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A Model to Study NMDA Receptors in Early Nervous System Development

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A Model to Study NMDA Receptors in Early Nervous System Development Research Articles: Behavioral/Cognitive A Model to Study NMDA Receptors in Early Nervous System Development https://doi.org/10.1523/JNEUROSCI.3025-19.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.3025-19.2020 Received: 22 December 2019 Revised: 5 March 2020 Accepted: 12 March 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 Zoodsma et al. 1 A model to study NMDA receptors in early nervous system development 2 3 Josiah D. Zoodsma*1,3, Kelvin Chan*1,2,3, Ashwin A. Bhandiwad6, David Golann3, Guangmei 4 Liu4, Shoaib Syed3, Amalia Napoli1.3, Harold A. Burgess6, Howard I. Sirotkin§3, Lonnie P. 5 Wollmuth§3,4,5 6 1Graduate Program in Neuroscience, 7 2Medical Scientist Training Program (MSTP), 8 3Department of Neurobiology & Behavior, 9 4Department of Biochemistry & Cell Biology, 10 5Center for Nervous System Disorders, 11 Stony Brook University, Stony Brook, NY 11794-5230 12 6Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health 13 and Human Development, 14 Bethesda, MD, USA 15 *These authors contributed equally to this manuscript. 16 §Co-senior authors 17 18 Abbreviated title: NMDA receptors in zebrafish 19 Key Words: Zebrafish; CRISPR-Cas9; Ca2+ permeability; visual acuity; locomotor behavior; 20 prey-capture; habituation. 21 Acknowledgments: We thank Dr. Jane Cox for the grin1 probes; Donna Schmidt, Diane Henry- 22 Vanisko, Abby Frenkel and Julianna Casella for technical assistance; Stephanie Flanagan, Neal 23 Bhattacharji and the undergraduate fish room aides for fish care; and the Intramural Research 24 Program of the Eunice Kennedy Shriver National Institute for Child Health and Human 25 Development (HAB). 26 This work was supported by NIH Grants NRSA F30MH115618 (KC) and R01EY003821 27 (LPW), a Targeted Research Opportunity (TRO) Grant from Stony Brook University (LPW & 28 HIS), and a Hudson River Foundation Grant (HIS). 29 Author Contributions: J.D.Z., K.C., A.A.B, H.A.B, H.I.S., and L.P.W. designed the 30 experiments. J.D.Z., K.C., A.A.B, D.G., G.L. S.S., and A.N. performed the experiments. J.D.Z., 31 K.C., A.A.B., D.G., G.L., S.S., H.A.B, H.I.S., and L.P.W. analyzed the data. J.D.Z., K.C., 32 A.A.B, H.A.B, H.I.S., and L.P.W. wrote the paper. 33 Conflicts of Interest: We declare no financial conflicts of interest. 34 35 Address for correspondence: Dr. Lonnie P. Wollmuth 36 Depts. of Neurobiology & Behavior and Biochemistry & Cell 37 Biology 38 Center for Nervous System Disorders 39 Stony Brook University 40 Stony Brook, New York 11794-5230 41 Tel: (631) 632-4186 42 ORCID: 0002-8179-1259 43 E-mail: [email protected] 44 ABSTRACT 45 NMDA receptors (NMDARs) are glutamate-gated ion channels that play critical roles in 46 neuronal development and nervous system function. Here, we developed a model to study 47 NMDARs in early development in zebrafish, by generating CRISPR-mediated lesions in the 48 NMDAR genes, grin1a and grin1b, which encode the obligatory GluN1 subunits. While 49 receptors containing grin1a or grin1b show high Ca2+ permeability, like their mammalian 50 counterpart, grin1a is expressed earlier and more broadly in development than grin1b. Both 51 grin1a-/- and grin1b-/- zebrafish are viable. Unlike in rodents, where the grin1 knockout is 52 embryonic lethal, grin1 double mutant fish (grin1a-/-; grin1b-/-), which lack all NMDAR- 53 mediated synaptic transmission, survive until about 10 days post fertilization, providing a unique 54 opportunity to explore NMDAR function during development and in generating behaviors. Many 55 behavioral defects in the grin1 double mutant larvae, including abnormal evoked responses to 56 light and acoustic stimuli, prey capture deficits and a failure to habituate to acoustic stimuli, are 57 replicated by short-term treatment with the NMDAR antagonist MK-801, suggesting they arise 58 from acute effects of compromised NMDAR-mediated transmission. Other defects, however, 59 such as periods of hyperactivity and alterations in place preference, are not phenocopied by MK- 60 801, suggesting a developmental origin. Taken together, we have developed a unique model to 61 study NMDARs in the developing vertebrate nervous system. 62 2 63 SIGNIFICANCE 64 Rapid communication between cells in the nervous system depends on ion channels that are 65 directly activated by chemical neurotransmitters. One such ligand-gated ion channel, the N- 66 methyl-D-aspartate receptor (NMDAR), impacts nearly all forms of nervous system function. It 67 has been challenging, however, to study the prolonged absence of NMDARs in vertebrates, and 68 hence their role in nervous system development, due to experimental limitations. Here, we 69 demonstrate that zebrafish lacking all NMDAR transmission are viable through early 70 development and are capable of a wide range of stereotypic behaviors. As such, this zebrafish 71 model provides a unique opportunity to study the role of NMDAR in the development of the 72 early vertebrate nervous system. 