Articles in PresS. Am J Physiol Cell Physiol (February 4, 2015). doi:10.1152/ajpcell.00353.2014
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A Neuroprotective Role of the NMDA Receptor Subunit GluN3A (NR3A) in
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Ischemic Stroke of the Adult Mouse
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Jin Hwan Lee1, Zheng Z. Wei1,2, Dongdong Chen1,2, Xiaohuan Gu1,2, Ling Wei1,2
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and Shan Ping Yu1,2
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Departments of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322; 2
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Atlanta VA Medical Center, Center for Center for Visual
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and Neurocognitive Rehabilitation, Decatur, GA 30033
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Running title: Neuroprotective role of GluN3A in adult ischemic stroke
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Corresponding author
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Shan Ping Yu, M.D., Ph.D.
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101 Woodruff Circle
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Woodruff Memorial Research Building, Suite 620
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Emory University School of Medicine
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Atlanta, GA 30322
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E-mail: [email protected]
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Tel. 404-712-8678
1 Copyright © 2015 by the American Physiological Society.
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Abstract
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GluN3A or NR3A is a developmentally regulated N-methyl-D-aspartate receptor
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(NMDAR) subunit, showing a unique inhibitory role that decreases NMDAR current and
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the receptor mediated Ca2+ influx. In the neonatal brain, GluN3A is shown to associate
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with synaptic maturation, spine formation, and plays a neuroprotective role. Its functional
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role in the adult brain, however, is largely unknown. We tested the hypothesis that,
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disrespecting the relatively lower expression level of GluN3A in the adult brain, this
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inhibitory NMDAR subunit shows a protective action against ischemia-induced brain
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injury. In littermate wild type (WT) and GluN3A knockout (KO) mice, focal cerebral
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ischemia was induced by permanent occlusion of right distal branches of the middle
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cerebral artery (MCA) plus 10 min ligation of both common carotid arteries (CCAs).
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Twenty-four hrs after focal cerebral ischemia, the infarction volume assessed using 2,3,5-
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triphenyltetrazolium chloride (TTC) staining was significantly larger in GluN3A KO
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mice compared to WT mice. Terminal deoxynucleotidyl transferase dUTP nick end
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labeling (TUNEL) staining demonstrated enhanced cell death in GluN3A KO mice.
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Moreover, the deletion of GluN3A hindered sensorimotor functional recovery after stroke.
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It is suggested that, although the expression level is relatively lower in the adult brain,
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GluN3A is still a noteworthy regulator in ischemia-induced excitotoxicity and brain
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injury.
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Key words: Stroke, NMDA receptors, GluN3A, Adult brain, Functional recovery
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Abbreviations
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NMDA: N-methyl-D-aspartate
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NMDAR: NMDA receptor
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MCA: middle cerebral artery
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CCAs: common carotid arteries
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WT: wild type
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KO: knockout
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GluN1: glutamate NMDA receptor 1 (NR1)
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GluN2: glutamate NMDA receptor 2A (NR2A)
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GluN2B: glutamate NMDA receptor 2B (NR2B)
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GluN3A: glutamate NMDA receptor 3A (NR3A)
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GluN3B: glutamate NMDA receptor 3B (NR3B)
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TTC: 2,3,5-triphenyltetrazolium chloride
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TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling
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Introduction The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves
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critical roles in physiological and pathological processes in the central nervous system
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(CNS) (7, 9, 17, 20). The receptor activity regulates fast excitatory neurotransmission,
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contribute to the regulation of neuronal gene expression and play key roles in the
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formation of neural networks and the formation of long-term potentiation (LTP) and
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memory (18). NMDAR is composed of proteins derived from the GluR1 (NR1) family
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and the GluN2 (NR2) family of genes. The GluN1 family has a variety of splice variants,
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and GluN2 family is comprised of four subunit classes, GluN2A, GluN2B, GluN2C and
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GluN2D. The gene expression of both families shows temporal and spatial distributions
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in the brain (13, 15, 22). NMDAR may also contain an inhibitory subunit, GluN3A
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(NR3A, NMDAR-L, or chi-1) and GluN3B (NR3B) (2, 6, 19, 26). Incorporation of
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GluN3A with GluN1/GluN2 subunits suppresses NMDAR activity. For example, co-
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expression of GluN3A results in decreased NMDAR single channel conductance, Ca2+
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permeability and the receptor Mg2+ sensitivity, which may limit synaptic strengthening
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and stabilization (3, 6, 8, 25, 26). On the other hand, genetic knockout (KO) of GluN3A
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in mice results in enhanced NMDA currents and increased dendritic spines in early
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postnatal cerebrocortical neurons compared to wild type (WT) mice (8). In addition,
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increases of intracellular Ca2+ concentration in the neuronal cells from GluN3A KO mice
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contribute to cell death (3, 5, 14).
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It is well known that GluN3A is a developmentally regulated NMDAR subunit.
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GluN3A mRNA and protein levels peak at the end of the first postnatal week but
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decreases to a low level by adulthood (6, 26). Based on these findings, studies on
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GluN3A have been almost exclusively focused on the developing brain of neonates (26,
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30). In a hypoxic-ischemic stroke model of neonatal mice, deletion of GluN3A caused
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enlarged infarct formation while over-expression of GluN3A decreased the brain damage
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(24). On the other hand, the significance of endogenous GluN3A in the adult brain has
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been largely ignored, most likely due to the fact that the GluN3A level is down-regulated
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in the adult brain and due to the assumption that the low level GluN3A has no or little
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functional role. The pathological role of GluN3A in the adult brain, ironically, has never
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been directly tested and no quantified experimental data from adult animals has been
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reported before.
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We recently demonstrated that even though the GluN3A expression level declines
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to lower levels from the neonatal period to the adulthood, significant protein levels of
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GluN3A are still detectable in the adult brain (21). An important role of GluN3A in
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locomotion, pain perception, and cognitive functions was observed in adult animals (21).
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The results indicate that even if at relatively lower expression level, GluN3A still can act
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as an imperative player in the adult brain. In the present investigation, we extended the
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examination into the pathological condition of ischemic stroke and tested the hypothesis
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that a role of GluN3A in the adult stroke brain might have been overlooked before. We
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also hypothesized that a neuroprotective role of GluN3A in the adult brain could indicate
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a receptor subunit target for the development of therapeutic treatment of stroke.
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Methods
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Animals
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NMDA receptor GluN3A (NR3A) constitutive knockout (KO) mice (kindly
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provided by Dr. Stuart A. Lipton and Dr. Nobuki Nakanishi at Stanford University) and
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littermate wild type (WT) mice at different postnatal days were used in the experiments.
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The animals were maintained at a constant temperature of 21°C with a 12 hrs light/dark
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cycle in the Laboratory Animal Center for Research, Medicine School of South Carolina.
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All experimental and surgical procedures were approved by the Institutional Animal Care
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and Use Committee (IACUC) at Emory University (Animal protocol number: 208-2008).
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No human tissue or sample was tested in this study.
