Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Apr 1;104(4):947–956. doi: 10.1083/jcb.104.4.947

gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

PMCID: PMC2114432  PMID: 3031086

Abstract

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.

Full Text

The Full Text of this article is available as a PDF (4.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschuler R. A., Betz H., Parakkal M. H., Reeks K. A., Wenthold R. J. Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res. 1986 Mar 26;369(1-2):316–320. doi: 10.1016/0006-8993(86)90542-1. [DOI] [PubMed] [Google Scholar]
  2. Barker J. L., McBurney R. N. GABA and glycine may share the same conductance channel on cultured mammalian neurones. Nature. 1979 Jan 18;277(5693):234–236. doi: 10.1038/277234a0. [DOI] [PubMed] [Google Scholar]
  3. Barker J. L., Ransom B. R. Amino acid pharmacology of mammalian central neurones grown in tissue culture. J Physiol. 1978 Jul;280:331–354. doi: 10.1113/jphysiol.1978.sp012387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belin M. F., Weisman-Nanopoulos D., Steinbusch H., Verhofstad A., Maître M., Jouvet M., Pujol J. F. Mise en évidence de glutamate décarboxylase et de sérotonine dans un même neurone au niveau du noyau du raphé dorsalis du rat par des méthodes de double marquage immunocytochimique. C R Seances Acad Sci III. 1981 Oct 19;293(6):337–341. [PubMed] [Google Scholar]
  5. Bisti S., Iosif G., Marchesi G. F., Strata P. Pharmacological properties of inhibitions in the cerebellar cortex. Exp Brain Res. 1971;14(1):24–37. doi: 10.1007/BF00234908. [DOI] [PubMed] [Google Scholar]
  6. Brennan M. J. GABA autoreceptors are not coupled to benzodiazepine receptors in rat cerebral cortex. J Neurochem. 1982 Jan;38(1):264–266. doi: 10.1111/j.1471-4159.1982.tb10879.x. [DOI] [PubMed] [Google Scholar]
  7. Chan-Palay V., Nilaver G., Palay S. L., Beinfeld M. C., Zimmerman E. A., Wu J. Y., O'Donohue T. L. Chemical heterogeneity in cerebellar Purkinje cells: existence and coexistence of glutamic acid decarboxylase-like and motilin-like immunoreactivities. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7787–7791. doi: 10.1073/pnas.78.12.7787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan-Palay V., Palay S. L. Ultrastructural localization of gamma-aminobutyric acid receptors in the mammalian central nervous system by means of [3H]muscimol binding. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2977–2980. doi: 10.1073/pnas.75.6.2977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Changeux J. P. Coexistence of neuronal messengers and molecular selection. Prog Brain Res. 1986;68:373–403. doi: 10.1016/s0079-6123(08)60252-6. [DOI] [PubMed] [Google Scholar]
  10. Changeux J. P., Devillers-Thiéry A., Chemouilli P. Acetylcholine receptor: an allosteric protein. Science. 1984 Sep 21;225(4668):1335–1345. doi: 10.1126/science.6382611. [DOI] [PubMed] [Google Scholar]
  11. Curtis D. R., Hösli L., Johnston G. A., Johnston I. H. The hyperpolarization of spinal motoneurones by glycine and related amino acids. Exp Brain Res. 1968;5(3):235–258. doi: 10.1007/BF00238666. [DOI] [PubMed] [Google Scholar]
  12. Curtis D. R., Johnston G. A. Amino acid transmitters in the mammalian central nervous system. Ergeb Physiol. 1974;69(0):97–188. doi: 10.1007/3-540-06498-2_3. [DOI] [PubMed] [Google Scholar]
