TTX-Resistant NMDA Receptor-Mediated Membrane Potential Oscillations in Neonatal Mouse Hb9 Interneurons

Harris-Warrick RM (2012) TTX-Resistant NMDA Receptor-Mediated Membrane Potential Oscillations in Neonatal Mouse Hb9 Interneurons. PLoS ONE 7(10): e47940. doi:10.1371/journal.pone.0047940 TTX-Resistant NMDA Receptor-Mediated Membrane Potential Oscillations in Neonatal Mouse Hb9 Interneurons Mark A. Masino 0 Matthew D. Abbinanti 0 John Eian 0 Ronald M. Harris-Warrick 0 Gennady Cymbalyuk, Georgia State University, United States of America 0 1 Department of Neuroscience, University of Minnesota , Minneapolis , Minnesota, United States of America, 2 Department of Neurobiology and Behavior, Cornell University , Ithaca, New York , United States of America Conditional neuronal membrane potential oscillations have been identified as a potential mechanism to help support or generate rhythmogenesis in neural circuits. A genetically identified population of ventromedial interneurons, called Hb9, in the mouse spinal cord has been shown to generate TTX-resistant membrane potential oscillations in the presence of NMDA, serotonin and dopamine, but these oscillatory properties are not well characterized. Hb9 interneurons are rhythmically active during fictive locomotor-like behavior. In this study, we report that exogenous N-Methyl-D-Aspartic acid (NMDA) application is sufficient to produce membrane potential oscillations in Hb9 interneurons. In contrast, exogenous serotonin and dopamine application, alone or in combination, are not sufficient. The properties of NMDA-induced oscillations vary among the Hb9 interneuron population; their frequency and amplitude increase with increasing NMDA concentration. NMDA does not modulate the T-type calcium current (ICa(T)), which is thought to be important in generating locomotor-like activity, in Hb9 neurons. These results suggest that NMDA receptor activation is sufficient for the generation of TTX-resistant NMDA-induced membrane potential oscillations in Hb9 interneurons. - Funding: This work was supported by National Institutes of Health Grants R01-NS065054 (MAM) and R01-NS35631 and R01-NS17323 (to RMHW), Minnesota Medical Foundation Grant 4052-9238-11 (MAM) and Office of the Dean of the Graduate School of the University of Minnesota (Grant-in-Aid of Research, Artistry and Scholarship 21934 (MAM)).NIH: www.nih.gov MMF: www.mmf.umn.edu GIA: www.research.umn.edu/advance/gia.html#.T_2YrGDcFBw. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In vertebrates, neural circuits called central pattern generators (CPGs) that produce rhythmic motor output during locomotion are located in the ventromedial region of the spinal cord [19]. Motor activity in many rhythm-generating networks is determined, in part, by the intrinsic properties of the constituent neurons [1017]. These intrinsic properties are determined by the repertoire of ionic currents expressed by individual neurons [16,1820]. For example, NMDA receptors in vertebrates are activated in spinal circuits during locomotor activity [1,2125] and NMDA is known to induce TTX-resistant membrane potential oscillations in a number of neuron types in various regions of the central nervous system [2636]. Although the identities of the neural components and the cellular mechanisms that participate in driving rhythmic locomotor activity in vertebrate CPGs are not well understood, conditional neuronal membrane potential oscillations have been identified as a potential mechanism to help support rhythmogenesis in neural circuits [2634,36]. A recent strategy to characterize the cellular properties that promote voltage oscillations has been to monitor the activity of identified neuronal populations from transgenic lines of mice that express fluorescent proteins under the control of specific promoter constructs, such as transcription factors [35,3744]. Using this approach, it has been shown that the Hb9 interneurons, a class of ventromedial excitatory interneurons, are rhythmically active during fictive locomotion in neonatal mice [40,41]. Further, Hb9 interneurons appear to be conditional oscillators since they generate TTX-resistant membrane potential oscillations in the presence of NMDA, serotonin and dopamine [35,41]. Although Hb9 interneurons are unlikely to be, by themselves, responsible for producing the locomotor rhythm in neonatal mice [35,45,46], their bursting properties, location, and rhythmicity during locomotor activity have led to the suggestion that these interneurons play a role in generation of the locomotor pattern [40,41,4750]. Thus, we studied the membrane properties underlying their rhythmicity. We find that NMDA receptor activation can induce strong Hb9 oscillations, and does not do so via activation of low threshold calcium currents. Materials and Methods Animals All procedures were approved by the Institutional Animal Care and Use Committees at the University of Minnesota and Cornell University and were in accordance with National Institutes of Health guidelines. Experiments were performed on spinal cords isolated from transgenic mice (Hb9::eGFP provided by Robert Brownstone, Dalhousie University) from post-natal day 3 (P3) to P9. Animals were euthanized by acute decapitation, as recommended by the AMVA Panel on Euthanasia. Spinal Cord Preparation The spinal cord from T9-S1 was removed by laminectomy in ice-cold (4uC), oxygenated (95% O2/5% CO2) low calcium Ringers solution (in mM: 128 NaCl, 4.7 KCl, 1.2 KH2PO4, 0.25 CaCl2, 1.3 MgCl2, 3.25 MgSO4, 25 NaHCO3, and 22 Dglucose). The meninges were removed and the cord was imbedded in 3.7% agarose (Invitrogen; UltraPure Agarose) in either HEPES Ringers solution (in mM: 101 NaCl, 3.8 KCl, 18.7 MgCl2, 1.3 MgSO4, 1.2 KH2PO4, 1.0 CaCl2, 10 HEPES and 25 D-glucose; pH to 7.4 with NaOH) or sucrose solution (in mM: 188 sucrose, 25 D-glucose, 26 NaHCO3, 25 NaCl, 10 MgSO4, 1.2 NaH2PO4 and 1.9 KCl; pH to 7.4 with NaOH). The imbedded spinal section was transferred to a vibrating microtome (Leica, VT1000S or VT1200S), and transverse sections (200300 mm) of the L1-L3 region were cut in HEPES Ringers or sucrose solution at 0uC, transferred immediately to an incubation chamber containing prewarmed (30uC), oxygenated (95% O2/5% CO2) normal Ringers solution and allowed to equilibrate for at least 30 minutes before starting the experiment. Pharmacology The following channel blockers were used: TTX (1 mM, to block voltage-gated sodium current), tetraethylammonium chloride (TEA-Cl, 30 mM to block voltage-gated potassium current), 4-aminopyridine, (4-AP, 4 mM, to block fast transient potassium current), CsCl (2 mM, to block hyperpolarization activated inward current). Pharmacological agents used to evoke endogenous membrane potential oscillations in slice preparations were NMethyl-D-aspartic acid (NMDA 321 mM), serotonin creatinine sulfate complex (5-HT 321 mM) and dopamine hydrochloride (DA (...truncated)