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Genotyping The Genotyping of GluN3A KO and WT animals was performed as described
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previously (21). DNA for genotyping was extracted from tail snips (approximately 2-4
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mm). The separately primers were 5’-CCA CGG TGA GCT TGG GGA AG (NN494),
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5’-GCC TGA AGA ACG AGA TCA GC (NN506) and 5’-TTG GGG AGC GCC CTG
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CAT GG (NN507) (Sigma-Aldrich, St. Louis, MO, USA). The 251 bp product (NN494
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and NN506) confutes GluN3A knockout mice and 200 bp product (NN494 and NN507)
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confutes WT mice. DNA (2 µl) was amplified on a thermocycler (MJ minim, Personal
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Thermal Cycler, Bio-Rad, CA, USA) for 40 cycles (95°C for 60 sec, 58°C for 30 sec,
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72°C for 60 sec). After additional incubation at 72°C for 10 min and transferred to 4°C,
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PCR products were subjected to electrophoresis in 1.5% agarose gel with ethidium
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bromide. Relative intensity of PCR bands was analyzed using the InGenius3 manual gel
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documentation system (Syngene, Frederick, MD, USA).
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Neuronal cell cultures
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The preparation of nearly pure cortical neurons was previously described (31).
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Briefly, neurons were harvested from the cortex of gestational 14–16 day mouse embryos.
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The pregnant mouse was sacrificed using overdosed isoflurane and brain dissection was
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performed soon after heartbeat stopped and no response to pawn stimuli. The cortex was
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dissociated in trypsin and plated on poly-D-lysine and laminin coated tissue culture
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dishes. Cells were maintained in neurobasal media (Invitrogen, Carlsbad, CA) with B27
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supplement and L-glutamine until the time of the experiment. Cytarabine (Ara-C) was
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added on the third day to prevent the proliferation of glial cells. Mature neuronal cultures
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(12-14 days in vitro (DIV)) were used for cell death assays.
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NMDA toxicity and assessment of neuronal cell death Excitotoxicity was induced by 10 min exposure to NMDA (300 µM) alone or in
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the presence of z-VAD-fmk (100 µM), carried out at room temperature in a HEPES
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buffer solution (HBSS) containing (in mM): NaCl, 120; KCl, 5.4; MgCl2, 0.8; CaCl2,
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1.8; HEPES, 20; glucose, 15; glycine, 0.01 (pH 7.4). The exposure solution was then
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washed away and replaced by media stock (MS) supplemented with 10 µM glycine
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before returning cultures to the incubator for 20-24 hrs.
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Neuronal cell death was estimated by examination of the cultures under phase-
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contrast microscopy, and quantitated by measurement of lactate dehydrogenase (LDH)
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release by damaged cells into the bathing medium 24 hrs following the experimentation
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(14). The LDH level corresponding to near-complete neuron death (without glial death)
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was determined by assaying sister cultures exposed to 300 µM NMDA for 24 hrs in MS
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supplemented with glycine. The percent neuronal death was normalized to the mean LDH
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value released 24 hrs after a sham wash (defined as 0%) and continuous exposure to 300
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µM NMDA (defined as 100%). Background LDH levels were determined in sister
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cultures subjected to sham washing and subtracted from experimental values to yield the
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signal specific to experimentally induced injury.
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Focal cerebral ischemia in mice Focal cerebral ischemia, confined to the sensorimotor cortex in the right middle
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cerebral artery (MCA) territory of WT and GluN3A KO male mice were induced by
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permanent occlusions of distal branches of the right MCA and temporary (10 min)
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ligations of both common carotid arteries (CCAs) (29). In brief, 8–10 week old male
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mice (25-28 g) were anesthetized by 4% chloral hydrate (100 mg/kg, i.p.). Chloral
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hydrate has been widely used as a sedation agent in human and animal
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surgeries/procedures for a number of years. It was chosen in this investigation due to its
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stable and well controlled anesthetic action and negligible effect on NMDA receptors (4).
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The right MCA branches were permanently ligated using a 10-0 suture (Surgical
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Specialties Co., Reading, PA) accompanied by a bilateral 10-min ligation of CCAs. Body
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temperature was monitored during surgery and maintained at 37.0 0.3°C using a
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temperature control unit and heating pads. The mortality rate due to ischemic surgery
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and/or anesthesia failure was around 7% in these experiments. Free access to food and
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water was allowed after recovery from anesthesia. All mice were kept in air-ventilated
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cages with room temperature maintained at 24 0.5°C. Animals were euthanized and
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decapitated at different time points after ischemic stroke. Brains were immediately
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removed, mounted in optimal cutting temperature compound (Sakura Finetek USA, Inc.,
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Torrance, CA) and stored at −80°C for further processing.
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TTC staining of infarct volume measurement Twenty-four hrs after onset of MCAO, animals in different groups were sacrificed
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for assessment of brain infarct formation. 2,3,5-triphenyltetrazolium chloride (TTC;
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Sigma-Aldrich) staining was used to reveal damaged/dead brain tissue as previously
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described (29). Brains were removed and placed in a brain matrix then sliced into 1-mm
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coronal sections. Slices were incubated in 2% TTC solution at 37°C for 5 min, then
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stored in 10% buffered formalin for 24 hrs. Digital images of the caudal aspect of each
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slice were obtained by a flatbed scanner. Infarct, ipsilateral hemisphere, and contralateral
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hemisphere areas were measured using ImageJ software (NIH, Bethasda, MD, USA). The
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indirect method (subtraction of residual right hemisphere cortical volume from cortical
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volume of the intact left hemisphere) will be used for infarct volumes calculation. Infarct
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measurements were performed under double-blind conditions.
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Western blot analysis Western blot analysis was used to detect the expression of NMDAR subunits.
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Brain cortical tissue was lysed in a lysis buffer containing 0.02 M Na4P2O7, 10 mM Tris-
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HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton, 1 mM EGTA, 2 mM
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Na3VO4, and a protease inhibitor cocktail (Sigma-Aldrich). The supernatant was
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collected after centrifugation at 15,000 g for 10 min at 4°C. Protein concentration was
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determined with a bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL, USA).
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Equivalent amounts of total protein were separated by molecular weight on an SDS-
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polyacrylamide gradient gel, and then transferred to a PVDF membrane. The blot was
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incubated in 5% bovine serum albumin for at least 1 hr and then reacted with primary
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antibodies at 4°C for overnight. The primary antibodies used in this investigation
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included: and the dilutions for each were rabbit anti-GluN2A antibody (Cell Signaling,
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Danvers, MA, USA) at 1:2000, rabbit anti-GluN2B antibody (Cell Signaling) at 1:2000,
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rabbit anti-GluN3A antibody (Abcam, Cambridge, MA, USA) at 1:1000, and mouse anti-
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-actin (Sigma-Aldrich) at 1:5000 (Supporting Table 1). After washing with Tris-
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buffered saline with Tween (TBST), membranes were incubated with AP-conjugated or
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HRP-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA) for 1-2 hrs
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at room temperature. After final washing with TBST, the signals were detected with
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bromochloroidolylphosphate/nitroblue tetrazolium (BCIP/NBP) solution (Sigma-Aldrich)
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or film. Signal intensity was measured by ImageJ and normalized to the actin signal
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intensity.
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Immunohistochemical assessment Frozen brain tissues were sliced into 10 µm-thick coronal sections using a
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cryostat vibratome (Leica CM 1950; Leica Microsystems, Buffalo Grove, IL, USA).