  13. De Camilli P., Harris S. M., Jr, Huttner W. B., Greengard P. Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J Cell Biol. 1983 May;96(5):1355–1373. doi: 10.1083/jcb.96.5.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Diamond J., Roper S. Analysis of Mauthner cell responses to iontophoretically delivered pulses of GABA, glycine and L-glutamate. J Physiol. 1973 Jul;232(1):113–128. doi: 10.1113/jphysiol.1973.sp010259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Diamond J., Roper S., Yasargil G. M. The membrane effects, and sensitivity to strychnine, of neural inhibition of the Mauthner cell, and its inhibition by glycine and GABA. J Physiol. 1973 Jul;232(1):87–111. doi: 10.1113/jphysiol.1973.sp010258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eränkö O., Pickel V. M., Härkönen M., Eränko L., Joh T. H., Reis D. J. Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion. Histochem J. 1982 May;14(3):461–478. doi: 10.1007/BF01011857. [DOI] [PubMed] [Google Scholar]
  17. Faber D. S., Korn H. Single-shot channel activation accounts for duration of inhibitory postsynaptic potentials in a central neuron. Science. 1980 May 9;208(4444):612–615. doi: 10.1126/science.6245449. [DOI] [PubMed] [Google Scholar]
  18. Graham D., Pfeiffer F., Simler R., Betz H. Purification and characterization of the glycine receptor of pig spinal cord. Biochemistry. 1985 Feb 12;24(4):990–994. doi: 10.1021/bi00325a027. [DOI] [PubMed] [Google Scholar]
  19. Hamill O. P., Bormann J., Sakmann B. Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA. 1983 Oct 27-Nov 2Nature. 305(5937):805–808. doi: 10.1038/305805a0. [DOI] [PubMed] [Google Scholar]
  20. Hökfelt T., Johansson O., Ljungdahl A., Lundberg J. M., Schultzberg M. Peptidergic neurones. Nature. 1980 Apr 10;284(5756):515–521. doi: 10.1038/284515a0. [DOI] [PubMed] [Google Scholar]
  21. Hökfelt T., Ljungdahl A. Light and electron microscopic autoradiography on spinal cord slices after incubation with labeled glycine. Brain Res. 1971 Sep 10;32(1):189–194. doi: 10.1016/0006-8993(71)90163-6. [DOI] [PubMed] [Google Scholar]
  22. Korn H., Mallet A. Transformation of binomial input by the postsynaptic membrane at a central synapse. Science. 1984 Sep 14;225(4667):1157–1159. doi: 10.1126/science.6474167. [DOI] [PubMed] [Google Scholar]
  23. Kosaka T., Hataguchi Y., Hama K., Nagatsu I., WU J. Y. Coexistence of immunoreactivities for glutamate decarboxylase and tyrosine hydroxylase in some neurons in the periglomerular region of the rat main olfactory bulb: possible coexistence of gamma-aminobutyric acid (GABA) and dopamine. Brain Res. 1985 Sep 16;343(1):166–171. doi: 10.1016/0006-8993(85)91172-2. [DOI] [PubMed] [Google Scholar]
  24. Krnjević K., Schwartz S. The action of gamma-aminobutyric acid on cortical neurones. Exp Brain Res. 1967;3(4):320–336. doi: 10.1007/BF00237558. [DOI] [PubMed] [Google Scholar]
  25. Logan W. J., Snyder S. H. High affinity uptake systems for glycine, glutamic and aspaspartic acids in synaptosomes of rat central nervous tissues. Brain Res. 1972 Jul 20;42(2):413–431. doi: 10.1016/0006-8993(72)90540-9. [DOI] [PubMed] [Google Scholar]
  26. Matus A. I., Dennison M. E. An autoradiographic study of uptake of exogenous glycine by vertebrate spinal cord splices in vitro. J Neurocytol. 1972 Jul;1(1):27–34. doi: 10.1007/BF01098643. [DOI] [PubMed] [Google Scholar]
  27. Matus A. I., Dennison M. E. Autoradiographic localisation of tritiated glycine at 'flat-vesicle' synapses in spinal cord. Brain Res. 1971 Sep 10;32(1):195–197. doi: 10.1016/0006-8993(71)90164-8. [DOI] [PubMed] [Google Scholar]
  28. McLaughlin B. J., Barber R., Saito K., Roberts E., Wu J. Y. Immunocytochemical localization of glutamate decarboxylase in rat spinal cord. J Comp Neurol. 1975 Dec 1;164(3):305–321. doi: 10.1002/cne.901640304. [DOI] [PubMed] [Google Scholar]
