron diversity. To 95 49 The importance of interneuron-expressed iGluRs investigate this, we conditionally deleted Grin1 96 50 is most notable in psychiatric disorders exhibiting in MGE progenitors that give rise to cortical and 97 51 impaired NMDAR-associated systems [24, 25]. In hippocampal PV, SST, and NGFC subsets, us- 98 52 the adult brain, acute pharmacological NMDAR ing the Nkx2-1-Cre mouse line [3, 4, 42]. In 99 53 blockade results in circuit disinhibition and psy- this model, Nkx2-1 -driven Cre expression is re- 100 54 chotic symptoms [26], mediated in-part, by the ported in cycling/proliferating MGE cells, well 101 55 enhanced sensitivity of interneuronal NMDARs to before the cells become postmitotic, allowing for 102 56 their antagonists [27]. Indeed, direct blockade of assessment of the developmental impact of em- 103 57 interneuron activity also precipitates distinct be- bryonic loss of Grin1 activity across all subsets of 104 58 havioral deficits relevant to schizophrenia [28]. In MGE-derived interneurons. Applying a combina- 105 59 particular, ablation of the obligate NMDAR sub- tion of high-throughput single-cell RNA sequenc- 106 60 unit gene Grin1 in interneuron-specific early post- ing (scRNAseq), MGE-interneuron-specific ribo- 107 61 natal mouse [29], but not PV-specific [30], or glu- somal tagging, and quantitative immunostaining 108 62 tamatergic neuron-specific Grin1 ablation [31], and in situ RNAscope analyses we establish that 109 Mahadevan et al., 2021 2/49 NMDAR-mediated control of gene expression in MGE-derived interneurons bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 110 NMDAR-mediated transcriptional cascades reg- markers Pvalb, Sst, and Lamp5, marking PV and 155 111 ulate MGE subtype abundances, by regulating SST, NGFC subsets respectively (Figure1B, 156 112 the expression of diverse transcriptional, synap- Figure1-Supplement3A). While we did not 157 113 togenic and membrane excitability genetic pro- recover cells expressing the CGE-markers Prox1, 158 114 grams. Notably, we identify numerous disease- Htr3a or Vip, we recovered a minor fraction of 159 115