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Sections were dried on the slide warmer for 30 min, fixed with 10% formalin buffer,
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washed with -20°C precooled ethanol: acetic acid (2:1) solution for 10 min, and finally
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permeabilized with 0.2% Triton-X 100 solution for 5 min. All slides were washed 3 times
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with PBS (5 min each) after each step. Then, tissue sections were blocked with 1% fish
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gelatin (Sigma-Aldrich) in PBS for 1 hr at room temperature, and subsequently incubated
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with the primary antibody: mouse anti-GluN1 antibody (1:200; Life Technologies, Grand
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Island, NY, USA), rabbit anti-GluN2A antibody (1:200; Abcam), rabbit anti-GluN2B
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(1:200; Abcam), rabbit anti-GluN3A (1:200; Abcam), and mouse anti-NeuN (1:400;
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Millipore, Billerica, MA, USA) overnight at 4°C (Supporting Table 1). Next day, the
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slides were washed 3 times with PBS for 5 min, then reacted with the secondary
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antibodies Alexa Fluor®488 goat anti-mouse or rabbit (1:300; Life Technologies) and
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Cy3-conjugated donkey anti-rabbit (1:300; Jackson ImmunoResearch Laboratories, West
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Grove, PA, USA) or Cy5-conjugated donkey anti-mouse or rabbit (1:400; Jackson
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ImmunoResearch Laboratories) for 80 min at room temperature. After 3 washes with
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PBS, nuclei were stained with Hoechst 33342 (1:20,000; Life Technologies) for 5 min as
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a counterstain. And then the brain sections were mounted, coverslipped, imaged, and
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photographed under a fluorescent microscope (BX51, Olympus, Japan) and laser
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scanning confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA).
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TUNEL staining and positive cell quantification
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A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
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kit was used to examine cell death by detecting fragmented DNA in 10-µm-thick coronal
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fresh frozen sections as described previously (n=8 per group) (16). After fixation (10%
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buffered formalin for 10 min and then ethanol:acetic acid (2:1) solution for 5 min) and
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permeabilization (0.2% Triton X-100 solution), brain sections were incubated in the
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equilibration buffer for 10 min. Recombinant terminal deoxynucleotidyl transferase
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(rTdT) and nucleotide mixture were then added on the slide at 37°C for 60 min in the
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dark. Reactions were terminated by 2x SSC solution for 15 min. Nuclei were
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counterstained with Hoechst 33342 (1:20,000; Molecular Probes) for 5 min.
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Cell counting Cell counting was performed following the principles of design based stereology
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(16). Systematic random sampling was employed to ensure accurate and non-redundant
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cell count. Every section under analysis was at least 90 µm apart from the next. Three 10
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µm thick sections spanning the entire region of interest were selected for cell
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quantification. Counting was performed on 5 to 8 non-overlapping randomly selected 40x
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fields per section in the specified region of the cortex. ImageJ (NIH) was used to analyze
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each picture. All analysis was performed in a double-blinded fashion.
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Adhesive removal test The adhesive removal test measures sensorimotor function as previously
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described (1, 10). Briefly, a small adhesive dot was placed on each forepaw, and the
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amount of time (seconds) needed to contact and remove the tape from each forepaw was
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recorded. Mice were trained three times for three days (1–2 trials per day) before
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ischemia induction to ensure that they were able to remove the tape within 12 seconds to
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get a basal level of performance. Mice that show no response or prolonged time to
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remove the tape (>20 sec) during training period were excluded from future experiments.
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Animals were tested before ischemia and 3 and 7 days after ischemia by an investigator
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who was blind to the experimental groups. The test was performed four times per mouse,
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and the average time was used in the analysis. In this behavioral study, 16 animals were
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randomly assigned to WT and GluN3A KO groups before ischemia and 3 days after
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ischemia. At 7 days after stroke, 6 animals were used in each group. All testing trials
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were conducted during the day time.
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Locomotor activity test Behavioral changes of experimental rats were monitored and analyzed using the
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TopScan System (Clever Sys Inc., Reston, VA, USA). Mice were allowed to freely
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move in an open field container (50 cm × 50 cm × 50 cm). For locomotor activity,
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travelled distance and velocity were recorded for 1 hr before surgery and at 3 and 7 days
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after focal cerebral ischemia. After the recording, the videos were analyzed by the
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TopScan Realtime Option Version 3.0 (Clever Sys Inc.).
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Cylinder test A unilateral injury to the motor cortex results in an asymmetry in the forelimb
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used for support the body during rearing, which can be measured using the cylinder test.
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The mice were placed in a glass cylinder (9.5 cm diameter and 11 cm height), and the
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number of times each forelimb or both forelimbs were used to support the body on the
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wall of the cylinder was counted for 5 min. The animals were evaluated before surgery
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and at 3 and 7 days after focal cerebral ischemia. Two mirrors were placed behind the
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cylinder to view all directions. The numbers of impaired and non-impaired forelimb
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contacts were calculated as a percentage of total contacts.
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Statistical Analysis
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All results are expressed as mean ± S.E.M. Statistical comparisons, using Graph
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Pad Prism 6 (Graph Pad Software, Inc., San Diego, CA), were analyzed by unpaired
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Student’s t-test and Fisher’s test with least significant difference to identify significant
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differences. P < 0.05 was considered significant for all comparisons.
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Results
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GluN3A expression patterns and cellular distribution in the mouse brain
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To characterize the expression of GluN3A, we measured the expression level of
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GluN3A at postnatal day 5, 15, 30, and 70 in WT mice using Western blot analysis. As
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reported before, the highest level of GluN3A was observed in the P5 brain and the
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GluN3A expression was then declined to lower levels in the following days (Fig. 1A and
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1B). However, even if the GluN3A level was relatively lower in the adult brain (P30-
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P70) than that in the neonatal brain (P5-P15), the GluN3A expression was readily
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detectable. In WT and GluN3A KO brains, Western blotting confirmed that this NMDAR
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subunit was largely eliminated or undetectable in the KO brain tissues (Fig. 1D).
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Immunohistochemical analysis revealed that GluN3A was observed in the neuron of
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cortex including sensorimotor cortex from WT adult brain (Fig. 1C). We and others
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showed before that deletion of GluN3A does not affect the expression levels of GluN1
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and GluN2 (21, 24). Since GluN2 subunits especially GluN2B are specifically linked to
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excitotoxicity (27), we measured the GluN2A and GluN2B protein levels in the adult WT
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and GluN3A KO brains and the expression in cortical neurons. There was no change in
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GluN2A and GluN2B levels in the absence of GluN3A (Fig. 1D and 1E). Also, the
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number of GluN2B-postive cells didn’t show any difference between WT and GluN3A
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KO mice (Fig. 1F).
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Using immunocytochemical staining, we specifically examined the cellular
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distribution of GluN3A in cultured cortical neurons. It was clear that GluN3A localized
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to the cell membrane and dendrites (Fig. 2A-2C). Moreover, they were colocalized with
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the GluN1 subunit, suggesting formation of functional NMDARs in these locations (Fig.
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2D).
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Neuroprotective effects of GluN3A expression against in vitro ischemic insult in
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cortical cultures
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We next examined NMDA-induced neuronal cell death in cortical neuronal
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cultures of 12-14 DIV. The neuronal culture was exposed to 300 µM NMDA for 10 min
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and washed out with normal culture media. This short-term NMDA insult caused marked
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cell death 24 hrs later. The neuronal cell death showed apoptotic features such as cell
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body shrinkage and sensitivity to the block by the caspase inhibitor z-VAD-fmk (Fig. 3A
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and 3B). Compared to WT neurons, GluN3A KO neurons showed enhanced vulnerability
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to the NMDA insult, there was significant more neuronal death in GluN3A KO cultures
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(Fig. 3B).
