title: ALX 1393 inhibits spontaneous network activity by inducing glycinergic tonic currents in the spinal ventral horn.
abstract: BACKGROUND
  Strychnine-sensitive glycine receptors are activated by glycine and facilitate chloride influx into neurons. Glycinergic transmission might be either mediated by synaptic or extrasynaptic glycine receptors. While phasic neurotransmission is provided by a synaptic pathway, activation of extrasynaptic glycine receptors induces tonic inhibition. The glycine transporter 2 (GlyT2) regulates the uptake of glycine into presynaptic boutons. It is not determined yet, whether inhibition of GlyT2 by ALX 1393 can produce inhibition of spinal motoric networks and, whether phasic or tonic glycinergic inhibition is mostly enhanced.
  
  
  METHODS
  We investigated the effect of ALX 1393 on spontaneous action potential firing activity by extracellular recordings in the ventral horn area of organotypic spinal cultures. Additionally, using the whole-cell patch-clamp technique, we defined the influence of GlyT2 inhibition on tonic and phasic glycinergic transmission in commissural interneurons of the ventral horn.
  
  
  RESULTS
  GlyT2 inhibition by ALX 1393 potently reduced neuronal action potential activity in a concentration-dependent manner (n=211). The half maximal effect of ALX 1393 was observed at 100 ± 31 nM. Moreover, 88.3 ± 2.6% of the action potential activity was suppressed at 1 μM. Whole-cell patch-clamp recordings unveiled that ALX 1393 (200 nM) induced a tonic current (-45.7 ± 11.6 pA, n=5) that was significantly reversed by application of the competitive glycine receptor antagonist strychnine. Contrastingly, phasic glycinergic transmission was not augmented by GlyT2 inhibition (charge transferred per time period for control conditions: 1.1 ± 0.1 pC, n=7, for ALX 1393: 0.9 ± 0.2 pC, n=7, p>0.05).
  
  
  CONCLUSIONS
  GlyT2 inhibition induced glycinergic tonic currents, which might be the underlying mechanism for the observed suppression of spontaneous action potential activity by ALX 1393 in the spinal ventral horn. Silencing neuronal action potential activity by blocking GlyT2 might be a novel principle to inhibit locomotor circuits in the ventral horn area and to induce muscle relaxation.
authors.affiliation: Section of Experimental Anesthesiology, Department of Anesthesiology & Intensive Care Medicine, Eberhard-Karls-University, Waldhörnlestr. 22, D-72072 Tübingen, Germany. Electronic address: veit-simon.eckle@uni-tuebingen.de.
authors.forename: V-S
authors.initials: VS
authors.lastname: Eckle
authors.forename: B
authors.initials: B
authors.lastname: Antkowiak
mesh_terms: D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects*; D000818:Animals; D000870:Anterior Horn Cells / Q000187:drug effects; D055592:Biophysical Phenomena / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism*; D018684:Glycine Agents / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors*; D051379:Mice; D008810:Mice, Inbred C57BL; D009415:Nerve Net / Q000187:drug effects* / Q000378:metabolism; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D012694:Serine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013116:Spinal Cord / Q000166:cytology*; D013997:Time Factors; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C539864:ALX 1393; D018691:Excitatory Amino Acid Antagonists; D018684:Glycine Agents; D050487:Glycine Plasma Membrane Transport Proteins; D012694:Serine; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; C056639:2-amino-5-phosphopentanoic acid; D014633:Valine; D005998:Glycine
keywords: ACSF; ALX 1393; GlyT2; IPSCs; artificial cerebrospinal fluid; glycine receptor; glycine transporter 2; glycine transporter type 2; inhibitory postsynaptic currents; organotypic culture; spinal cord; ventral horn