  29. Möhler H., Battersby M. K., Richards J. G. Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1666–1670. doi: 10.1073/pnas.77.3.1666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakajima Y. Fine structure of the synaptic endings on the Mauthner cell of the goldfish. J Comp Neurol. 1974 Aug 15;156(4):379–402. [PubMed] [Google Scholar]
  31. Nelson P. G., Ransom B. R., Henkart M., Bullock P. N. Mouse spinal cord in cell culture. IV. Modulation of inhibitory synaptic function. J Neurophysiol. 1977 Sep;40(5):1178–1187. doi: 10.1152/jn.1977.40.5.1178. [DOI] [PubMed] [Google Scholar]
  32. Oertel W. H., Graybiel A. M., Mugnaini E., Elde R. P., Schmechel D. E., Kopin I. J. Coexistence of glutamic acid decarboxylase- and somatostatin-like immunoreactivity in neurons of the feline nucleus reticularis thalami. J Neurosci. 1983 Jun;3(6):1322–1332. doi: 10.1523/JNEUROSCI.03-06-01322.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oertel W. H., Schmechel D. E., Tappaz M. L., Kopin I. J. Production of a specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Neuroscience. 1981;6(12):2689–2700. doi: 10.1016/0306-4522(81)90113-5. [DOI] [PubMed] [Google Scholar]
  34. Ottersen O. P., Storm-Mathisen J. Glutamate- and GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique. J Comp Neurol. 1984 Nov 1;229(3):374–392. doi: 10.1002/cne.902290308. [DOI] [PubMed] [Google Scholar]
  35. Peck E. J., Jr Receptors for amino acids. Annu Rev Physiol. 1980;42:615–627. doi: 10.1146/annurev.ph.42.030180.003151. [DOI] [PubMed] [Google Scholar]
  36. Pfeiffer F., Betz H. Solubilization of the glycine receptor from rat spinal cord. Brain Res. 1981 Dec 7;226(1-2):273–279. doi: 10.1016/0006-8993(81)91099-4. [DOI] [PubMed] [Google Scholar]
  37. Pfeiffer F., Graham D., Betz H. Purification by affinity chromatography of the glycine receptor of rat spinal cord. J Biol Chem. 1982 Aug 25;257(16):9389–9393. [PubMed] [Google Scholar]
  38. Pfeiffer F., Simler R., Grenningloh G., Betz H. Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7224–7227. doi: 10.1073/pnas.81.22.7224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. REXED B. A cytoarchitectonic atlas of the spinal cord in the cat. J Comp Neurol. 1954 Apr;100(2):297–379. doi: 10.1002/cne.901000205. [DOI] [PubMed] [Google Scholar]
  40. Ransom B. R., Bullock P. N., Nelson P. G. Mouse spinal cord in cell culture. III. Neuronal chemosensitivity and its relationship to synaptic activity. J Neurophysiol. 1977 Sep;40(5):1163–1177. doi: 10.1152/jn.1977.40.5.1163. [DOI] [PubMed] [Google Scholar]
  41. Ribak C. E., Vaughn J. E., Saito K., Barber R., Roberts E. Glutamate decarboxylase localization in neurons of the olfactory bulb. Brain Res. 1977 Apr 22;126(1):1–18. doi: 10.1016/0006-8993(77)90211-6. [DOI] [PubMed] [Google Scholar]
  42. Richards J. G., Möhler H. Benzodiazepine receptors. Neuropharmacology. 1984 Feb;23(2B):233–242. doi: 10.1016/0028-3908(84)90064-9. [DOI] [PubMed] [Google Scholar]
  43. Rotter A., Schultz C. M., Frostholm A. Regulation of glycine receptor binding in the mouse hypoglossal nucleus in response to axotomy. Brain Res Bull. 1984 Oct;13(4):487–492. doi: 10.1016/0361-9230(84)90029-7. [DOI] [PubMed] [Google Scholar]
  44. Saito K., Barber R., Wu J., Matsuda T., Roberts E., Vaughn J. E. Immunohistochemical localization of glutamate decarboxylase in rat cerebellum. Proc Natl Acad Sci U S A. 1974 Feb;71(2):269–273. doi: 10.1073/pnas.71.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schoch P., Richards J. G., Häring P., Takacs B., Stähli C., Staehelin T., Haefely W., Möhler H. Co-localization of GABA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies. Nature. 1985 Mar 14;314(6007):168–171. doi: 10.1038/314168a0. [DOI] [PubMed] [Google Scholar]