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Neuroprotective effects of GluN3A expression against ischemic damage in the adult
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brain
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The focal cerebral ischemia in our experiments selectively damaged the somatosensory cortex of adult mice. Twenty four hrs after the ischemic insult, brain
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sections were stained with TTC solution to identify the damaged/died brain tissue (Fig.
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4A). The infarction volume and ratio of the cerebral cortex in GluN3A KO mice were
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significantly larger than that in WT mice (Fig. 4B and 4C). Using Western blotting, we
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compared the levels of GluN3A before and after ischemia in WT mice and did not
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observe significant change (data not shown).
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To confirm the difference in the ischemia-induced injury between the two groups
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at cellular levels, TUNEL staining was applied to evaluate the cell death in the penumbra
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region 24 hrs after focal cerebral ischemia. The number of dead/injured cells measured as
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TUNEL-positive cells was noticeable higher in GluN3A brain than that in WT stroke
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mice (Fig. 4D and 4E).
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Effect of GluN3A on functional recovery after focal cerebral ischemia Several behavior tests measured the functional outcomes after focal cerebral
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ischemia. In the adhesive dot removal test, the latency times to contact and remove the
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sticky dot from the right and left forelimbs were recorded at 3 and 7 days after focal
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cerebral ischemia (Fig. 5A and 5B). Before ischemic insult, the contact and removal
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times were similar between WT and GluN3A KO mice. After focal cerebral ischemia,
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animals showed prolonged times in response to the sticky dot attached to their left paws
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due to impaired sensorimotor function on the right side of the sensorimotor cortical area.
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GluN3A KO mice showed significantly hindered performance at 3 days after ischemia on
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both time to contact and time to remove sticky dot activities (Fig. 5A and 5B). TopScan
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system analysis was used to measure the locomotor activity after focal cerebral ischemia.
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GluN3A KO mice showed significant less travelled distance and lower velocity than WT
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mice at 3 days after focal cerebral ischemia (Fig. 5C and 5D). Consistently, GluN3A KO
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mice showed significant worse performance in cylinder test at 3 days after focal cerebral
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ischemia (Fig. 5E).
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Discussion This investigation reveals that, event at a relatively lower level than in the
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neonatal brain, the NMDAR inhibitory subunit GluN3A in the adult brain exhibits a
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neuroprotective effect in ischemic stroke. We demonstrate that deletion of this subunit
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results in enlarged infarct volume and increased cell death in the ischemic cortex of the
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adult mouse brain. Also, GluN3A KO mice exhibited hindered functional recovery
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compared to WT mice. These observations, on one hand, are surprising based on the
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common belief that GluN3A has little, if any, functional role in the adult brain due to its
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low expression level (26, 30). On the other hand, the result is consistent with our previous
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investigation showing that GluN3A plays an imperative role in pain perception,
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locomotion, and cognitive function in adult mice (21). Taking together, it is suggested
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that although the expression level of GluN3A in the adult brain is down-regulated from
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its peak level at the neonatal stage, this subunit in the adult brain still have significant
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impacts on multiple brain activities under physiological and pathological conditions such
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as in ischemic stroke. We noticed that the difference in the infarct volume between WT
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and GluN3A KO mice is relative moderate, which is consistent with the low level of
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GluN3A in the adult brain. The present investigation, however, is not an investigation of
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stroke treatment, which is largely judged by how big the protective effect is. Instead, we
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aimed to reveal that whether GluN3A could play a functional role in cerebral ischemia of
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the adult brain. The observation provides important information for a previously
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unidentified function of the GluN3A subunit after ischemic stroke in adults. It is expected
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that genetic manipulations and other strategies of increasing the GluN3A level in the
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adult brain can amplify the endogenous defense mechanism against ischemic stroke.
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A neuroprotective role of the GluN3A subunit was demonstrated in an earlier
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investigation in cortical cultures subjected to excitotoxic insults and in a neonatal
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hypoxia-ischemia model of mice (24). It was mentioned in the abstract of the report that a
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neuroprotective effect against ischemic stroke was only observed in neonatal but not in
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adult GluN3A KO mice (24). Unfortunately, no experimental data on the adult stroke
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experiment was presented in the report (24). On the other hand, the authors did show a
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neuroprotective effect of the GluN3A subunit in adult animals subjected to NMDA-
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induced retina injury and in the GluN3A-overexpressing transgenic mice after transient
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ischemic stroke (24). The protective effect of overexpressing GluN3A was also shown in
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a recent study on cultured hippocampal neuronal cell death induced by oxygen glucose
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deprivation (OGD) (28). We show in adult animals that the basal level of this subunit
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already shows a moderate neuroprotection, supporting that increasing the GluN3A level
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can be an effective therapeutic target for the treatment of stroke.
396
Recent studies raise an intriguing possibility that NMDAR hypofunction in
397
schizophrenia and bipolar disorder may arise from an excess increase or decrease of
398
GluN3A expression. GluN3A mRNA levels are significantly elevated in the dorsolateral
399
prefrontal cortex of schizophrenic patients and decreased in that of bipolar disorder
400
patients (23). Altered GluN3A expression levels regulate the development of spine
18
401
densities in schizophrenic patients (11, 12). Taken together, these findings suggest that
402
GluN3A expression can be an important regulator on the development of schizophrenia
403
and bipolar disorder and a key target gene for treating the patients with psychotic
404
disorders.
405
In conclusion, the present study shows a NMDAR subunit-mediated endogenous
406
neuroprotective action after focal cerebral ischemia in the adult brain. Together with the
407
previous reports of the GluN3A regulation on pain sensation, cognitive activities,
408
locomotion and retina excitotoxicity in adult animals (21, 24), the expression of GluN3A
409
thus shows significant physiological and pathophysiological roles in adults. In this study,
410
we used conventional GluN3A KO mice. It is possible that the loss of GluN3A activity
411
during the development stage may mask and change the role of the gene in the adult stage.
412
Therefore, further studies using conditional KO approach may be needed to provide more
413
inside information on the developmentally regulated neuroprotective mechanism.
414
19
415 416
Acknowledgement This work was supported by the NIH grants NS062097, NS073378, NS075338, a VA
417
Merit grant, an American Heart Association Grant-in-Aid grant 12GRNT12060222 and an AHA
418
Established Investigator Award.
419 420 421
Disclosures The authors have no disclosure of conflicts of interest related to this investigation.
422
20
423
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25
518
Figure Legends
519
Figure 1. Expression patterns of GluN3A in the mouse brain
520
Expression patterns of GluN3A and its effect on other NMDAR subunits were
521
examined using Western blot analysis and immunohistochemical method in the mouse
522
brain. A and B. The peak level of GluN3A was detected in the P5 brain and its expression
523
declines to lower levels in the following days. However, GluN3A expression was still
524
detectable in even P30 and P70 brain. N = 4-5 animals in each group; * P
1
A Neuroprotective Role of the NMDA Receptor Subunit GluN3A (NR3A) in
2
Ischemic Stroke of the Adult Mouse
3 4
Jin Hwan Lee1, Zheng Z. Wei1,2, Dongdong Chen1,2, Xiaohuan Gu1,2, Ling Wei1,2
5
and Shan Ping Yu1,2
6 7
1
Departments of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322; 2
8
Atlanta VA Medical Center, Center for Center for Visual
9
and Neurocognitive Rehabilitation, Decatur, GA 30033
10 11
Running title: Neuroprotective role of GluN3A in adult ischemic stroke
12 13 14
Corresponding author
15
Shan Ping Yu, M.D., Ph.D.
