Skip to main content
SpringerLink
Log in

An in situ hybridization and immunofluorescence study of glycinergic receptors and gephyrin in the vestibular nuclei of the intact and unilaterally labyrinthectomized rat

  • Research Article
  • Published:
Experimental Brain Research Aims and scope Submit manuscript

Abstract

We investigated whether the expression of glycinergic receptor (GLYR) subunits of gephyrin and of their mRNAs in the medial vestibular nuclei are affected following unilateral labyrinthectomy. Specific radioactive oligonucleotide probes recognizing the sequences encoding α1–3 and ß subunits of GLYR and the anchoring protein gephyrin were used to probe sections of vestibular nuclei. Signals in these in situ hybridization experiments were detected with film or by emulsion photography. Animals were killed at various times following the lesion: 5 h, 1, 3, 8, 30 and 60 days. Specific monoclonal GLYR and gephyrin antibodies were also used to determine GLYR and gephyrin immunoreactivity in control and operated rats (5 h, 1, 3 and 8 days post-lesion). In normal animals, several brainstem regions including the lateral, medial, superior and inferior vestibular nuclei contained mRNAs for gephyrin and the α1 and β subunits of GLYR, and expressed the GLYR and gephyrin polypeptides. In unilaterally labyrinthectomized rats, no asymmetry was detected on autoradiographs between the two medial vestibular nuclei with any of the oligonucleotide probes used, or at any time following the lesion. No difference in the immunofluorescence staining was observed between the intact and deafferented medial vestibular nuclei of lesioned animals or between the vestibular nuclei of lesioned and controls rats. Thus, deafferentation of the vestibular nuclei did not affect the expression of gephyrin, of the various GLYR subunits, or of their mRNAs in the deafferented and intact medial vestibular nuclei. It is therefore unlikely that GLYR and gephyrin modulation contribute significantly to the recovery of the resting discharge of the deafferented medial vestibular neurons and consequently to the restoration of a normal posture and eye position.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1A–F
Fig. 2A, B
Fig. 3A–C
Fig. 4A–D
Fig. 5

Similar content being viewed by others

Abbreviations

AMPA:

α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

AF:

Arithmetic fluorescence

CNS:

Central nervous system

DTT:

Dithiothreitol

EGTA:

Ethylene glycol tetraacetic acid

GABA:

γ-aminobutyric acid

GLY:

Glycine

GLYR:

Glycine receptors

MVN:

Medial vestibular nucleus

MVNn:

Medial vestibular nucleus neurons

OD:

Optical density

PBS:

Phosphate buffered saline

mRNA:

Messenger ribonucleic acid

MVNn:

Medial vestibular nuclei neurons

NMDA:

N-methyl-D-aspartate

rAF:

Relative arithmetic fluorescence

rOD:

Relative optical density

tRNA:

Transfer ribonucleic acid

SD:

Standard deviation

SSC:

Standard saline citrate

VNn:

Vestibular nuclei neurons

References

  • Alvarez FJ, Harrington DA, Dewey DE, Fyffe REW (1997) Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord. J Comp Neurol 379:150–170

    Google Scholar 

  • Balaban CD, Romero GG (1998) A role of climbing fibers in regulation of flocculonodular lobe protein kinase C expression during vestibular compensation. Brain Res 804:253–265

    CAS  PubMed  Google Scholar 

  • Betz H, Araki T, Yamamoto M, Murakami T, Wanaka A, Tohyama M (1988) Localization of glycine receptors in the rat central nervous system: an immunocytochemical analysis using monoclonal antibody. Neuroscience 25:613–624

    Article  PubMed  Google Scholar 

  • Betz H, Kuhse J, Schmieden V, Malosio ML, Langosch D, Prior P, Schmitt B, Kirsch J (1991) How to build a glycinergic postsynaptic membrane. J Cell Sci Suppl 15:23–25

    CAS  PubMed  Google Scholar 

  • Cabot JB, Bushnell V, Alessi V, Mendell NR (1995) Postsynaptic gephyrin immunoreactivity exhibits a nearly one-to-one correspondence with gamma-aminobutyric acid-like immunogold-labeled synaptic inputs to sympathetic preganglionic neurons. J Comp Neurol 356:418–432