  46. Somogyi P., Hodgson A. J., Chubb I. W., Penke B., Erdei A. Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system. J Histochem Cytochem. 1985 Mar;33(3):240–248. doi: 10.1177/33.3.2579123. [DOI] [PubMed] [Google Scholar]
  47. Somogyi P., Hodgson A. J., Smith A. D., Nunzi M. G., Gorio A., Wu J. Y. Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material. J Neurosci. 1984 Oct;4(10):2590–2603. doi: 10.1523/JNEUROSCI.04-10-02590.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Takeda N., Inagaki S., Shiosaka S., Taguchi Y., Oertel W. H., Tohyama M., Watanabe T., Wada H. Immunohistochemical evidence for the coexistence of histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities in nerve cells of the magnocellular nucleus of the posterior hypothalamus of rats. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7647–7650. doi: 10.1073/pnas.81.23.7647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ticku M. K., Huang A., Barker J. L. GABA receptor binding in cultured mammalian spinal cord neurons. Brain Res. 1980 Jan 20;182(1):201–206. doi: 10.1016/0006-8993(80)90846-x. [DOI] [PubMed] [Google Scholar]
  50. Toggenburger G., Wiklund L., Henke H., Cuénod M. Release of endogenous and accumulated exogenous amino acids from slices of normal and climbing fibre-deprived rat cerebellar slices. J Neurochem. 1983 Dec;41(6):1606–1613. doi: 10.1111/j.1471-4159.1983.tb00871.x. [DOI] [PubMed] [Google Scholar]
  51. Triller A., Cluzeaud F., Pfeiffer F., Betz H., Korn H. Distribution of glycine receptors at central synapses: an immunoelectron microscopy study. J Cell Biol. 1985 Aug;101(2):683–688. doi: 10.1083/jcb.101.2.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Triller A., Korn H. Morphologically distinct classes of inhibitory synapses arise from the same neurons: ultrastructural identification from crossed vestibular interneurons intracellularly stained with HRP. J Comp Neurol. 1981 Nov 20;203(1):131–155. doi: 10.1002/cne.902030111. [DOI] [PubMed] [Google Scholar]
  53. Triller A., Korn H. Transmission at a central inhibitory synapse. III. Ultrastructure of physiologically identified and stained terminals. J Neurophysiol. 1982 Sep;48(3):708–736. doi: 10.1152/jn.1982.48.3.708. [DOI] [PubMed] [Google Scholar]
  54. Werman R., Davidoff R. A., Aprison M. H. Inhibition of motoneurones by iontophoresis of glycine. Nature. 1967 May 13;214(5089):681–683. doi: 10.1038/214681a0. [DOI] [PubMed] [Google Scholar]
  55. Werman R., Davidoff R. A., Aprison M. H. Inhibitory of glycine on spinal neurons in the cat. J Neurophysiol. 1968 Jan;31(1):81–95. doi: 10.1152/jn.1968.31.1.81. [DOI] [PubMed] [Google Scholar]
  56. Wilkin G. P., Csillag A., Balázs R., Kingsbury A. E., Wilson J. E., Johnson A. L. Localization of high affinity [3H]glycine transport sites in the cerebellar cortex. Brain Res. 1981 Jul 6;216(1):11–33. doi: 10.1016/0006-8993(81)91275-0. [DOI] [PubMed] [Google Scholar]
  57. Young A. B., Snyder S. H. Strychnine binding associated with glycine receptors of the central nervous system. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2832–2836. doi: 10.1073/pnas.70.10.2832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zarbin M. A., Wamsley J. K., Kuhar M. J. Glycine receptor: light microscopic autoradiographic localization with [3H]strychnine. J Neurosci. 1981 May;1(5):532–547. doi: 10.1523/JNEUROSCI.01-05-00532.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES