Brain Structure and Function

, Volume 220, Issue 5, pp 2835–2849 | Cite as

Genetic ablation of VIAAT in glycinergic neurons causes a severe respiratory phenotype and perinatal death

  • Jamilur Rahman
  • Stefanie Besser
  • Christian Schnell
  • Volker Eulenburg
  • Johannes Hirrlinger
  • Sonja M. WojcikEmail author
  • Swen HülsmannEmail author
Original Article


Both glycinergic and GABAergic neurons require the vesicular inhibitory amino acid transporter (VIAAT) for synaptic vesicle filling. Presynaptic GABA concentrations are determined by the GABA-synthesizing enzymes glutamate decarboxylase (GAD)65 and GAD67, whereas the presynaptic glycine content depends on the plasma membrane glycine transporter 2 (GlyT2). Although severely impaired, glycinergic transmission is not completely absent in GlyT2-knockout mice, suggesting that other routes of glycine uptake or de novo synthesis of glycine exist in presynaptic terminals. To investigate the consequences of a complete loss of glycinergic transmission, we generated a mouse line with a conditional ablation of VIAAT in glycinergic neurons by crossing mice with loxP-flanked VIAAT alleles with a GlyT2-Cre transgenic mouse line. Interestingly, conditional VIAAT knockout (VIAAT cKO) mice were not viable at birth. In addition to the dominant respiratory failure, VIAAT cKO showed an umbilical hernia and a cleft palate. Immunohistochemistry revealed an almost complete depletion of VIAAT in the brainstem. Electrophysiology revealed the absence of both spontaneous glycinergic and GABAergic inhibitory postsynaptic currents from hypoglossal motoneurons. Our results demonstrate that the deletion of VIAAT in GlyT2-Cre expressing neurons also strongly affects GABAergic transmission and suggest a large overlap of the glycinergic and the GABAergic neuron population during early development in the caudal parts of the brain.


Embryonic development Transmitter release Vesicular filling Electrophysiology Brainstem 



This work was funded by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB). SH and JH received additional support from the DFG (Hu797/7-1, 8-1; Hi1414/2-1). The authors are grateful to Anja-Annett Grützner, Astrid Zeuch, Astrid Ohle and Annette Fahrenholz for technical assistance.


  1. Aubrey KR, Rossi FM, Ruivo R, Alboni S, Bellenchi GC, Le Goff A, Gasnier B, Supplisson S (2007) The transporters GlyT2 and VIAAT cooperate to determine the vesicular glycinergic phenotype. J Neurosci 27(23):6273–6281. doi: 10.1523/JNEUROSCI.1024-07.2007 CrossRefPubMedGoogle Scholar
  2. Bentzen BH, Grunnet M (2011) Central and peripheral GABA(A) receptor regulation of the heart rate depends on the conscious state of the animal. Adv Pharmacol Sci 2011:578273. doi: 10.1155/2011/578273 PubMedCentralPubMedGoogle Scholar
  3. Beyoglu D, Idle JR (2012) The glycine deportation system and its pharmacological consequences. Pharmacol Ther 135(2):151–167. doi: 10.1016/j.pharmthera.2012.05.003 PubMedCentralCrossRefPubMedGoogle Scholar
  4. Butera RJ Jr, Rinzel J, Smith JC (1999) Models of respiratory rhythm generation in the pre-Botzinger complex. I. Bursting pacemaker neurons. J Neurophysiol 82(1):382–397PubMedGoogle Scholar
