Summary
Single and sequential double immunocytochemical techniques were applied to localize gamma-aminobutyric acid (GABA)-and choline acetyltransferase (ChAT)-like immunoreactivity (-LI) in the hypoglossal nucleus of the rat. After subsequential double staining a relatively high number of hypoglossal motor neurons showed the coexistence of both ChAT-and GABA-LI. Coexistence of both substances was also revealed in the axons of the hypoglossal nerve situated within the medulla oblongata. Cells showing only ChAT-or GABA-LI were also observed. Differences in immunostaining between the different cell groups of the hypoglossal nucleus were established.
Following axotomy of the right hypoglossal nerve, a decrease or loss of the immunoreactivity for both ChAT and GABA in the motor neurons was established until the 3rd week after the operation. The results obtained do not give evidence on the origin of the GABA-like immunoreactive material and its functional significance in the cholinergie neurons. It can be only speculated that the GABA-like material is either taken up from the intercellular space or is synthesized by the ChAT-LI nerve cells. Functionally, the importance of GABA for the synthesis of gamma-hydroxybutyrate (a novel neurotransmitter candidate) and its postsynaptic transmitter action or presynaptic regulatory action (through autoreceptors in the membrane of the nerve endings) on the release of acetylcholine (ACh) should be taken into consideration.
Similar content being viewed by others
References
Agardh E, Yeh HH, Herrmann R, Puro DG (1985) γ-Aminobutyric acid-mediated inhibition at cholinergic synapses formed by cultured retinal neurons. Brain Res 330:323–328
Agardh E, Brunn A, Ehinger B, Storm-Mathisen J (1986) GABA immunoreactivity in the retina. Ophthalmol Vis Sci 27:674–678
Agardh E, Ehinger B, Wu J-Y (1987) GABA and GAD-like immunoreactivity in the primate retina. Histochemistry 86:485–490
Altschuler RA, Parakkal MH, Fex J (1983) Localization of enkephalin-like immunoreactivity in acetylcholinesterase-positive cells in the guinea-pig lateral superior olivary complex that project to the cochlea. Neuroscience 9:621–630
Barber RP, Vaughn JE, Roberts E (1982) The cytoarchitecture of GABAergic neurons in rat spinal cord. Brain Res 238:305–328
Basbaum AI, Glazer EJ, Oertel W (1986) Immunoreactive glutamic acid decarboxylase in the trigeminal nucleus caudalis of the cat: a light and electron-microscopic analysis. Somatosens Res 4:77–94
Belin MF, Nanopoulos D, Didier M, Aguera M, Steinbush H, Verhofstad A, Maitre M, Pujol JF (1983) Immunocytochemical evidence for the presence of γ-aminobutyric acid and serotonin in one nerve cell. A study on the raphe nuclei of the rat using antibodies to glutamate decarboxylase and serotonin. Brain Res 275:329–339
Bonnano G, Raiteri M (1986) GABA enhances acetylcholine release from hippocampal nerve endings through a mechanism blocked by a GABA uptake inhibitor. Neurosci Lett 70:360–363
Bowery NG, Hill DR, Hudson AL, Price GW, Turnbull MJ, Wilkin GP (1984) Heterogeneity of mammalian GABA receptors. In: Bowery NG (ed) Actions and interactions of GABA and benzodiazepines. Raven Press, New York, pp 81–108
Brennan MJW (1982) GABA autoreceptors are not coupled to benzodiazepine receptors in the rat cerebral cortex. J Neurochem 38:264–266
Burnstock G (1983) Autonomic neurotransmitters and trophic factors. J Autonom Nerv Syst 7:213–217
Celio M (1985) Most GABA-ergic neurons in cerebral cortex and hippocampus contain the calcium-binding protein parvalbumin. Acta Anat 121:247
Chan-Palay V, Nilaver G, Palay SL, Beinfeld MC, Zimmerman EA, Wu J-Y, O'Donohue TL (1981) Chemical heterogeneity in cerebellar Purkinje cells: existence and coexistence of glutamic acid decarboxylase-like and motilin-like immunoreactivities. Proc Natl Acad Sci USA 78:7787–7791
Chia-Sheng Ling SLM, Schmechel DE (1986) Glutamic acid decarboxylase and somatostatin immunoreactivities in rat visual cortex. J Comp Neurol 244:369–383
Contamina P, Sáenz de Cabezón A, Parra P, Ramón y Cajal-Agüeras S, Ramo C, Pinilla MJ, Arino MP (1987) Distribution of neural elements containing GABA-like immunoreactivity in visual centres of the rabbit. Acta Anat 130:19
Cruz L, Basbaum AI (1985) Multiple opioid peptides and the modulation of pain: Immunohistochemical analysis of dynorphin and enkephalin in the trigeminal nucleus caudalis and spinal cord of the cat. J Comp Neurol 230:331–348
