Journal of Neurocytology

, Volume 33, Issue 3, pp 265–276 | Cite as

Alpha-adrenergic receptor (α2A) is colocalized in basal forebrain cholinergic neurons: A light and electron microscopic double immunolabeling study

  • L. Zaborszky
  • D. L. Rosin
  • J. Kiss


A variety of data suggest that noradrenaline and acetylcholine may interact in the basal forebrain, however no morphological studies have addressed whether indeed cholinergic neurons express adrenergic receptors. We have investigated the presence of alpha-adrenergic receptor subtype α2A -AR in cholinergic neurons of the basal forebrain. Cholinergic neurons were identified with an antibody against choline acetyltransferase and the receptor with a polyclonal antibody raised against a 47 amino acid fragment of the third intracellular loop of the α2A-AR. For double labeling at the light microscopic level the Ni-DAB/DAB technique was used, and for electron microscopy an immunoperoxidase/immunogold method was applied. We detected the α2A-AR protein in cholinergic as well as in non-cholinergic neurons. Almost half of all cholinergic neurons contained this adrenergic receptor. Double-labeled neurons were distributed throughout the rostro-caudal extent of the basal forebrain cholinergic continuum, including the medial septum, vertical and horizontal diagonal band nuclei, pallidal regions, substantia innominata and the internal capsule. Non-cholinergic neurons that expressed the α2A-AR outnumbered cholinergic/α2A-AR neurons by several factors. Electron microscopy confirmed the presence of α2A-AR in cholinergic neurons in the medial septum, vertical and horizontal diagonal band nuclei. Gold particles (10 nm) indicative of α2A-AR were diffusely distributed in the cytoplasm and accumulated in cytoplasmic areas near the Golgi complex and cysterns of the endoplasmic reticulum and were associated with the cellular membranes at synaptic and non-synaptic locations. Since many of the α2A-AR+/non-cholinergic neurons we detected are likely to be GABAergic cells, our data support the hypothesis that noradrenaline may act via basal forebrain cholinergic and non-cholinergic neurons to influence cortical activity.


Adrenergic Receptor Cholinergic Neuron Basal Forebrain Medial Septum GABAergic Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. AOKI, C., GO, C.-G., VENKATESAN, C. & KUROSE, H. (1994) Perikaryal and synaptic localization of á 2A-adrenergic receptor-like immunoreactivity. Brain Research 650, 181–204.PubMedGoogle Scholar
  2. AOKI, C., VENKATESAN, C., GO, C. G., FORMAN, R. & KUROSE, K. (1998) Cellular and subcellular sites for noradrenergic action in the monkey dorsolateral prefrontal cortex as revealed by the immunocytochemical localization of noradrenergic receptors and axons. Cerebral Cortex 8, 269–277.PubMedGoogle Scholar
  3. BERRIDGE, C. W., BOLEN, S. J., MANLEY, M. S., & FOOTE, S. L. (1996) Modulation of forebrain electroencephalographic activity in Halothane-anesthetized rat via actions of noradrenergic â-receptors within the medial septal region. Journal of Neuroscience 16, 7010–7020.PubMedGoogle Scholar
  4. BOYAJIAN, C. L., LOUGHLIN, S. E. & LESLIE, F. M. (1987) Anatomical evidence for alpha-2 adrenoreceptor heterogeneity: Differential autoradiographic distributions of [ 3 H] Rauwolscine and [ 3 H] Idazoxan in rat brain. Journal of Pharmacology and Experimental Therapeutics 241, 1079–1091.PubMedGoogle Scholar