  5. Condie BG, Bain G, Gottlieb DI, Capecchi MR (1997) Cleft palate in mice with a targeted mutation in the gamma-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67. Proc Natl Acad Sci USA 94(21):11451–11455PubMedCentralCrossRefPubMedGoogle Scholar
  6. Davies LP, Johnston GA (1974) Postnatal changes in the levels of glycine and the activities of serine hydroxymethyltransferase and glycine:2-oxoglutarate aminotransferase in the rat central nervous system. J Neurochem 22(1):107–112CrossRefPubMedGoogle Scholar
  7. Delpy A, Allain AE, Meyrand P, Branchereau P (2008) NKCC1 cotransporter inactivation underlies embryonic development of chloride-mediated inhibition in mouse spinal motoneuron. J Physiol 586(4):1059–1075. doi: 10.1113/jphysiol.2007.146993 PubMedCentralCrossRefPubMedGoogle Scholar
  8. Ding R, Tsunekawa N, Obata K (2004) Cleft palate by picrotoxin or 3-MP and palatal shelf elevation in GABA-deficient mice. Neurotoxicol Teratol 26(4):587–592. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  9. Drorbaugh JE, Fenn WO (1955) A barometric method for measuring ventilation in newborn infants. Pediatrics 16(1):81–87PubMedGoogle Scholar
  10. Feng G, Tintrup H, Kirsch J, Nichol MC, Kuhse J, Betz H, Sanes JR (1998) Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity. Science 282(5392):1321–1324CrossRefPubMedGoogle Scholar
  11. Ferguson C, Hardy S, Werner D, Hileman S, DeLorey T, Homanics G (2007) New insight into the role of the beta3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout. BMC Neurosci 8(1):85PubMedCentralCrossRefPubMedGoogle Scholar
  12. Fujii M, Arata A, Kanbara-Kume N, Saito K, Yanagawa Y, Obata K (2007) Respiratory activity in brainstem of fetal mice lacking glutamate decarboxylase 65/67 and vesicular GABA transporter. Neuroscience 146(3):1044–1052. doi: 10.1016/j.neuroscience.2007.02.050 CrossRefPubMedGoogle Scholar
  13. Gomeza J, Hulsmann S, Ohno K, Eulenburg V, Szoke K, Richter D, Betz H (2003a) Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition. Neuron 40(4):785–796CrossRefPubMedGoogle Scholar
  14. Gomeza J, Ohno K, Hulsmann S, Armsen W, Eulenburg V, Richter DW, Laube B, Betz H (2003b) Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron 40(4):797–806CrossRefPubMedGoogle Scholar
  15. Gray PA, Janczewski WA, Mellen N, McCrimmon DR, Feldman JL (2001) Normal breathing requires preBotzinger complex neurokinin-1 receptor-expressing neurons. Nat Neurosci 4(9):927–930. doi: 10.1038/nn0901-927 PubMedCentralCrossRefPubMedGoogle Scholar
  16. Gunther U, Benson J, Benke D, Fritschy JM, Reyes G, Knoflach F, Crestani F, Aguzzi A, Arigoni M, Lang Y et al (1995) Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors. Proc Natl Acad Sci USA 92(17):7749–7753PubMedCentralCrossRefPubMedGoogle Scholar
  17. Hubner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, Jentsch TJ (2001) Disruption of KCC2 reveals an essential role of K–Cl cotransport already in early synaptic inhibition. Neuron 30(2):515–524CrossRefPubMedGoogle Scholar
  18. Hulsmann S, Oku Y, Zhang W, Richter DW (2000) Metabotropic glutamate receptors and blockade of glial Krebs cycle depress glycinergic synaptic currents of mouse hypoglossal motoneurons. Eur J Neurosci 12(1):239–246CrossRefPubMedGoogle Scholar
  19. Ishihara N, Armsen W, Papadopoulos T, Betz H, Eulenburg V (2010) Generation of a mouse line expressing Cre recombinase in glycinergic interneurons. Genesis 48(7):437–445. doi: 10.1002/dvg.20640 CrossRefPubMedGoogle Scholar
  20. Janczewski WA, Onimaru H, Homma I, Feldman JL (2002) Opioid-resistant respiratory pathway from the preinspiratory neurones to abdominal muscles: in vivo and in vitro study in the newborn rat. J Physiol 545(Pt 3):1017–1026. doi: 10.1113/jphysiol.2002.023408 PubMedCentralCrossRefPubMedGoogle Scholar
  21. Jo YH (2012) Endogenous BDNF regulates inhibitory synaptic transmission in the ventromedial nucleus of the hypothalamus. J Neurophysiol 107(1):42–49. doi: 10.1152/jn.00353.2011 CrossRefPubMedGoogle Scholar
  22. Jursky F, Nelson N (1995) Localization of glycine neurotransmitter transporter (GLYT2) reveals correlation with the distribution of glycine receptor. J Neurochem 64(3):1026–1033. doi: 10.1046/j.1471-4159.1995.64031026.x CrossRefPubMedGoogle Scholar
  23. Jursky F, Nelson N (1996) Developmental expression of the glycine transporters GLYT1 and GLYT2 in mouse brain. J Neurochem 67(1):336–344CrossRefPubMedGoogle Scholar
  24. Kikuchi G (1973) The glycine cleavage system: composition, reaction mechanism, and physiological significance. Mol Cell Biochem 1(2):169–187. doi: 10.1007/BF01659328 CrossRefPubMedGoogle Scholar
  25. Kling C, Koch M, Saul B, Becker CM (1997) The frameshift mutation oscillator [Glra1(spd-ot)] produces a complete loss of glycine receptor alpha1-polypeptide in mouse central nervous system. Neuroscience 78(2):411–417CrossRefPubMedGoogle Scholar
  26. Kuwana S, Tsunekawa N, Yanagawa Y, Okada Y, Kuribayashi J, Obata K (2006) Electrophysiological and morphological characteristics of GABAergic respiratory neurons in the mouse pre-Botzinger complex. Eur J Neurosci 23(3):667–674. doi: 10.1111/j.1460-9568.2006.04591.x CrossRefPubMedGoogle Scholar
  27. Lal A, Oku Y, Hulsmann S, Okada Y, Miwakeichi F, Kawai S, Tamura Y, Ishiguro M (2011) Dual oscillator model of the respiratory neuronal network generating quantal slowing of respiratory rhythm. J Comput Neurosci 30(2):225–240. doi: 10.1007/s10827-010-0249-0 PubMedCentralCrossRefPubMedGoogle Scholar
  28. Latal AT, Kremer T, Gomeza J, Eulenburg V, Hulsmann S (2010) Development of synaptic inhibition in glycine transporter 2 deficient mice. Mol Cell Neurosci 44(4):342–352. doi: 10.1016/j.mcn.2010.04.005 CrossRefPubMedGoogle Scholar
  29. Liu P, Jenkins NA, Copeland NG (2003) A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 13(3):476–484Google Scholar
  30. Luque JM, Nelson N, Richards JG (1995) Cellular expression of glycine transporter 2 messenger RNA exclusively in rat hindbrain and spinal cord. Neuroscience 64(2):525–535. doi: 10.1016/0306-4522(94)00404-S CrossRefPubMedGoogle Scholar
  31. McIntire SL, Reimer RJ, Schuske K, Edwards RH, Jorgensen EM (1997) Identification and characterization of the vesicular GABA transporter. Nature 389(6653):870–876. doi: 10.1038/39908 CrossRefPubMedGoogle Scholar
  32. Medrihan L, Rohlmann A, Fairless R, Andrae J, Doring M, Missler M, Zhang W, Kilimann MW (2009) Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses. J Physiol 587(Pt 21):5095–5106. doi: 10.1113/jphysiol.2009.178236 PubMedCentralCrossRefPubMedGoogle Scholar