Curtis EM, Stewart MG (1986) Development of γ-aminobutyric acid immunoreactivity in chick hyperstriatum ventrale and cerebellum: light and electron microscopical observations. Dev Brain Res 30:189–199
Dale HH, Feldberg W, Vogt M (1936) Release of acetylcholine at voluntary motor preve endings. J Physiol (Lond) 86:353–380
Davidoff M (1973) Über die Glia im Hypoglossuskern der Ratte nach Axotomy. Z Zellforsch 141:427–442
Davidoff MS, Irintehev AM (1986) Acetylcholinesterase activity and type C synapses in the hypoglossal, facial and spinal cord motor nuclei of rats. An electron-microscope study. Histochemistry 84:515–524
DeGroat WC (1970) The actions of γ-aminobutyric acid on mammalian autonomic ganglia. J Pharmacol Exp Ther 172:384–386
Desarmenien M, Feltz P, Occhipiniti G, Santangelo E, Schlihter R (1984) Coexistence of GABA a and GABA b receptors on A and C primary afferents. Br J Pharmacol 81:327–333
Eckenstein F, Thoenen H (1982) Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons. EMBO J 1:363–368
Eckenstein F, Thoenen H (1983) Cholinergic neurons in the rat cerebral cortex demonstrated by immunohistochemical localization of choline acetyltransferase. Neurosci Lett 36:211–215
Eugène D (1987) Fast non-cholinergic depolarizing postsynaptic potentials in neurons of rat superior cervical ganglia. Neurosci Lett 78:51–56
Feldberg W, Gaddum JH (1934) The chemical transmitter at synapses in a sympathetic ganglion. J Physiol (Lond) 81:305–319
Geffard M, Vieillemaringe J, Heinrich-Rock A-M, Duris P (1985) Anti-acetylcholine antibodies and first immunocytochemical application in insect brain. Neurosci Lett 57:1–6
Hallanger AE, Wainer BH, Rye DB (1986) Colocalization of gamma-aminobutyric acid and acetylcholinesterase in rodent cortical neurons. Neuroscience 19:763–770
Hendry SHC, Jones EG (1986) Reduction in number of immunostained GABAergic neurons in deprived-eye dominance columns of monkey area 17. Nature 320:750–753
Hendry SHC, Jones EG, DeFelipe J, Schmechel D, Brandon C, Emson PC (1984) Neuropeptide containing neurons of the cerebral cortex are also GABAergic. Proc Natl Acad Sci USA 81:6526–6530
Houser CR, Crawford GD, Barber RP, Salvaterra PM, Vaughn JE (1983a) Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase. Brain Res 266:97–119
Houser CR, Lee M, Vaughn JE (1983b) Immunocytochemical localization of glutamic acid decarboxylase in normal and deafferented superior colliculus. Evidence for reorganization of γ-aminobutyric acid synapses. J Neurosci 3:2030–2042
Kaneko T, Tashiro K, Sugimoto T, Konishi A, Mizuno N (1985) Identification of thalamic neurons with vasoactive intestinal polypeptide-like immunoreactivity in the rat. Brain Res 347:390–393
Kato E, Kuba K, Koketsu K (1978) Presynaptic inhibition by γ-aminobutyric acid in bullfrog sympathetic ganglion cells. Brain Res 153:398–402
Katsumaru H, Murakami F, Wu J-V, Tsukaharn N (1986) Sprouting of GABAergic synapses in the red nucleus after lesions of the nucleus interpositus in the cat. J Neurosci 6:2864–2874
Kimura H, McGeer PL, Peng J-H, McGeer EG (1981a) The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat. J Comp Neurol 200:151–201
Kimura H, McGeer PL, Peng JH, McGeer EG (1981b) Mapping of cholinergic system in rostral forebrain of the rodent. In: Pepeu G, Ladinsky H (eds) Cholinergic mechanisms Plenum Press, New York London, pp 695–704
Kimura H, McGeer PL, Peng J-H (1984) Choline acetyltransferase containing neurons in the rat brain. In: Björklung A, Hökfelt T, Kuhar MJ (eds) Handbook of chemical neuroanatomy. Vol 3: Classical transmitters and transmitter receptors in the CNS, part II. Elsevier, Amsterdam, pp 51–67
Krnjevic K (1984) Some functional consequences of GABA uptake by brain cells. Neurosci Lett 47:282–287
Leránth C, Fehér E (1983) Synaptology and sources of vasoactive intestinal polypeptide and substance P containing axons of the rat celiac ganglion. An experimental electron microscopic immunohistochemical study. Neuroscience 10:947–958
Levey AI, Bolam JP, Rye DB, Hallanger AE, Demuth RM, Mesulam MM, Wainer BH (1986) A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride. J Histochem Cytochem 34:1449–1457
Lundberg JM, Hökfelt T (1986) Multiple co-existence of peptides and classical transmitters in peripheral autonomic and sensory neurons — functional and pharmacological implications. Prog Brain Res 68:241–262
