The Journal of Neuroscience, December 2, 2015 • 35(48):15971–15983 • 15971 Cellular/Molecular NMDA Receptors Containing the GluN2D Subunit Control Neuronal Function in the Subthalamic Nucleus X Sharon A. Swanger,1* Katie M. Vance,1* Jean-Franc¸ois Pare,3 Florence Sotty,4 Karina Fog,4 Yoland Smith,2,3 and X Stephen F. Traynelis1 1Department of Pharmacology and 2Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 30322, 3Yerkes National Primate Research Center and Morris K. Udall Center of Excellence for Parkinson’s Disease Research, Emory University, Atlanta, Georgia 30329, and 4H. Lundbeck A/S, Division of Neurodegeneration and Biologics, Ottiliavej 9, DK-2500 Valby, Denmark The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)–P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson’s disease. Key words: excitatory synapse; GluN2B; GluN2D; glutamate receptor; NMDA; subthalamic nucleus Significance Statement The subthalamic nucleus (STN) is a key component of the basal ganglia, a group of subcortical nuclei that control movement and are dysregulated in movement disorders such as Parkinson’s disease. Subthalamic neurons receive direct excitatory input, but the pharmacology of excitatory synaptic transmission in the STN has been understudied. Here, we show that GluN2B- and GluN2D- containing NMDA receptors mediate the NMDA receptor component of EPSCs in subthalamic neurons. Moreover, our results demonstrate that pharmacologic modulation of GluN2D-containing receptors alters the time course of EPSCs and controls the in vivo spike-firing rate in the STN. This study identifies GluN2D as a potential target for modulating subthalamic neuron activity. Introduction nuclei that are critical for movement control. Neuronal activity in The subthalamic nucleus (STN) is the lone glutamatergic mem- the STN provides the primary excitatory drive for the basal gan- ber of the basal ganglia, a group of interconnected subcortical glia output nuclei, the globus pallidus internal, and substantia nigra pars reticulata (SNr; Bolam et al., 2000; Bevan et al., 2002; Wilson and Bevan, 2011). STN neurons exhibit autonomous fir- Received April 30, 2015; revised Oct. 20, 2015; accepted Oct. 29, 2015. ing that is driven by voltage-dependent sodium channels (Beur- Author contributions: S.A.S., K.M.V., J.-F.P., F.S., K.F., Y.S., and S.F.T. designed research; S.A.S., K.M.V., J.-F.P., and F.S. performed research; S.A.S., K.M.V., J.-F.P., F.S., K.F., Y.S., and S.F.T. analyzed data; S.A.S., K.M.V., F.S., K.F., rier et al., 2000; Do and Bean, 2003; Surmeier et al., 2005; Y.S., and S.F.T. wrote the paper. Atherton et al., 2008). Spike firing of subthalamic neurons is also WethankSusanJenkinsforherhelpwithimmunocytochemistryandtissueprocessing,andStefanieRitterforher help with the GluN2D antibody. We also thank Jing Zhang, Anel Tankovic, and Phuong Le for excellent technical assistance. This work was supported by the NIH-NINDS [NS036654 and NS065371 (SFT), F32-NS086368 (SAS), *S.A.S. and K.M.V. contributed equally to this work. T32-NS007480 (KMV)], NIH-NIDA T32-DA015040 (SAS and KMV), The Michael J. Fox Foundation (SFT), and a re- Correspondence should be addressed to Dr. Stephen F. Traynelis, Department of Pharmacology, Rollins Research search grant to Emory University from Lundbeck A/S (SFT). Center, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322. E-mail: [email protected]. S.F.T. is a paid consultant for NeurOp and Janssen Pharmaceuticals and holds an equity position at NeurOp. The DOI:10.1523/JNEUROSCI.1702-15.2015 remaining authors declare no competing financial interests. Copyright © 2015 the authors 0270-6474/15/3515971-13$15.00/0 15972 • J. Neurosci., December 2, 2015 • 35(48):15971–15983 Swanger, Vance et al. • GluN2D Function in the Subthalamic Nucleus regulated by several types of afferents, including excitatory inputs Materials and Methods from the cortex, thalamus, pedunculopontine nucleus, and supe- Tissue punches and Western blotting. All procedures using animals were rior colliculus (Afsharpour, 1985; Canteras et al., 1988; Fe´ger et approved by the Institutional Animal Care and Use Committee of Emory al., 1994; Nambu et al., 1996; Fe´ger, 