  5. CAPE, E. G. & JONES, B. E. (1998) Differential modulation of high-frequency gamma-electroencephalogram activity and sleep-wake state by noradrenaline and serotonin microinjections into the region of cholinergic basalis neurons. Journal of Neuroscience 18, 2653–2666.PubMedGoogle Scholar
  6. CELESIA, G. G. & JASPER, H. H. (1966) Acetylcholine released from cerebral cortex in relation to state of activation. Neurology 16, 1053–1064.PubMedGoogle Scholar
  7. DECKER, M. W. & MCGAUGH, J. L. (1991) The role of interactions between the cholinergic system and other neuromodulatory systems in learning and memory. Synapse 7, 151–168.PubMedGoogle Scholar
  8. DOMYANCIC, A. V. & MORILAK, D. A. (1997) Distribution of á 1A adrenergic receptor mRNA in the rat brain visualized by in situ hybridization. Journal of Comparative Neurology 386, 358–378.PubMedGoogle Scholar
  9. DRINGENBERG, H. C. & VANDERWOLF, C. H. (1997) Neocortical activation: Modulation by multiple pathways acting on central cholinergic and serotonergic systems. Experimental Brain Research 116, 160–174.Google Scholar
  10. DRINGENBERG, H. C. & VANDERWOLF, C. H. (1998) Involvement of direct and indirect pathways in electrocorticographic activation. Neuroscience and Biobehavioral Reviews 22, 243–257.PubMedGoogle Scholar
  11. DUQUE, A., BALATONI, B., DETARI, L. & ZABORSZKY, L. (2000) EEG correlation of the discharge properties of identified neurons in the basal forebrain. Journal of Neurophysiology 84, 1627–1635.PubMedGoogle Scholar
  12. FOOTE, S. L. & ASTONJONES, G. S. (1995) Pharmacology and physiology of central nordarenergic systems. In Psychopharmacology: The Fourth Generation of Progress. (edited by BLOOM, F.E. & KUPFER, D. J.) pp. 335–345. New York: Raven Press.Google Scholar
  13. FORT, P. KHATEB, A. PEGNA, A., MUHLETHALER, M. & JONES, B. E. (1995) Noradrenergic modulation of cholinergic nucleus basalis neurons demonstrated by in vitro pharmacological and immunohistochemical evidence in the guinea-pig brain. European Journal of Neuroscience 7, 1502–1511.PubMedGoogle Scholar
  14. GERVASONI, D., DARRACQ, L., FORT, P., SOULIERE, F., CHOUVET, G. & LUPPI, P.-H. (1988) Electrophysiological evidence that noradrenergic neurons of the rat locus coeruleus are tonically inhibited by GABA during sleep. European Journal of Neuroscience10, 964–970.Google Scholar
  15. GROVE, E. A. (1988). Neural associations of the substantia innominata in the rat: Afferent connections. Journal of Comparative Neurology 277, 315–346.PubMedGoogle Scholar
  16. HAROUTUNIAN, V., KANOF, P. D., TSUBOYAMA, G. & DAVIS, K. L. (1990) Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats. Brain Research 507, 261–266.PubMedGoogle Scholar
  17. HEIDER, M., SCHLIEBS, R., ROSSNER, S. & BIGL, L. (1997) Basal forebrain cholinergic immunolesion by 192IgG-saporin: Evidence for a presynaptic location of subpopulations of alpha2-and beta-adrenergic as well as 5-HT2A receptors on cortical cholinergic terminals. Neurochemistry Research 22, 957–966.Google Scholar
  18. HUDSON, A. L., MALLARD, N. J., TYACKE, R. & NUTT, D. J. (1992) [ 3 H]-RX821002: A highly selective ligand for the identification of alfa2-adrenoreceptors in the rat brain. Molecular Neuropharmacology 1, 219–229.Google Scholar