Lundberg JM, Hökfelt T, Schultzberg M, Uvuaes-Wallenstein K, Köhler C, Said ST (1979) Occurrence of vasoactive intestinal polypeptide in certain cholinergic neurons of the cat: evidence from combined immunocytochemistry and acetylcholinesterase staining. Neuroscience 4:1539–1559
Maley B, Newton BW (1985) Immunohistochemistry of γ-aminobutyric acid in the cat nucleus tractus solitarius. Brain Res 330:364–368
Matute C, Streit P (1986) Monoclonal antibodies demonstrating GABA-like immunoreactivity. Histochemistry 86:147–157
Melander T, Staines WA (1986) A galanin-like peptide coexists in putative cholinergic somata of the septum-basal forebrain complex and in acetylcholinesterase-containing fibers and varicosities within the hippocampus in the owl monkey (Aotus triirgatus). Neurosci Lett 68:17–32
Mesulam M-M, Mufson EJ, Wainer BH, Levey AI (1983) Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1–Ch6). Neuroscience 10:1185–1201
Mesulam M-M, Mufson EJ, Levey AI, Wainer BH (1984) Atlas of cholinergic neurons in the forebrain and upper brainstem of the Macaque based on monoclonal choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry. Neuroscience 12:669–686
Miller JA, Richter JA (1986) Effects of GABAergic drugs in vivo on high-affinity choline uptake in vitro in mouse hippocampal synaptosomes. J Neurochem 47:1916–1918
Montero VM (1986) Localization of γ-aminobutyric acid (GABA) in type 3 cells and demonstration of their source to F2 terminals in the cat lateral geniculate nucleus: a Golgi-electron microscopic GABA-immunocytochemical study. J Comp Neurol 254:228–245
Mugnaini E, Oertel WH (1985) An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy Vol 4: GABA and neuropeptides in the CNS part I. Elsevier, Amsterdam, pp 436–608
Naumann RE, Wong RKS (1984) Voltage-clamp study on GABA response desensitization in single pyramidal cells from the hippocampus of adult guinea pigs. Neurosci Lett 47:289–294
Nishimura Y, Schwartz ML, Rakic P (1986) GABA and GAD immunoreactivity of photoreceptor terminals in primate retina. Nature 320:753–756
Oertel WH, Schmechel DE, Mugnaini E, Tappaz ML, Kopin IJ (1981a) Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neuroscience 6:2715–2735
Oertel WH, Schmechel DE, Tappaz ML, Kopin IJ (1981b) Production of a specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Neuroscience 6:2689–2700
Oertel WH, Mugnaini E, Schmechel DE, Tappaz M, Kopin IJ (1982) The immunocytochemical demonstration of gamma-aminobutyric acid-ergic neurons. Methods and application. In: Palay SL, Chan-Palay V (eds) Cytochemical methods in chemical neuroanatomy Liss, New York, pp 297–329
Oertel WH, Reithmüller G, Mugnaini E, Schmechel DE, Weindl A, Gramsch C, Herz A (1983) Opioid peptide like immunoreactivity localized in GABAergic neurons of rat neostriatum and central amygdaloid nucleus. Life Sci 33 (Suppl I):73–76
Ottersen OP, Storm-Mathisen J (1984) Glutamate-and GABA-containing neurons in the mouse and rat brain as demonstrated with a new immunocytochemical technique. J Comp Neurol 229:374–392
Phelps P, Vaughn JE (1986) Immunocytochemical localization of choline acetyltransferase in rat ventral striatum: a light and electron microscopic study. J Neurocytol 15:595–617
Roberts E (1984) GABA neurons in the mammalian central nervous system: model for a minimal basic unit. Neurosci Lett 47:195–200
Roberts E, Chase TN, Tower DB (eds) (1976) GABA in nervous system function. Raven Press, New York
Seguela P, Geffard M, Buijis RM, LeMoal M (1984) Antibodies against gamma-aminobutyric acid: specificity studies and immunocytochemical results. Proc Natl Acad Sci USA 81:3888–3892
Senba E, Daddona PE, Watanabe T, Wu J-Y, Nagy JI (1985) Coexistence of adenosine deaminase, histidine decarboxylase and glutamate decarboxylase in hypothalamic neurons of the rat. J Neuroscience 5:3393–3402
Somogyi P, Hodgson AJ (1985) Antisera to γ-aminobutyric acid. III. Demonstration of GABA in Golgi-impregnated neurons and in conventional electron microscopic sections of the cat striate cortex. J Histochem Cytochem 33:249–257
Somogyi P, Takagi H (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7:1779–1783
Somogyi P, Hodgson AJ, Smith AD, Nunzi MG, Gorio A, Wu HY (1984) Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatinor cholecystokinin-immunoreactive material. J Neurosci 4:2590–2603