16
101 Woodruff Circle
17
Woodruff Memorial Research Building, Suite 620
18
Emory University School of Medicine
19
Atlanta, GA 30322
20
E-mail: [email protected]
21
Tel. 404-712-8678
1 Copyright © 2015 by the American Physiological Society.
22
Abstract
23
GluN3A or NR3A is a developmentally regulated N-methyl-D-aspartate receptor
24
(NMDAR) subunit, showing a unique inhibitory role that decreases NMDAR current and
25
the receptor mediated Ca2+ influx. In the neonatal brain, GluN3A is shown to associate
26
with synaptic maturation, spine formation, and plays a neuroprotective role. Its functional
27
role in the adult brain, however, is largely unknown. We tested the hypothesis that,
28
disrespecting the relatively lower expression level of GluN3A in the adult brain, this
29
inhibitory NMDAR subunit shows a protective action against ischemia-induced brain
30
injury. In littermate wild type (WT) and GluN3A knockout (KO) mice, focal cerebral
31
ischemia was induced by permanent occlusion of right distal branches of the middle
32
cerebral artery (MCA) plus 10 min ligation of both common carotid arteries (CCAs).
33
Twenty-four hrs after focal cerebral ischemia, the infarction volume assessed using 2,3,5-
34
triphenyltetrazolium chloride (TTC) staining was significantly larger in GluN3A KO
35
mice compared to WT mice. Terminal deoxynucleotidyl transferase dUTP nick end
36
labeling (TUNEL) staining demonstrated enhanced cell death in GluN3A KO mice.
37
Moreover, the deletion of GluN3A hindered sensorimotor functional recovery after stroke.
38
It is suggested that, although the expression level is relatively lower in the adult brain,
39
GluN3A is still a noteworthy regulator in ischemia-induced excitotoxicity and brain
40
injury.
41 42
Key words: Stroke, NMDA receptors, GluN3A, Adult brain, Functional recovery
43
2
44
Abbreviations
45
NMDA: N-methyl-D-aspartate
46
NMDAR: NMDA receptor
47
MCA: middle cerebral artery
48
CCAs: common carotid arteries
49
WT: wild type
50
KO: knockout
51
GluN1: glutamate NMDA receptor 1 (NR1)
52
GluN2: glutamate NMDA receptor 2A (NR2A)
53
GluN2B: glutamate NMDA receptor 2B (NR2B)
54
GluN3A: glutamate NMDA receptor 3A (NR3A)
55
GluN3B: glutamate NMDA receptor 3B (NR3B)
56
TTC: 2,3,5-triphenyltetrazolium chloride
57
TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling
3
58 59
Introduction The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves
60
critical roles in physiological and pathological processes in the central nervous system
61
(CNS) (7, 9, 17, 20). The receptor activity regulates fast excitatory neurotransmission,
62
contribute to the regulation of neuronal gene expression and play key roles in the
63
formation of neural networks and the formation of long-term potentiation (LTP) and
64
memory (18). NMDAR is composed of proteins derived from the GluR1 (NR1) family
65
and the GluN2 (NR2) family of genes. The GluN1 family has a variety of splice variants,
66
and GluN2 family is comprised of four subunit classes, GluN2A, GluN2B, GluN2C and
67
GluN2D. The gene expression of both families shows temporal and spatial distributions
68
in the brain (13, 15, 22). NMDAR may also contain an inhibitory subunit, GluN3A
69
(NR3A, NMDAR-L, or chi-1) and GluN3B (NR3B) (2, 6, 19, 26). Incorporation of
70
GluN3A with GluN1/GluN2 subunits suppresses NMDAR activity. For example, co-
71
expression of GluN3A results in decreased NMDAR single channel conductance, Ca2+
72
permeability and the receptor Mg2+ sensitivity, which may limit synaptic strengthening
73
and stabilization (3, 6, 8, 25, 26). On the other hand, genetic knockout (KO) of GluN3A
74
in mice results in enhanced NMDA currents and increased dendritic spines in early
75
postnatal cerebrocortical neurons compared to wild type (WT) mice (8). In addition,
76
increases of intracellular Ca2+ concentration in the neuronal cells from GluN3A KO mice
77
contribute to cell death (3, 5, 14).
78
It is well known that GluN3A is a developmentally regulated NMDAR subunit.
79
GluN3A mRNA and protein levels peak at the end of the first postnatal week but
80
decreases to a low level by adulthood (6, 26). Based on these findings, studies on
4
81
GluN3A have been almost exclusively focused on the developing brain of neonates (26,
82
30). In a hypoxic-ischemic stroke model of neonatal mice, deletion of GluN3A caused
83
enlarged infarct formation while over-expression of GluN3A decreased the brain damage
84
(24). On the other hand, the significance of endogenous GluN3A in the adult brain has
85
been largely ignored, most likely due to the fact that the GluN3A level is down-regulated
86
in the adult brain and due to the assumption that the low level GluN3A has no or little
87
functional role. The pathological role of GluN3A in the adult brain, ironically, has never
88
been directly tested and no quantified experimental data from adult animals has been
89
reported before.
90
We recently demonstrated that even though the GluN3A expression level declines
91
to lower levels from the neonatal period to the adulthood, significant protein levels of
92
GluN3A are still detectable in the adult brain (21). An important role of GluN3A in
93
locomotion, pain perception, and cognitive functions was observed in adult animals (21).
94
The results indicate that even if at relatively lower expression level, GluN3A still can act
95
as an imperative player in the adult brain. In the present investigation, we extended the
96
examination into the pathological condition of ischemic stroke and tested the hypothesis
97
that a role of GluN3A in the adult stroke brain might have been overlooked before. We
98
also hypothesized that a neuroprotective role of GluN3A in the adult brain could indicate
99
a receptor subunit target for the development of therapeutic treatment of stroke.
100 101
Methods
102
Animals
103
NMDA receptor GluN3A (NR3A) constitutive knockout (KO) mice (kindly
5
104
provided by Dr. Stuart A. Lipton and Dr. Nobuki Nakanishi at Stanford University) and
105
littermate wild type (WT) mice at different postnatal days were used in the experiments.
106
The animals were maintained at a constant temperature of 21°C with a 12 hrs light/dark
107
cycle in the Laboratory Animal Center for Research, Medicine School of South Carolina.
108
All experimental and surgical procedures were approved by the Institutional Animal Care
109
and Use Committee (IACUC) at Emory University (Animal protocol number: 208-2008).
110
No human tissue or sample was tested in this study.
111 112 113
Genotyping The Genotyping of GluN3A KO and WT animals was performed as described
114
previously (21). DNA for genotyping was extracted from tail snips (approximately 2-4
115
mm). The separately primers were 5’-CCA CGG TGA GCT TGG GGA AG (NN494),
116
5’-GCC TGA AGA ACG AGA TCA GC (NN506) and 5’-TTG GGG AGC GCC CTG
117
CAT GG (NN507) (Sigma-Aldrich, St. Louis, MO, USA). The 251 bp product (NN494
118
and NN506) confutes GluN3A knockout mice and 200 bp product (NN494 and NN507)
119
confutes WT mice. DNA (2 µl) was amplified on a thermocycler (MJ minim, Personal
120
Thermal Cycler, Bio-Rad, CA, USA) for 40 cycles (95°C for 60 sec, 58°C for 30 sec,
121
72°C for 60 sec). After additional incubation at 72°C for 10 min and transferred to 4°C,
122
PCR products were subjected to electrophoresis in 1.5% agarose gel with ethidium
123
bromide. Relative intensity of PCR bands was analyzed using the InGenius3 manual gel
124
documentation system (Syngene, Frederick, MD, USA).