    Google Scholar 

  • Calza L, Giardino L, Zanni M, Galetti G (1992) Muscarinic and gamma-aminobutyric acid-ergic receptor changes during vestibular compensation. A quantitative autoradiographic study of the vestibular nuclei complex in the rat. Eur Arch Otorhinolaryngol 249:34–39

    CAS  PubMed  Google Scholar 

  • Curthoys IS, Halmagyi GM (1995) Vestibular compensation: a review of the oculomotor, neural, and clinical consequences of unilateral vestibular loss. J Vestib Res 5:67–107

    CAS  PubMed  Google Scholar 

  • Darlington CL, Dutia MB, Smith PF (2002) The contribution of the intrinsic excitability of vestibular nucleus neurons to recovery from vestibular damage. Eur J Neurosci 15:1719–1727

    Article  PubMed  Google Scholar 

  • de Waele C, Lapeyre PN (1995) Glycinergic inhibition of spontaneously active guinea-pig medial vestibular nucleus in vitro. Neurosci Lett 188:155–158

    Article  PubMed  Google Scholar 

  • de Waele C, Serafin M, Mühlethaler M, Vidal PP (1988) Vestibular compensation: an in vivo and in vitro study of second order vestibular neurons. Soc Neurosci Abstr 14:331

    Google Scholar 

  • de Waele C, Chat M, Arbitol M, Menini C, Mallet J, Vidal PP (1994) Distribution of glutamatergic receptors and GAD mRNA-containing neurons in the vestibular nuclei of normal and hemilabyrinthectomized rats. Eur J Neurosci 6:565–576

    PubMed  Google Scholar 

  • de Waele C, Muhlethaler M, Vidal PP (1995) Neurochemistry of the central vestibular pathways. Brain Res Rev 20:24–46

    PubMed  Google Scholar 

  • de Waele C, Loquet G, Campos Torres A, Vidal PP (2000) Lack of growth-associated protein-43 reemergence or of growth-associated protein-43 mRNA modulation in deafferented vestibular nuclei during the first 6 weeks after unilateral inner ear lesion. Exp Brain Res 132:464–475

    Article  PubMed  Google Scholar 

  • Dieringer N (1995) Vestibular compensation: neural plasticity and its relation to functional recovery after labyrinthine lesion in frog and other vertebrate. Prog Neurobiol 46:97–129

    CAS  PubMed  Google Scholar 

  • Fish U (1973) The vestibular responses following unilateral vestibular neurectomy. Acta Otolaryng 76:229–238

    Google Scholar 

  • Friauf E, Hammerschmidt B, Kirsh J (1997) Developement of adult-type inhibitory glycine receptors in the central auditory system of rats. J Comp Neurol 385:117–134

    Google Scholar 

  • Frostholm A, Rotter A (1985) Glycine receptor distribution in mouse CNS: autoradiographic localization of [3H]strychnine binding sites. Brain Res Bull 15:473–486

    Article  PubMed  Google Scholar 

  • Fujita M, Sato K, Sato M, Inoue T, Kozuka T, Tohyhama M (1991) Regional distribution of the cells expressing glycine receptor β subunit mRNA in rat brain. Brain Res 560:23–27

    Article  CAS  PubMed  Google Scholar 

  • Furuya N, Koizumi T (1998) Neurotransmitters of vestibular commissural inhibition in the cat. Acta Otolaryngol 18:64–69

    Google Scholar 

  • Furuya N, Yabe T, Koizumi T (1992) Neurotransmitters in the vestibular commissural system of the cat. Ann N Y Acad Sci 656:594–601

    CAS  PubMed  Google Scholar 

  • Gacek RR, Lyon MJ, Schoonmaker J (1988) Ultrastructural changes in vestibulo-ocular neurons following vestibular neurectomy in the cat. Ann Otol Rhinol Laryngol 97:42–51

    Google Scholar 

  • Goto MM, Romero GG, Balaban CD (1997) Transient changes in flocculonodular lobe protein kinase C expression during vestibular compensation. Neuroscience 17:4367–4381

    CAS  PubMed  Google Scholar 

  • Grenningloh G, Pribilla I, Prior P, Muthaup G, Beyreuther K, Taleb O, Betz H (1990) Cloning and expression of the 58 Kd beta subunit of inhibitory glycine receptor. Neuron 4:963–970

    CAS  PubMed  Google Scholar 

  • Hironaka T, Morita Y, Hagihira S, Tateno E, Kita H, Tohyama M (1990) Localization of GABAA-receptor α1 subunit mRNA-containing neurons in the lower brainstem of the rat. Mol Brain Res 7:335–345

    Article  CAS  PubMed  Google Scholar 

  • Horii A, Smith PF, Darlington CL (2001) Quantitative changes in gene expression of glutamate receptor subunits/subtypes in the vestibular nucleus, inferior olive and flocculus before and following unilateral labyrinthectomy in the rat: real-time quantitative PCR method. Exp Brain Res 139:188–200

    Article  CAS  PubMed  Google Scholar 

  • Kerr DR, Sansom AJ, Smith PF, Darlington CL (2000) Comparison of protein kinase activity and protein phosphorylation in the medial vestibular nucleus and prepositus hypoglossi in labyrinthine-intact and labyrinthectomized guinea pigs. J Vestib Res 10:107–117

    CAS  PubMed  Google Scholar 

  • King J, Zheng Y, Liu P, Darlington CL, Smith PF (2002) NMDA and AMPA receptor subunit protein expression in the rat vestibular nucleus following unilateral labyrinthectomy. Neuroreport 13:1541–1545

    CAS  PubMed  Google Scholar 

  • Kirsh J, Betz H (1993) Widespread distribution of gephyrin, a putative glycine receptor-tubulin linker protein, in rat brain. Brain Res 621:301–310

    Article  PubMed  Google Scholar 

  • Kirsh J, Betz H, Triller A, Wolters I (1993a) Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons. Nature 366:745–748

    CAS  PubMed  Google Scholar 

  • Kirsh J, Malosio ML, Wolters I, Betz H (1993b) Distribution of gephyrin transcripts in the adult and developing rat brain. Eur J Neurosci 5:1109–1117

    PubMed  Google Scholar 

  • Kneussel M, Hermann A, Kirsh J, Betz H (1999) Hydrophobic interactions mediate binding of the glycine receptor beta-subunit to gephyrin. J Neurochem 72:1323–1326

    Article  CAS  PubMed  Google Scholar 

  • Krenning J, Hughes LF, Caspary DM, Helfert RH (1998) Age-related glycine receptor subunit changes in the cochlear nucleus of Fischer-344 rats. Laryngoscope 108:26–31

    Google Scholar 

  • Kuhse J, Betz H, Kirsch J (1995) The inhibitory glycine receptor: architecture, synaptic localization, and molecular pathology of a post-synaptic ion-channel complex. Curr Opin Neurobiol 5:318–323

    Article  CAS  PubMed  Google Scholar 

  • Laurie DJ, Seeburg PH, Wisden W (1992) The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II Olfactory bulb and cerebellum. J Neurosci 12:1063–1076

    CAS  PubMed  Google Scholar 

  • Li H, Godfrey TG, Godfrey DA, Rubin AM (1996) Quantitative changes of amino acid distributions in the rat vestibular nuclear complex after unilateral vestibular ganglionectomy. J Neurochem 66:1550–1564

    CAS  PubMed  Google Scholar 

  • Magnusson AK, Ulfendahl M, Tham R (2002) Early compensation of vestibulo-oculomotor symptoms after unilateral vestibular loss in rats is related to GABA(B) receptor function. Neuroscience 111:625–634

    Article  CAS  PubMed  Google Scholar 

  • Malosio ML, Marqueze-Pouey B, Kushe J, Betz H (1991) Widespread expression of glycine receptor subunit mRNAs in adult and developing rat brain. EMBO J 10:2401–2409

    CAS  PubMed  Google Scholar 

  • Matzenbach B, Maulet Y, Sefton L, Courtier B, Avner P, Guenet JL, Betz H (1994) Structural analysis of mouse glycine receptor alpha subunit genes. J Biol Chem 269:2607–2612

    CAS  PubMed  Google Scholar 

  • Meyer G, Kirsh J, Betz H, Langosch D (1995) Identification of a gephyrin binding motif on the glycine receptor beta subunit. Neuron 15:563–572

    CAS  PubMed  Google Scholar 

  • Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. Academic press, San Diego

  • Peyret D, Campistron G, Geffard M, Aran JM (1987) Glycine immunoreactivity in the brainstem auditory and vestibular nuclei of the guinea pig. Acta Otolaryngol 104:71–76

    Google Scholar 

  • Piechotta K, Weth F, Harvey RJ, Friauf E (2001) Localization of rat glycine receptor alpha1 and alpha2 subunits transcripts in the developing auditory brainstem. J Comp Neurol 438:336–352

    Google Scholar 

  • Precht W, Scwindt PC, Backer R (1973) Removal of vestibular commissural inhibition by antagonists of GABA and glycine. Brain Res 62:222–226

    Article  CAS  PubMed  Google Scholar 

  • Prior P, Schmitt B, Grenningloh G, Pripilla I, Multhaup G, Beyreither K, Maulet Y, Werner P, Langloscg D, Kirsh J (1992) Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. Neuron 8:1161–1170

    CAS  PubMed  Google Scholar 

  • Rabbath G, de Waele C, Vassias I, Vidal PP (2002) GluR2-R4 AMPA subunit study in rat vestibular nuclei after unilateral labyrinthectomy: an in situ and immunohistochemical study. Neuroscience 111:189–206

    Article  CAS  PubMed  Google Scholar 

  • Racca C, Gardiol A, Triller A (1998) Cell-specific dendritic localization of glycine receptor alpha subunit messenger RNAs. Neuroscience 84:997–1012

    Article  CAS  PubMed  Google Scholar 

  • Ris L, de Waele C, Serafin M, Vidal PP, Godaux E (1995) Neuronal activity in the ipsilateral vestibular nucleus following unilateral labyrinthectomy in the alert guinea pig. J Neurophysiol 74:2087–2099

    CAS  PubMed  Google Scholar 

  • Ris L, Capron B, de Waele C, Vidal PP, Godaux E (1997) Dissociation between behavioral recovery and restoration of vestibular activity in the unilabyrinthectomized guinea-pig. J Physiol 500:509–522

    CAS  PubMed  Google Scholar 

  • Sans N, Sans A, Raymond J (1997) Regulation of NMDA receptor subunit mRNA expression in the guinea pig vestibular nuclei following unilateral labyrinthectomy. Eur J Neurosci 9:2019–2034

    CAS  PubMed  Google Scholar 

  • Sansom AJ, Brent VA, Jarvie PE, Darlington CL, Smith PF, Laverty R, Rostas JA (1997) In vitro phosphorylation of medial vestibular nucleus and prepositus hypoglossi proteins during behavioural recovery from unilateral vestibular deafferentation in the guinea pig. Brain Res 778:166–177

    CAS  PubMed  Google Scholar 

  • Sansom AJ, Smith PF, Darlington CL, Laverty R (2000) The effects of protein kinase C and calmodulin kinase II inhibitors on vestibular compensation in the guinea pig. Brain Res 882:45–54

    Article  CAS  PubMed  Google Scholar 

  • Sassoe-Pognetto M, Kirsch J, Grünert U, Greferath U, Fritschy JM, Mohler H, Betz H, Wässle H (1995) Colocalization of gephyrin and GABA(A)-receptor subunits in the rat retina. J Comp Neurol 357:1–14

    Google Scholar 

  • Sato K, Zhang JH, Saika T, Sato M, Tada K, Tohyama M (1991) Localization of glycine receptor α1 subunit mRNA-containing neurons in the rat brain: an analysis using in situ hybridization histochemistry. Neuroscience 43:381–395

    Article  CAS  PubMed  Google Scholar 

  • Sato K, Kiyama H, Tohyama M (1992) Regional distribution of cells expressing glycine receptor α2 subunit mRNA in rat brain. Brain Res 590:95–108

    Article  CAS  PubMed  Google Scholar 

  • Schaeffer KP, Meyer DL (1974) Compensation of vestibular lesions. In: Kornhuber HH (ed) Handbook of sensory physiology, vol VI, part 2. Springer, Berlin Heidelberg New York, pp 463–490