  33. Morgado-Valle C, Baca SM, Feldman JL (2010) Glycinergic pacemaker neurons in preBotzinger complex of neonatal mouse. J Neurosci 30(10):3634–3639. doi: 10.1523/JNEUROSCI.3040-09.2010 PubMedCentralCrossRefPubMedGoogle Scholar
  34. Nakauchi J, Matsuo H, Kim DK, Goto A, Chairoungdua A, Cha SH, Inatomi J, Shiokawa Y, Yamaguchi K, Saito I, Endou H, Kanai Y (2000) Cloning and characterization of a human brain Na+-independent transporter for small neutral amino acids that transports d-serine with high affinity. Neurosci Lett 287(3):231–235. doi: 10.1016/s0304-3940(00)01169-1 CrossRefPubMedGoogle Scholar
  35. Oh WJ, Westmoreland JJ, Summers R, Condie BG (2010) Cleft palate is caused by CNS dysfunction in Gad1 and VIAAT knockout mice. PLoS ONE 5(3):e9758. doi: 10.1371/journal.pone.0009758 PubMedCentralCrossRefPubMedGoogle Scholar
  36. Onimaru H, Arata A, Homma I (1990) Inhibitory synaptic inputs to the respiratory rhythm generator in the medulla isolated from newborn rats. Pflug Arch 417(4):425–432CrossRefGoogle Scholar
  37. Paxinos G (2007) Atlas of the developing mouse brain: at E17.5, PO, and P6. Elsevier, Acad. Press, Amsterdam [u.a.]Google Scholar
  38. Rahman J, Latal AT, Besser S, Hirrlinger J, Hulsmann S (2013) Mixed miniature postsynaptic currents resulting from co-release of glycine and GABA recorded from glycinergic neurons in the neonatal respiratory network. Eur J Neurosci 37(8):1229–1241. doi: 10.1111/ejn.12136 CrossRefPubMedGoogle Scholar
  39. Sagne C, El Mestikawy S, Isambert MF, Hamon M, Henry JP, Giros B, Gasnier B (1997) Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases. FEBS Lett 417(2):177–183CrossRefPubMedGoogle Scholar
  40. Saito K, Kakizaki T, Hayashi R, Nishimaru H, Furukawa T, Nakazato Y, Takamori S, Ebihara S, Uematsu M, Mishina M, Miyazaki J, Yokoyama M, Konishi S, Inoue K, Fukuda A, Fukumoto M, Nakamura K, Obata K, Yanagawa Y (2010) The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice. Mol Brain 3:40. doi: 10.1186/1756-6606-3-40 PubMedCentralCrossRefPubMedGoogle Scholar
  41. Shimizu Y, Thumkeo D, Keel J, Ishizaki T, Oshima H, Oshima M, Noda Y, Matsumura F, Taketo MM, Narumiya S (2005) ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles. J Cell Biol 168(6):941–953. doi: 10.1083/jcb.200411179 PubMedCentralCrossRefPubMedGoogle Scholar
  42. Thoby-Brisson M, Karlen M, Wu N, Charnay P, Champagnat J, Fortin G (2009) Genetic identification of an embryonic parafacial oscillator coupling to the preBotzinger complex. Nat Neurosci 12(8):1028–1035. doi: 10.1038/nn.2354 CrossRefPubMedGoogle Scholar
  43. Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in fetal mice lacking glutamate decarboxylase 67. Eur J Neurosci 21(1):173–178. doi: 10.1111/j.1460-9568.2004.03860.x CrossRefPubMedGoogle Scholar
  44. Turgeon B, Meloche S (2009) Interpreting neonatal lethal phenotypes in mouse mutants: insights into gene function and human diseases. Physiol Rev 89(1):1–26. doi: 10.1152/physrev.00040.2007 CrossRefPubMedGoogle Scholar
  45. Tyzio R, Cossart R, Khalilov I, Minlebaev M, Hubner CA, Represa A, Ben-Ari Y, Khazipov R (2006) Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery. Science 314(5806):1788–1792. doi: 10.1126/science.1133212 CrossRefPubMedGoogle Scholar
  46. Wallen-Mackenzie A, Gezelius H, Thoby-Brisson M, Nygard A, Enjin A, Fujiyama F, Fortin G, Kullander K (2006) Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation. J Neurosci 26(47):12294–12307. doi: 10.1523/JNEUROSCI.3855-06.2006 CrossRefPubMedGoogle Scholar
  47. Winter SM, Hirrlinger J, Kirchhoff F, Hulsmann S (2007) Transgenic expression of fluorescent proteins in respiratory neurons. Respir Physiol Neurobiol 159(1):108–114. doi: 10.1016/j.resp.2007.05.009 CrossRefPubMedGoogle Scholar
  48. Winter SM, Fresemann J, Schnell C, Oku Y, Hirrlinger J, Hulsmann S (2009) Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices. Pflug Arch 458(3):459–469. doi: 10.1007/s00424-009-0647-1 CrossRefGoogle Scholar
  49. Wittmeier S, Song G, Duffin J, Poon CS (2008) Pacemakers handshake synchronization mechanism of mammalian respiratory rhythmogenesis. Proc Natl Acad Sci USA 105(46):18000–18005. doi: 10.1073/pnas.0809377105 PubMedCentralCrossRefPubMedGoogle Scholar
  50. Wojcik SM, Katsurabayashi S, Guillemin I, Friauf E, Rosenmund C, Brose N, Rhee JS (2006) A shared vesicular carrier allows synaptic corelease of GABA and glycine. Neuron 50(4):575–587. doi: 10.1016/j.neuron.2006.04.016 CrossRefPubMedGoogle Scholar
  51. Yao D, Mackenzie B, Ming H, Varoqui H, Zhu H, Hediger MA, Erickson JD (2000) A novel system A isoform mediating Na+/neutral amino acid cotransport. J Biol Chem 275(30):22790–22797. doi: 10.1074/jbc.M002965200 CrossRefPubMedGoogle Scholar
  52. Zafra F, Aragon C, Olivares L, Danbolt NC, Gimenez C, Storm-Mathisen J (1995a) Glycine transporters are differentially expressed among CNS cells. J Neurosci 15(5 Pt 2):3952–3969PubMedGoogle Scholar
  53. Zafra F, Gomeza J, Olivares L, Aragon C, Gimenez C (1995b) Regional distribution and developmental variation of the glycine transporters GLYT1 and GLYT2 in the rat CNS. Eur J Neurosci 7(6):1342–1352. doi: 10.1111/j.1460-9568.1995.tb01125.x CrossRefPubMedGoogle Scholar
  54. Zeilhofer HU, Studler B, Arabadzisz D, Schweizer C, Ahmadi S, Layh B, Bosl MR, Fritschy JM (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482(2):123–141. doi: 10.1002/cne.20349 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jamilur Rahman
    • 3
  • Stefanie Besser
    • 4
  • Christian Schnell
    • 2
    • 3
    • 8
  • Volker Eulenburg
    • 5
  • Johannes Hirrlinger
    • 4
    • 7
  • Sonja M. Wojcik
    • 6
    Email author
  • Swen Hülsmann
    • 1
    • 2
    • 3
    Email author
  1. 1.Clinic for Anesthesiology, Laboratory for Experimental NeuroanesthesiologyUniversity Hospital GöttingenGöttingenGermany
  2. 2.Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB)GöttingenGermany
  3. 3.Institute for Neurophysiology and Cellular BiophysicsGeorg-August-University GöttingenGöttingenGermany
  4. 4.Carl-Ludwig-Institute for PhysiologyUniversity of LeipzigLeipzigGermany
  5. 5.Institute for Biochemistry and Molecular MedicineUniversity of ErlangenErlangenGermany
  6. 6.Department of Molecular NeurobiologyMax Planck Institute of Experimental MedicineGöttingenGermany
  7. 7.Department of NeurogeneticsMax Planck Institute of Experimental MedicineGöttingenGermany
  8. 8.Divisions of Pathophysiology and Repair and Neuroscience, School of BiosciencesCardiff UniversityCardiffUK

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