  19. JACOBS, B. L. (1987) Brain monoaminergic unit activity in behaving animals. Progress of Psychobiology, Physiology, Psychology 12, 171–206.Google Scholar
  20. JASPER, H. H. & TESSIER, J. (1971) Acetylcholine liberations from cerebral cortex during paradoxical (REM) sleep. Science 172, 601–602.PubMedGoogle Scholar
  21. KANAI, T. & SZERB, J. C. (1965) Mesencephalic reticular activating system and cortical acetylcholine output. Nature 205,80–82.Google Scholar
  22. KING, P. R., GUNDLACH, A. L. & LOUIS, W. J. (1995) Quantitative autoradiographic localization in rat brain of á 2-adrenergic and non-adrenergic I-receptor binding sites labelled by [ 3 H]rilmenidine. Brain Research 675, 264–278.PubMedGoogle Scholar
  23. KISS, J., PATEL, A. J. & HALASZ, B. (1993) Colocalization of NGF receptor with VIP in the rat suprachiasmatic neurons. Neuroreport 4, 1315–1318.PubMedGoogle Scholar
  24. LEE, A., ROSIN, D. L. & VAN BOCKSTAELE, E. J. (1998b) á 2A-adrenergic receptors in the rat nucleus locus coeruleus: Subcellular localization in catecholaminergic dendrites, astrocytes, and presynaptic axon terminals. Brain Research 795, 157–169.PubMedGoogle Scholar
  25. LEE, A., TALLEY, E., ROSIN, D. L. & LYNCH, K. R. (1995) Characterization of á 2A-adrenergic receptors in GT1neurosecretory cells. Neuroendocrinology 62, 215–225.PubMedGoogle Scholar
  26. LEE, A., WISSEKERKE, A. E., ROSIN, D. L. & LYNCH, K. L. (1998a) Localization of á 2C-adrenergic receptor immunoreactivity in catecholaminergic neurons in the rat central nervous system. Neuroscience 84, 1085–1096.PubMedGoogle Scholar
  27. LIPSCOMBE, D., KONGSAMUT, S. & TSIEN, R. W. (1989) Alfa2-adrenergic receptor inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating. Nature 340, 634–642.Google Scholar
  28. MANNS, I. D., ALONSO, A. & JONES, B. E. (2000a) Discharge properties of juxtacellularly labeled and immunohistochemically identified cholinergic basal fore-brain neurons recorded in association with the electroencephalogram in anesthetized rats. Journal of Neuroscience 20, 1505–1518.PubMedGoogle Scholar
  29. MANNS, I. D., ALONSO, A. & JONES, B. E. (2000b) Discharge profiles of juxtacellularly labeled and immunohistochemically identified GABAergic basal forebrain neurons recorded in association with the electroencephalogram in anesthetized rats. Journal of Neuroscience 20, 9252–9263.PubMedGoogle Scholar
  30. MANNS, I. D., LEE, M. G., MODIRROUSTA, M., HOU, Y.P. & JONES, B. E. (2003) Alpha 2 adrenergic receptors on GABAergic, putative sleep-promoting basal forebrain neuron. European Journal of Neuroscience18, 723–727.PubMedGoogle Scholar
  31. MARIGHETTO, A., DURKIN, T., TOUMANE, T., LEBRUN, C. & JAFFARD, R. (1989) Septal alpha-noradrenergic antagonism in vivo blocks the testing-induced activation of septohippocampal cholinergic neurons and produces a concomitant deficit in working memory performance of mice. Pharmacology, Biochemistry and Behaviour 34, 553–558.Google Scholar
  32. MESULAM, M. M., MUFSON, E. J., WAINER, B. H. & LEVEY, A. I. (1983) Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1-Ch6). Neuroscience 10,1185–1201.Google Scholar
  33. MILNER, T. A., LEE, A., AICHER, S. A. & ROSIN, D. L. (1998) Hippocampal á 2A-adrenergic receptors are located predominantly presynaptically but are also found post-synaptically and in selective astrocytes. Journal of Comparative Neurology 395, 310–327.PubMedGoogle Scholar