  • Schucknecht HF (1982) Behavior of the vestibular nerve following labyrinthectomy. Ann Otol Rhinol Laryngol Supp 97:16–32

    Google Scholar 

  • Seitanidou T, Nicola MA, Triller A, Korn H (1992) Partial glycinergic denervation induces transient changes in the distribution of the glycine receptor associated protein in a central neuron. J Neurosci 12:116–131

    CAS  PubMed  Google Scholar 

  • Simbürger E, Praschke M, Kirsh J, Nitsch R (2000) Distribution of the receptor-anchoring protein gephyrin in the rat dentate gyrus and changes following entorhinal cortex lesion. Cereb Cortex 10:422–432

    Article  PubMed  Google Scholar 

  • Smith PF, Curthoys IS (1988a) Neuronal activity in the ipsilateral medial vestibular nucleus of the guinea pig following unilateral labyrinthectomy. Brain Res 444:308–319

    CAS  PubMed  Google Scholar 

  • Smith PF, Curthoys IS (1988b) Neuronal activity in the contralateral medial vestibular nucleus of the guinea pig following unilateral labyrinthectomy. Brain Res 444:295–307

    CAS  PubMed  Google Scholar 

  • Smith PF, Curthoys IS (1989) Mechanisms of recovery following unilateral labyrinthectomy: a review. Brain Res Rev 14:155–180

    CAS  PubMed  Google Scholar 

  • Song Y, Huang LYM (1990) Modulation of glycine receptor chloride channels by cAMP-dependant protein kinase. Nature 348:242–245

    Article  CAS  PubMed  Google Scholar 

  • Todd AJ, Spike RC, Chong D, Neilson M (1995) The relationship between glycine and gephyrin in synapses of the rat spinal cord. Eur J Neurosci 7:1–11

    CAS  PubMed  Google Scholar 

  • Triller A, Cluzeaud F, Pfeiffer H, Beitz H, Korn H (1985) Distribution of glycine receptors at central synapses: an immunoelectron microscopy study. J Cell Biol 101:683–688

    CAS  PubMed  Google Scholar 

  • Vaello ML, Ruiz-Gomez A, Lermas J, Mayor FJ (1994) Modulation of inhibitory glycine receptors by phosphorylation by protein kinase. J Biol Chem 369:2002–2008

    Google Scholar 

  • Vibert N, Bantikyan A, Babalian A, Serafin M, Mûhlethaler M, Vidal PP (1999) Post-lesional plasticity in the central nervous system of the guinea-pig: a ” top-down ” adaptation process? Neuroscience 94:1–5

    Article  CAS  PubMed  Google Scholar 

  • Vibert N, Beraneck M, Bantikyan A, Vidal PP (2000) Vestibular compensation modifies the sensitivity to inhibitory amino acids. Neuroreport 11:1–7

    PubMed  Google Scholar 

  • Yamanaka T, Him A, Cameron SA, Dutia MB (2000) Rapid compensatory changes in GABA receptor efficacy in rat vestibular neurons after unilateral labyrinthectomy. J Physiol 523:413–424

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the ACI, France. We would like to thank Antoine Triller for his advice for the immunofluorescent study and Martine Pouradier for her excellent technical assistance.

Author information

Authors and Affiliations

  1. LNRS (CNRS-Paris V-Paris VII), ESA 7060, Centre Universitaire des Saints-Pères, 45 rue des Saints-Pères, 75270, Paris Cedex 06, France

    Lyndell Eleore, Isabelle Vassias, Pierre-Paul Vidal & Catherine de Waele

Authors
  1. Lyndell Eleore
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Isabelle Vassias
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pierre-Paul Vidal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Catherine de Waele
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Catherine de Waele.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Eleore, L., Vassias, I., Vidal, PP. et al. An in situ hybridization and immunofluorescence study of glycinergic receptors and gephyrin in the vestibular nuclei of the intact and unilaterally labyrinthectomized rat. Exp Brain Res 154, 333–344 (2004). https://doi.org/10.1007/s00221-003-1626-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00221-003-1626-y

Keywords

Search

Navigation