Somogyi P, Hodgson AJ, Chubb JW, Penke B, Erdei A (1985) Antisera to γ-aminobutyric acid. II. Immunocytochemical application to the central nervous system. J Histochem Cytochem 33:240–248
Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG (1970) The unlabeled antibody method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horse radish peroxidase-antiperoxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315–333
Storm-Mathisen J, Leknes AK, Bore AT, Vaaland JL, Edminson P, Haug FMS, Ottersen OP (1983) First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301:517–520
Takeda N, Inagaki S, Shiosaka T, Taguchi Y, Oertel W, Tohyama M, Watanabe T, Wada H (1984) Immunohistochemical evidence for the coexistence of GABA and histidine decarboxylase-like immunoreactivities in nerve cells of the magnocellular nucleus of the posterior hypothalamus of the rat. Proc Natl Acad Sci USA 81:7647–7650
Tunnicliff G, Ngo TT (1986) Regulation of γ-aminobutyric acid synthesis in the vertebrate nervous system. Neurochem Int 8:287–297
Verburg-Van Kemenade BML, Jenks BG, Driessen AGJ (1986) GABA and dopamine act directly on melanophores of Xenopus to inhibit MSH secretion. Brain Res Bull 17:697–704
Vincent S, Hökfelt T, Christensson I, Terenius L (1982) Immunohistochemical evidence for a dynorphin immunoreactive striatonigral pathway. Eur J Pharmacol 85:251–252
Vincent SR, Satoch K, Fibiger HC, Panula P, Armstrong DM (1984) Peptides in the ascending cholinergic reticular system. Abstracts of the VIIth International Congress of Histochemistry and Cytochemistry, Helsinki, August 5–11, p 466
Vuillez P, Pérez SC, Stoeckel ME (1987) Colocalization of GABA and tyrosine hydroxylase immunoreactivities in the axons innervaling the neurointermediate lobe of the rat pituitary: an ultrastructural immunogold study. Neurosci Lett 79:53–58
Wainer BH, Bolam JP, Freund TF, Henderson Z, Totterdell S, Smith AD (1984a) Cholinergic synapses in the rat brain: a correlated light and electron microscopic immunohistochemical study employing a monoclonal antibody against choline acetyltransferase. Brain Res 308:69–76
Wainer BH, Levey I, Mufson EJ, Mesulam MM (1984b) Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase. Neurochem Int 6:163–182
Wassef M, Simons J, Tappaz ML, Sotelo C (1986) Non-Purkinje cell GABAergic innervation of the deep cerebellar nuclei: A quantitative immunocytochemical study in C57BL and in Purkinje cell degeneration mutant mice. Brain Res 399:125–135
Watt CB, Su YT, Lam DM-K (1984) Interactions between enkephalin and GABA in avian retina. Nature 311:761–763
Weissman-Nanopoulos D, Belin MF, Mandel P, Maitre M (1984) Immunocytochemical evidence for the presence of enzymes synthesizing GABA and GHB in the same neuron. Neurochem Int 6:333–338
Wolff JR, Böttcher H, Zetzsche T, Oertel WH, Chronwall BM (1984) Development of GABAergic neurons in rat visual cortex as identified by glutamate decarboxylase-like immunoreactivity. Neurosci Lett 47:207–212
Wolff JR, Joo F, Kasa P, Storm-Mathisen J, Toldi J, Balcar VJ (1986) Presence of neurons with GABA-like immunoreactivity in the superior cervical ganglion of the rat. Neurosci Lett 71:157–162
Wu J-Y (1976) Purification and properties of l-Glutamate decarboxylase (GAD) and GABA-aminotransferase (GABA-T). In: Roberts E, Chase T, Tower D (eds) GABA in nervous system function. Raven Press, New York, pp 7–55
Wu J-Y (1983) Preparation of glutamic acid decarboxylase as immunogen for immunocytochemistry. In: Cuello AC (ed) IBRO handbook series: Methods in the neurosciences. Wiley, London New York, pp 159–191
Wu J-Y, Lin C-T, Brandon C, Chan T-S, Moehler H, Richards JG (1982) Regulation and immunocytochemical characterization of glutamic acid decarboxylase. In: Palay S, Chan-Palay V (eds) Cytochemical methods in neuroanatomy. Alan R Liss, New York, pp 279–296
Author information
Authors and Affiliations
Additional information
Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday
Rights and permissions
About this article
Cite this article
Davidoff, M.S., Schulze, W. Coexistence of GABA-and choline acetyltransferase (ChAT)-like immunoreactivity in the hypoglossal nucleus of the rat. Histochemistry 89, 25–33 (1988). https://doi.org/10.1007/BF00496580
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00496580