125 126
Neuronal cell cultures
6
127
The preparation of nearly pure cortical neurons was previously described (31).
128
Briefly, neurons were harvested from the cortex of gestational 14–16 day mouse embryos.
129
The pregnant mouse was sacrificed using overdosed isoflurane and brain dissection was
130
performed soon after heartbeat stopped and no response to pawn stimuli. The cortex was
131
dissociated in trypsin and plated on poly-D-lysine and laminin coated tissue culture
132
dishes. Cells were maintained in neurobasal media (Invitrogen, Carlsbad, CA) with B27
133
supplement and L-glutamine until the time of the experiment. Cytarabine (Ara-C) was
134
added on the third day to prevent the proliferation of glial cells. Mature neuronal cultures
135
(12-14 days in vitro (DIV)) were used for cell death assays.
136 137 138
NMDA toxicity and assessment of neuronal cell death Excitotoxicity was induced by 10 min exposure to NMDA (300 µM) alone or in
139
the presence of z-VAD-fmk (100 µM), carried out at room temperature in a HEPES
140
buffer solution (HBSS) containing (in mM): NaCl, 120; KCl, 5.4; MgCl2, 0.8; CaCl2,
141
1.8; HEPES, 20; glucose, 15; glycine, 0.01 (pH 7.4). The exposure solution was then
142
washed away and replaced by media stock (MS) supplemented with 10 µM glycine
143
before returning cultures to the incubator for 20-24 hrs.
144
Neuronal cell death was estimated by examination of the cultures under phase-
145
contrast microscopy, and quantitated by measurement of lactate dehydrogenase (LDH)
146
release by damaged cells into the bathing medium 24 hrs following the experimentation
147
(14). The LDH level corresponding to near-complete neuron death (without glial death)
148
was determined by assaying sister cultures exposed to 300 µM NMDA for 24 hrs in MS
149
supplemented with glycine. The percent neuronal death was normalized to the mean LDH
7
150
value released 24 hrs after a sham wash (defined as 0%) and continuous exposure to 300
151
µM NMDA (defined as 100%). Background LDH levels were determined in sister
152
cultures subjected to sham washing and subtracted from experimental values to yield the
153
signal specific to experimentally induced injury.
154 155 156
Focal cerebral ischemia in mice Focal cerebral ischemia, confined to the sensorimotor cortex in the right middle
157
cerebral artery (MCA) territory of WT and GluN3A KO male mice were induced by
158
permanent occlusions of distal branches of the right MCA and temporary (10 min)
159
ligations of both common carotid arteries (CCAs) (29). In brief, 8–10 week old male
160
mice (25-28 g) were anesthetized by 4% chloral hydrate (100 mg/kg, i.p.). Chloral
161
hydrate has been widely used as a sedation agent in human and animal
162
surgeries/procedures for a number of years. It was chosen in this investigation due to its
163
stable and well controlled anesthetic action and negligible effect on NMDA receptors (4).
164
The right MCA branches were permanently ligated using a 10-0 suture (Surgical
165
Specialties Co., Reading, PA) accompanied by a bilateral 10-min ligation of CCAs. Body
166
temperature was monitored during surgery and maintained at 37.0 0.3°C using a
167
temperature control unit and heating pads. The mortality rate due to ischemic surgery
168
and/or anesthesia failure was around 7% in these experiments. Free access to food and
169
water was allowed after recovery from anesthesia. All mice were kept in air-ventilated
170
cages with room temperature maintained at 24 0.5°C. Animals were euthanized and
171
decapitated at different time points after ischemic stroke. Brains were immediately
172
removed, mounted in optimal cutting temperature compound (Sakura Finetek USA, Inc.,
8
173
Torrance, CA) and stored at −80°C for further processing.
174 175 176
TTC staining of infarct volume measurement Twenty-four hrs after onset of MCAO, animals in different groups were sacrificed
177
for assessment of brain infarct formation. 2,3,5-triphenyltetrazolium chloride (TTC;
178
Sigma-Aldrich) staining was used to reveal damaged/dead brain tissue as previously
179
described (29). Brains were removed and placed in a brain matrix then sliced into 1-mm
180
coronal sections. Slices were incubated in 2% TTC solution at 37°C for 5 min, then
181
stored in 10% buffered formalin for 24 hrs. Digital images of the caudal aspect of each
182
slice were obtained by a flatbed scanner. Infarct, ipsilateral hemisphere, and contralateral
183
hemisphere areas were measured using ImageJ software (NIH, Bethasda, MD, USA). The
184
indirect method (subtraction of residual right hemisphere cortical volume from cortical
185
volume of the intact left hemisphere) will be used for infarct volumes calculation. Infarct
186
measurements were performed under double-blind conditions.
187 188 189
Western blot analysis Western blot analysis was used to detect the expression of NMDAR subunits.
190
Brain cortical tissue was lysed in a lysis buffer containing 0.02 M Na4P2O7, 10 mM Tris-
191
HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton, 1 mM EGTA, 2 mM
192
Na3VO4, and a protease inhibitor cocktail (Sigma-Aldrich). The supernatant was
193
collected after centrifugation at 15,000 g for 10 min at 4°C. Protein concentration was
194
determined with a bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL, USA).
195
Equivalent amounts of total protein were separated by molecular weight on an SDS-
9
196
polyacrylamide gradient gel, and then transferred to a PVDF membrane. The blot was
197
incubated in 5% bovine serum albumin for at least 1 hr and then reacted with primary
198
antibodies at 4°C for overnight. The primary antibodies used in this investigation
199
included: and the dilutions for each were rabbit anti-GluN2A antibody (Cell Signaling,
200
Danvers, MA, USA) at 1:2000, rabbit anti-GluN2B antibody (Cell Signaling) at 1:2000,
201
rabbit anti-GluN3A antibody (Abcam, Cambridge, MA, USA) at 1:1000, and mouse anti-
202
-actin (Sigma-Aldrich) at 1:5000 (Supporting Table 1). After washing with Tris-
203
buffered saline with Tween (TBST), membranes were incubated with AP-conjugated or
204
HRP-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA) for 1-2 hrs
205
at room temperature. After final washing with TBST, the signals were detected with
206
bromochloroidolylphosphate/nitroblue tetrazolium (BCIP/NBP) solution (Sigma-Aldrich)
207
or film. Signal intensity was measured by ImageJ and normalized to the actin signal
208
intensity.
209 210 211
Immunohistochemical assessment Frozen brain tissues were sliced into 10 µm-thick coronal sections using a
212
cryostat vibratome (Leica CM 1950; Leica Microsystems, Buffalo Grove, IL, USA).
213
Sections were dried on the slide warmer for 30 min, fixed with 10% formalin buffer,
214
washed with -20°C precooled ethanol: acetic acid (2:1) solution for 10 min, and finally
215
permeabilized with 0.2% Triton-X 100 solution for 5 min. All slides were washed 3 times
216
with PBS (5 min each) after each step. Then, tissue sections were blocked with 1% fish
217
gelatin (Sigma-Aldrich) in PBS for 1 hr at room temperature, and subsequently incubated
218
with the primary antibody: mouse anti-GluN1 antibody (1:200; Life Technologies, Grand
10
219
Island, NY, USA), rabbit anti-GluN2A antibody (1:200; Abcam), rabbit anti-GluN2B
220
(1:200; Abcam), rabbit anti-GluN3A (1:200; Abcam), and mouse anti-NeuN (1:400;
221
Millipore, Billerica, MA, USA) overnight at 4°C (Supporting Table 1). Next day, the
222
slides were washed 3 times with PBS for 5 min, then reacted with the secondary
223
antibodies Alexa Fluor®488 goat anti-mouse or rabbit (1:300; Life Technologies) and
224
Cy3-conjugated donkey anti-rabbit (1:300; Jackson ImmunoResearch Laboratories, West
225
Grove, PA, USA) or Cy5-conjugated donkey anti-mouse or rabbit (1:400; Jackson
226
ImmunoResearch Laboratories) for 80 min at room temperature. After 3 washes with
227
PBS, nuclei were stained with Hoechst 33342 (1:20,000; Life Technologies) for 5 min as
228
a counterstain. And then the brain sections were mounted, coverslipped, imaged, and
229
photographed under a fluorescent microscope (BX51, Olympus, Japan) and laser
230
scanning confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA).
231 232
TUNEL staining and positive cell quantification
233
A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
234
kit was used to examine cell death by detecting fragmented DNA in 10-µm-thick coronal
235
fresh frozen sections as described previously (n=8 per group) (16). After fixation (10%
236
buffered formalin for 10 min and then ethanol:acetic acid (2:1) solution for 5 min) and
237
permeabilization (0.2% Triton X-100 solution), brain sections were incubated in the
238
equilibration buffer for 10 min. Recombinant terminal deoxynucleotidyl transferase
239
(rTdT) and nucleotide mixture were then added on the slide at 37°C for 60 min in the
240
dark. Reactions were terminated by 2x SSC solution for 15 min. Nuclei were
241
counterstained with Hoechst 33342 (1:20,000; Molecular Probes) for 5 min.
11
242 243 244
Cell counting Cell counting was performed following the principles of design based stereology
245
(16). Systematic random sampling was employed to ensure accurate and non-redundant
246
cell count. Every section under analysis was at least 90 µm apart from the next. Three 10
247
µm thick sections spanning the entire region of interest were selected for cell
248
quantification. Counting was performed on 5 to 8 non-overlapping randomly selected 40x
249
fields per section in the specified region of the cortex. ImageJ (NIH) was used to analyze
250
each picture. All analysis was performed in a double-blinded fashion.
251 252 253
Adhesive removal test The adhesive removal test measures sensorimotor function as previously
254
described (1, 10). Briefly, a small adhesive dot was placed on each forepaw, and the
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amount of time (seconds) needed to contact and remove the tape from each forepaw was
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recorded. Mice were trained three times for three days (1–2 trials per day) before
257
ischemia induction to ensure that they were able to remove the tape within 12 seconds to
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get a basal level of performance. Mice that show no response or prolonged time to
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remove the tape (>20 sec) during training period were excluded from future experiments.
260
Animals were tested before ischemia and 3 and 7 days after ischemia by an investigator
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who was blind to the experimental groups. The test was performed four times per mouse,
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and the average time was used in the analysis. In this behavioral study, 16 animals were
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randomly assigned to WT and GluN3A KO groups before ischemia and 3 days after
12
264
ischemia. At 7 days after stroke, 6 animals were used in each group. All testing trials
265
were conducted during the day time.
266 267 268
Locomotor activity test Behavioral changes of experimental rats were monitored and analyzed using the
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TopScan System (Clever Sys Inc., Reston, VA, USA). Mice were allowed to freely
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move in an open field container (50 cm × 50 cm × 50 cm). For locomotor activity,
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travelled distance and velocity were recorded for 1 hr before surgery and at 3 and 7 days
272
after focal cerebral ischemia. After the recording, the videos were analyzed by the
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TopScan Realtime Option Version 3.0 (Clever Sys Inc.).
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Cylinder test A unilateral injury to the motor cortex results in an asymmetry in the forelimb
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used for support the body during rearing, which can be measured using the cylinder test.
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The mice were placed in a glass cylinder (9.5 cm diameter and 11 cm height), and the
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number of times each forelimb or both forelimbs were used to support the body on the
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wall of the cylinder was counted for 5 min. The animals were evaluated before surgery
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and at 3 and 7 days after focal cerebral ischemia. Two mirrors were placed behind the
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cylinder to view all directions. The numbers of impaired and non-impaired forelimb
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contacts were calculated as a percentage of total contacts.
284 285
Statistical Analysis
13
286
All results are expressed as mean ± S.E.M. Statistical comparisons, using Graph
287
Pad Prism 6 (Graph Pad Software, Inc., San Diego, CA), were analyzed by unpaired
288
Student’s t-test and Fisher’s test with least significant difference to identify significant
289
differences. P < 0.05 was considered significant for all comparisons.
290 291 292
Results
293
GluN3A expression patterns and cellular distribution in the mouse brain
294
To characterize the expression of GluN3A, we measured the expression level of
295
GluN3A at postnatal day 5, 15, 30, and 70 in WT mice using Western blot analysis. As
296
reported before, the highest level of GluN3A was observed in the P5 brain and the
297
GluN3A expression was then declined to lower levels in the following days (Fig. 1A and
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1B). However, even if the GluN3A level was relatively lower in the adult brain (P30-
299
P70) than that in the neonatal brain (P5-P15), the GluN3A expression was readily
300
detectable. In WT and GluN3A KO brains, Western blotting confirmed that this NMDAR
301
subunit was largely eliminated or undetectable in the KO brain tissues (Fig. 1D).
302
Immunohistochemical analysis revealed that GluN3A was observed in the neuron of
303
cortex including sensorimotor cortex from WT adult brain (Fig. 1C). We and others
304
showed before that deletion of GluN3A does not affect the expression levels of GluN1
305
and GluN2 (21, 24). Since GluN2 subunits especially GluN2B are specifically linked to
306
excitotoxicity (27), we measured the GluN2A and GluN2B protein levels in the adult WT
307
and GluN3A KO brains and the expression in cortical neurons. There was no change in
308
GluN2A and GluN2B levels in the absence of GluN3A (Fig. 1D and 1E). Also, the
14
309
number of GluN2B-postive cells didn’t show any difference between WT and GluN3A
310
KO mice (Fig. 1F).
311
Using immunocytochemical staining, we specifically examined the cellular
312
distribution of GluN3A in cultured cortical neurons. It was clear that GluN3A localized
313
to the cell membrane and dendrites (Fig. 2A-2C). Moreover, they were colocalized with
314
the GluN1 subunit, suggesting formation of functional NMDARs in these locations (Fig.
315
2D).
316 317
Neuroprotective effects of GluN3A expression against in vitro ischemic insult in
318
cortical cultures
319
We next examined NMDA-induced neuronal cell death in cortical neuronal
320
cultures of 12-14 DIV. The neuronal culture was exposed to 300 µM NMDA for 10 min
321
and washed out with normal culture media. This short-term NMDA insult caused marked
322
cell death 24 hrs later. The neuronal cell death showed apoptotic features such as cell
323
body shrinkage and sensitivity to the block by the caspase inhibitor z-VAD-fmk (Fig. 3A
324
and 3B). Compared to WT neurons, GluN3A KO neurons showed enhanced vulnerability
325
to the NMDA insult, there was significant more neuronal death in GluN3A KO cultures
326
(Fig. 3B).