73 74 75 3 76 INTRODUCTION 77 Nervous system function depends on the development of brain circuits that integrate appropriate 78 cell types and establish proper connectivity. In the vertebrate nervous system, glutamate is the 79 major excitatory neurotransmitter. At synapses that mediate rapid transmission, glutamate is 80 converted into biological signals by ligand-gated or ionotropic glutamate receptors (iGluRs), 81 including AMPA (AMPAR), kainate, and NMDA (NMDAR) receptor subtypes (Traynelis et al., 82 2010). Because of its unique signaling properties, including high Ca2+ permeability, NMDARs 83 are central to higher brain functions such as the plasticity underlining learning and memory 84 (Hunt & Castillo, 2012; Paoletti et al., 2013) and brain development (Cline & Haas, 2008; 85 Gambrill & Barria, 2011; Chakraborty et al., 2017). Due to the experimental limitations of 86 pharmacological manipulation, especially in studies viewing long-term outcomes as occurs for 87 many neurodevelopmental disorders, studying NMDARs in early circuit development is 88 challenging. Adding to this challenge, the loss of NMDAR subunits critical to early brain 89 development in murine models are embryonic lethal or die perinatally (Forrest et al., 1994; 90 Kutsuwada et al., 1996; Sprengel et al., 1998). 91 Zebrafish are a powerful model to study early nervous system development as fertilization is 92 external and larvae are transparent, which facilitates imaging of cell migration and circuit 93 formation. Furthermore, zebrafish larvae exhibit complex behaviors that provide simple and 94 robust readouts of higher nervous system function. Previous studies of NMDARs in zebrafish 95 have generally used acute pharmacological manipulations, mainly MK-801, a specific antagonist 96 of NMDARs (Huettner & Bean, 1988; Traynelis et al., 2010). These studies have highlighted the 97 central role of NMDARs in complex brain functions and behaviors including associative learning 98 (Sison & Gerlai, 2011b), social interactions (Seibt et al., 2011; Dreosti et al., 2015), place 4 99 preference (Swain et al., 2004), and responses to acoustic stimuli and prepulse inhibition 100 (Bergeron et al., 2015). Although these studies are invaluable, they do not reveal how brain or 101 circuit development impacts specific behaviors because acute antagonist applications only 102 change brain function for a short developmental timepoint and can have off-target effects. 103 NMDARs are heterotetramers composed of two obligate GluN1 subunits and typically two 104 GluN2 (A-D) subunits (Paoletti et al., 2013). In zebrafish, the obligatory GluN1 subunit is 105 encoded by two paralogous genes: grin1a and grin1b. The expression patterns of these genes 106 have only been described prior to 2 days post-fertilization (dpf) (Cox et al., 2005) and functional 107 differences between paralogues are unknown. Here, we studied the developmental expression of 108 grin1a and grin1b and the effect of their knockouts on early nervous system function. While 109 their encoded proteins, GluN1a and GluN1b, show high Ca2+ permeability, like their mammalian 110 counterparts, grin1a is expressed earlier in development and tends to be more widely expressed. 111 Nevertheless, both grin1a-/- and grin1b-/- fish are viable as adults. Unlike rodents, grin1 double 112 mutant fish (grin1a-/-; grin1b-/-), which lack all NMDAR-mediated synaptic transmission, survive 113 until about 10 dpf, which is old enough to assess the impact of NMDAR deficits on larval 114 behaviors. We took advantage of these fish to study the role of NMDARs in the development of 115 numerous complex behaviors, including prey capture, spontaneous and evoked movements, 116 habituation, and prepulse inhibition. 5 117 MATERIALS AND METHODS 118 Whole-cell electrophysiology 119 Human embryonic kidney 293 (HEK293) cells were grown in Dulbecco’s modified Eagle’s 120 medium (DMEM), supplemented with 10% FBS, for 24 h before transfection (Yelshansky et al., 121 2004; Alsaloum et al., 2016). Zebrafish cDNA constructs were synthesized from GenScript in a 122 pcDNA3.1(+)-p2A-eGFP vector. Zebrafish (zGluN1a/b) and rat (rGluN2A) NMDAR-encoding 123 cDNA constructs, were co-transfected into HEK293 cells along with a separate peGFP-Cl 124 construct at a ratio of 4:4:1 (N1:N2:eGFP) for whole-cell or macroscopic recordings using X- 125 tremeGene HP (Roche, 06-366). HEK293 cells were bathed in medium containing the GluN2 126 competitive antagonist DL-2-amino-5-phosphopentanoic acid (APV, 100 μM, Tocris) and Mg2+ 127 (100 μM). All experiments were performed 24-48 h post-transfection. 128 Whole-cell currents were recorded at room temperature (20–23°C) using an EPC-10 129 amplifier with PatchMaster software (HEKA), digitized at 10 kHz and low-pass filtered at 2.9 130 kHz (−3 dB) using an 8 pole low pass Bessel filter 4 (Yelshansky et al., 2004; Alsaloum et al., 131 2016).
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