  34. MILNER, T. A., ROSIN, D. L., LEE, A. & AICHER, S. A. (1999) Alpha 2A-adrenergic receptors are primarily presynaptic heteroreceptors in the C1 area of the rat rostral ventrolateral medulla. Brain Research 821, 200–211.PubMedGoogle Scholar
  35. NICHOLAS, A. P., PIERIBONE, V. & HOKFELT, T. (1993) Distributions of mRNAs for alpha-2 adrenergic receptor subtypes in rat brain: An in situ hybridization study. The Journal of Comparative Neurology 328, 575–594.PubMedGoogle Scholar
  36. PAXINOS, G. & WATSON, C. (1998) The Rat Brain in Stereotaxic Coordinates. New York: Academic Press.Google Scholar
  37. PICKEL, V. M., NIRENBERG, M. J. & MILNER, T. A. (1996) Ultrastructural view of central catecholaminergic transmission: Immunocytochemical localization of synthesizing enzymes, transporters and receptors. Journal of Neurocytology 25, 843–856.PubMedGoogle Scholar
  38. PIERIBONE, V. A., NICHOLAS, A. P., DAGERLIND, A. & HOKFELT, T. (1994) Distribution of á 1 adrenoreceptors in rat brain revealed by in situ hybridization experiments utilizing subtype-specific probes. Journal of Neuroscience 14, 4252–4268.PubMedGoogle Scholar
  39. RAMESH, V., KUMAR, V. M., JOHN, J. & MALLICK, H. (1995) Medial preoptic alpha-2 adrenoceptors in the regulation of sleep-wakefulness. Physiology and Behavior 57, 171–175.PubMedGoogle Scholar
  40. ROSIN, D. L. (2000) Distribution of á 2A-and á 2C-adrenergic receptor immunoreactivity in the central nervous system. In Methods in Molecular Biology 126, Adrenergic Receptor Protocols (edited by MACHIDA, C. A.) pp. 475–505. Totowa, NJ: Humana Press Inc.Google Scholar
  41. ROSIN, D. L., ZENG, D., STORNETTA, R. L., NORTON, F. R., RILEY, T., OKUSA, M. D., GUYENET, P. G. & LYNCH, K. R. (1993) Immunohistochemical localization of á 2A-adrenergic receptors in catecholaminergic and other brainstem neurons in the rat. Neuroscience 56, 139–155.Google Scholar
  42. ROSIN, D. L., TALLEY, E. M., LEE, A., STORNETTA, R. L., GAYLINN, B. D., GUYENET, P. G. & LYNCH, K. R. (1996) Distribution of á 2C-adrenergic receptor-like immunoreactivity in the rat central nervous system. Journal of Comparative Neurology 372, 135–165.Google Scholar
  43. RUFFOLO, R. R., NICHOLS, A. J., STADEL, J. M. & HIEBLE J. P. (1991) Structure and function of alpha-adrenoreceptors. Pharmacology Review 43, 475–505.Google Scholar
  44. RYE, D. B., WAINER, B. H., MESULAM, N.-M., MUFSON, E. J. & SAPER, C. B. (1984). Cortical projections arising from the basal forebrain: A study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase. Neuroscience 13, 627–643.PubMedGoogle Scholar
  45. SCHEININ, M., LOMASNEY, J. W., HAYDEN-HIXSON, D. M., SCHAMBRA, U. B., CARON, M. G., LEFKOWITZ, R. J. & FREMEAU, R. T. (1994) Distribution of á 2-adrenergic receptor subtype gene expression in rat brain. Molecular Brain Research 21, 133–149.PubMedGoogle Scholar
  46. SOMOGYI, P. & TAKAGI, H. (1982) Anote on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocyto-chemistry. Neuroscience 7, 1779–1784.PubMedGoogle Scholar
  47. SUN, N. & CASSEL, M. D. (1993) Intrinsic GABAergic neurons in the rat central extended amygdala. Journal of Comparative Neurology 390, 381–404.Google Scholar
  48. TALLEY, E. M., ROSIN, D. L., LEE, A., GUYENET, P. G. & LYNCH, K. R. (1996) Distribution of á 2A-adrenergic receptor-like immunoreactivity in the rat central nervous system.Journal of Comparative Neurology 372, 111–134.PubMedGoogle Scholar