327 328
Neuroprotective effects of GluN3A expression against ischemic damage in the adult
329
brain
330 331
The focal cerebral ischemia in our experiments selectively damaged the somatosensory cortex of adult mice. Twenty four hrs after the ischemic insult, brain
15
332
sections were stained with TTC solution to identify the damaged/died brain tissue (Fig.
333
4A). The infarction volume and ratio of the cerebral cortex in GluN3A KO mice were
334
significantly larger than that in WT mice (Fig. 4B and 4C). Using Western blotting, we
335
compared the levels of GluN3A before and after ischemia in WT mice and did not
336
observe significant change (data not shown).
337
To confirm the difference in the ischemia-induced injury between the two groups
338
at cellular levels, TUNEL staining was applied to evaluate the cell death in the penumbra
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region 24 hrs after focal cerebral ischemia. The number of dead/injured cells measured as
340
TUNEL-positive cells was noticeable higher in GluN3A brain than that in WT stroke
341
mice (Fig. 4D and 4E).
342 343 344
Effect of GluN3A on functional recovery after focal cerebral ischemia Several behavior tests measured the functional outcomes after focal cerebral
345
ischemia. In the adhesive dot removal test, the latency times to contact and remove the
346
sticky dot from the right and left forelimbs were recorded at 3 and 7 days after focal
347
cerebral ischemia (Fig. 5A and 5B). Before ischemic insult, the contact and removal
348
times were similar between WT and GluN3A KO mice. After focal cerebral ischemia,
349
animals showed prolonged times in response to the sticky dot attached to their left paws
350
due to impaired sensorimotor function on the right side of the sensorimotor cortical area.
351
GluN3A KO mice showed significantly hindered performance at 3 days after ischemia on
352
both time to contact and time to remove sticky dot activities (Fig. 5A and 5B). TopScan
353
system analysis was used to measure the locomotor activity after focal cerebral ischemia.
354
GluN3A KO mice showed significant less travelled distance and lower velocity than WT
16
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mice at 3 days after focal cerebral ischemia (Fig. 5C and 5D). Consistently, GluN3A KO
356
mice showed significant worse performance in cylinder test at 3 days after focal cerebral
357
ischemia (Fig. 5E).
358 359 360 361
Discussion This investigation reveals that, event at a relatively lower level than in the
362
neonatal brain, the NMDAR inhibitory subunit GluN3A in the adult brain exhibits a
363
neuroprotective effect in ischemic stroke. We demonstrate that deletion of this subunit
364
results in enlarged infarct volume and increased cell death in the ischemic cortex of the
365
adult mouse brain. Also, GluN3A KO mice exhibited hindered functional recovery
366
compared to WT mice. These observations, on one hand, are surprising based on the
367
common belief that GluN3A has little, if any, functional role in the adult brain due to its
368
low expression level (26, 30). On the other hand, the result is consistent with our previous
369
investigation showing that GluN3A plays an imperative role in pain perception,
370
locomotion, and cognitive function in adult mice (21). Taking together, it is suggested
371
that although the expression level of GluN3A in the adult brain is down-regulated from
372
its peak level at the neonatal stage, this subunit in the adult brain still have significant
373
impacts on multiple brain activities under physiological and pathological conditions such
374
as in ischemic stroke. We noticed that the difference in the infarct volume between WT
375
and GluN3A KO mice is relative moderate, which is consistent with the low level of
376
GluN3A in the adult brain. The present investigation, however, is not an investigation of
377
stroke treatment, which is largely judged by how big the protective effect is. Instead, we
17
378
aimed to reveal that whether GluN3A could play a functional role in cerebral ischemia of
379
the adult brain. The observation provides important information for a previously
380
unidentified function of the GluN3A subunit after ischemic stroke in adults. It is expected
381
that genetic manipulations and other strategies of increasing the GluN3A level in the
382
adult brain can amplify the endogenous defense mechanism against ischemic stroke.
383
A neuroprotective role of the GluN3A subunit was demonstrated in an earlier
384
investigation in cortical cultures subjected to excitotoxic insults and in a neonatal
385
hypoxia-ischemia model of mice (24). It was mentioned in the abstract of the report that a
386
neuroprotective effect against ischemic stroke was only observed in neonatal but not in
387
adult GluN3A KO mice (24). Unfortunately, no experimental data on the adult stroke
388
experiment was presented in the report (24). On the other hand, the authors did show a
389
neuroprotective effect of the GluN3A subunit in adult animals subjected to NMDA-
390
induced retina injury and in the GluN3A-overexpressing transgenic mice after transient
391
ischemic stroke (24). The protective effect of overexpressing GluN3A was also shown in
392
a recent study on cultured hippocampal neuronal cell death induced by oxygen glucose
393
deprivation (OGD) (28). We show in adult animals that the basal level of this subunit
394
already shows a moderate neuroprotection, supporting that increasing the GluN3A level
395
can be an effective therapeutic target for the treatment of stroke.
396
Recent studies raise an intriguing possibility that NMDAR hypofunction in
397
schizophrenia and bipolar disorder may arise from an excess increase or decrease of
398
GluN3A expression. GluN3A mRNA levels are significantly elevated in the dorsolateral
399
prefrontal cortex of schizophrenic patients and decreased in that of bipolar disorder
400
patients (23). Altered GluN3A expression levels regulate the development of spine
18
401
densities in schizophrenic patients (11, 12). Taken together, these findings suggest that
402
GluN3A expression can be an important regulator on the development of schizophrenia
403
and bipolar disorder and a key target gene for treating the patients with psychotic
404
disorders.
405
In conclusion, the present study shows a NMDAR subunit-mediated endogenous
406
neuroprotective action after focal cerebral ischemia in the adult brain. Together with the
407
previous reports of the GluN3A regulation on pain sensation, cognitive activities,
408
locomotion and retina excitotoxicity in adult animals (21, 24), the expression of GluN3A
409
thus shows significant physiological and pathophysiological roles in adults. In this study,
410
we used conventional GluN3A KO mice. It is possible that the loss of GluN3A activity
411
during the development stage may mask and change the role of the gene in the adult stage.
412
Therefore, further studies using conditional KO approach may be needed to provide more
413
inside information on the developmentally regulated neuroprotective mechanism.
414
19
415 416
Acknowledgement This work was supported by the NIH grants NS062097, NS073378, NS075338, a VA
417
Merit grant, an American Heart Association Grant-in-Aid grant 12GRNT12060222 and an AHA
418
Established Investigator Award.
419 420 421
Disclosures The authors have no disclosure of conflicts of interest related to this investigation.
422
20
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Figure Legends
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Figure 1. Expression patterns of GluN3A in the mouse brain
520
Expression patterns of GluN3A and its effect on other NMDAR subunits were
521
examined using Western blot analysis and immunohistochemical method in the mouse
522
brain. A and B. The peak level of GluN3A was detected in the P5 brain and its expression
523
declines to lower levels in the following days. However, GluN3A expression was still
524
detectable in even P30 and P70 brain. N = 4-5 animals in each group; * P