1997). STN neurons also University. Female Sprague Dawley rats [age postnatal day 7 (P7), P14, Ϫ Ϫ recieve inhibitory inputs from the globus pallidus external P30, P60, and P110–P120] and WT and GluN2D / C57BL/6 mice of (Smith and Grace, 1992; Shink et al., 1996; Bolam et al., 2002; both sexes (age P28–P30) were killed by isoflurane overdose (Ikeda et al., ␮ Karachi et al., 2005) and dopaminergic inputs from the substan- 1995). The brains were removed and 200–300 m sagittal slices contain- ing the STN were prepared in ice-cold PBS using a vibrating microtome. tia nigra pars compacta (SNc) (Hassani et al., 1997; Prensa et al., Tissue punches of the STN were removed using a 0.5 or 0.75 mm tissue 2000; Cragg et al., 2004). The loss of SNc neurons in Parkinson’s punch (Stoelting) and then frozen on dry ice. The tissue was sonicated in disease leads to altered firing patterns in the STN, which corre- lysis buffer, pH 7.4, containing the following (in mM): 150 NaCl, 50 Tris, lates with the development of motor symptoms in patients and in 50 NaF, 5 EDTA, 5 EGTA, 1% Triton, 1% SDS, and protease inhibitors. animal models of the disease (Hamani et al., 2004; Wilson and Protein concentrations were determined by the Bradford assay and then Bevan, 2011). In addition, synchronous firing between the STN the samples were diluted with lysis buffer and 2ϫ Laemmli buffer to a and motor cortex increases in the disease state, suggesting that final concentration of 0.5 ␮g/␮l. After heating at 95°C for 5 min, 5–10 ␮g activity at glutamatergic cortical inputs to the STN may be altered of total protein was loaded on a 4–15% SDS-PAGE gel. Proteins were transferred to PVDF membranes for Western blotting. The antibodies (Magill et al., 2001; Magill et al., 2004; Sharott et al., 2005; Ku¨hn used were mouse anti-GluN2D (Millipore, MAB5578, 1:5000), mouse et al., 2006; Mallet et al., 2008; Litvak et al., 2011; Moran et al., anti-tubulin (Sigma-Aldrich, 1:50,000), and HRP-conjugated goat anti- 2011; Alegre and Valencia, 2013). Furthermore, exogenous acti- mouse (Jackson Immunoresearch, 1:10,000). Membranes were stripped vation of the corticosubthalamic projection can induce parkin- with Restore Stripping Buffer (Pierce) for 15 min and washed before sonian motor symptoms in rodents (Pan et al., 2014), indicating being reprobed with additional primary antibodies. Chemiluminescence that glutamatergic neurotransmission in the STN has an integral signals were detected with film, which were imaged with the Bio-Rad Gel role in basal ganglia function and movement. Doc Imager and analyzed by densitometry using ImageJ. Subthalamic neurons express mRNA encoding subunits in Immunohistochemistry and electron microscopy. Adult Sprague Dawley rats and adult WT and GluN2D Ϫ/Ϫ C57BL/6 mice of both sexes were each ionotropic glutamate receptor class, including AMPA, kai- used for histology experiments. The animals were anesthetized with pen- nate, and NMDA receptors (Sato et al., 1993; Bischoff et al., 1997; tobarbital and perfusion fixed with a mixture of 4% paraformaldehyde Wu¨llner et al., 1997; Clarke and Bolam, 1998; Jakowec et al., and 0.1% glutaraldehyde (Galvan et al., 2006; Kuwajima et al., 2007). The 1998). AMPA/kainate-type receptors mediate the fast compo- brains were then cut into 60-␮m-thick sections with a vibratome and nent of EPSCs, whereas NMDA receptors mediate the slow com- processed for the light and electron microscopic localization of the ponent, which carries the majority of charge transfer and calcium GluN2D subunit using well characterized commercially available anti- influx during excitatory neurotransmission (Traynelis et al., bodies: mouse anti-GluN2D (Millipore, MAB5578) or goat anti- 2010). NMDA receptors are typically composed of two glycine- GluN2C/D (Santa Cruz Biotechnology, sc-31551; Laurie et al., 1997). Ј binding GluN1 subunits and two glutamate-binding GluN2 sub- The avidin–biotin peroxidase method and 3,3 diaminobenzidine were used to visualize the reaction product of GluN2D immunoreactivity. units (Dingledine et al., 1999; Karakas and Furukawa, 2014; Lee et Sections of the STN were prepared for electron microscopy as previously al., 2014).