  49. UNNERSTALL, J. L., KOPAJTIC, T. A. & KUHAR, M. J. (1984) Distribution of alpha 2 agonist binding sites in the rat and human central nervous system: Analysis of some functional, anatomical correlates of the pharamacologic effects of clonidine and related adrenergic agents. Brain Research 19,69–101.Google Scholar
  50. VEINANTE, P., STOECKEL, M.-E. & FREUND-MERCIER, M.-J. (1997). GABA-and peptide-immunoreactivities co-localize in the rat central extended amygdala. NeuroReport 8, 2985–2989.PubMedGoogle Scholar
  51. VENKATESAN, C., SONG, X-Z., GO, C.-G., KUROSE, H. & AOKI, C. (1996) Cellular and subcellular distribution of á 2A-adrenergic receptors in the visual cortex of neonatal and adult rats. Journal of Comparative Neurology 365, 79–95.PubMedGoogle Scholar
  52. VIZI, E. S. (1980) Modulation of cortical release of acetyl-choline by noradrenaline released from nerves arising from the rat locus coeruleus. Neuroscience 5, 2139–2144.PubMedGoogle Scholar
  53. WILLIAMS, J. T. & REINER, P. B. (1993) Noradrenaline hyperpolarizes identified rat mesopontine cholinergic neurons in vitro. Journal of Neuroscience 13, 3878–3883.PubMedGoogle Scholar
  54. WINZER-SERHAN, U. H., RAYMON, H. K., BROIDE, R. S., CHEN, F. M. & LESLIE, F. M. (1997) Expression of á 2 adrenoceptors during rat brain development-I. á 2A messenger RNA expression. Neuroscience 76, 241–260.PubMedGoogle Scholar
  55. ZABORSZKY, L., HEIMER, L., ECKENSTEIN, F. & LERANTH, C. (1986) GABAergic input to cholinergic forebrain neurons: An ultrastructural study using retrograde tracing of HRP and double immunolabeling. Journal of Comparative Neurology 250, 282–295.PubMedGoogle Scholar
  56. ZABORSZKY, L., PANG, K., SOMOGYI, J., NADASDY, Z. & KALLO, I. (1999) The basal forebrain corticopetal system revisited. Annals of the New York Academy of Science 877, 339–367.Google Scholar
  57. ZABORSZKY, L., CULLINAN, W. E. & LUINE, V. N. (1993) Catecholaminergic-cholinergic interaction in the basal forebrain. Progress in Brain Research 98,31–49.PubMedGoogle Scholar
  58. ZABORSZKY, L. & CULLINAN, W. E. (1996) Direct catecholaminergic-cholinergic interactions in the basal forebrain. I. Dopamine-â-hydroxylase-and tyrosine hydroxylase input to cholinergic neurons. Journal of Comparative Neurology 374, 535–554.PubMedGoogle Scholar
  59. ZABORSZKY, L., KISS, J. & ROSIN, D. L. (1995) Alpha2A-adrenergic receptors are present in basal forebrain cholinergic projection neurons. Society for Neuroscience 21, 69.Google Scholar
  60. ZABORSZKY, L. & DUQUE, A. (2003) Sleep-wake mechanisms and basal forebrain circuitry. Frontiers in Bioscience 8,d1146–1169. [PubMed#:12957822] URL: /current/vol8.htmGoogle Scholar
  61. ZENG, D. & LYNCH, K. R. (1991) Distribution of alpha2-adrenergic receptor mRNAs in the rat CNS. Molecular Brain Research 10, 219–225.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • L. Zaborszky
    • 1
  • D. L. Rosin
    • 2
  • J. Kiss
    • 3
  1. 1.Rutgers UniversityCenter for Molecular and Behavioral NeuroscienceNewarkUSA
  2. 2.Department of Pharmacology, University of Virginia Health Sciences CenterCharlottesvilleUSA
  3. 3.Hungarian Academy of Sciences and Semmelweis UniversityNeuroendocrine Research LaboratoryBudapestHungary

Personalised recommendations