Abstract
Immunodetection of catecholamine biosynthetic enzymes is frequently used for the visualization of central nervous catecholaminergic systems. Because of the method's limited specificity for the different catecholamines, interpretation of the results often presents difficulties. To determine criteria for the identification of dopaminergic, noradrenergic, and adrenergic afferents to the rat amygdaloid complex, comparative immunolabelling for tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), and phenylethanolamine-n-methyltransferase (PNMT) was carried out using single- and double-labelling for fluorescence, light- and electron microscopy. The observations were complemented by findings in brainstem and hypothalamic areas. The results indicated that. TH-labelling detected preferentially dopaminergic afferents in the lateral central and intercalated amygdaloid nuclei. DBH-labelling detected noradrenergic axons in nuclei lacking PNMT-immunoreactive fibres, and PNMT was a marker for adrenergic axons in the entire complex. For nuclei with combined dense dopaminergic, noradrenergic and/or adrenergic innervation, morphological and immunolabelling characteristics were described which, to a certain extent, enabled identification of the different afferents in anti-TH or anti-DBH-preparations. Using a monoclonal TH-antiserum, noradrenergic and adrenergic axons displayed weaker immunoreactivity than dopaminergic ones, and possessed characteristic morphological features. TH-immunoreactivity in noradrenergic axons differed depending on their origin, and showed intra-axonal compartmentalization. The present study provides a basis for the use of the detection of biosynthetic enzymes in future investigations into the ultrastructure and connectivity of the catecholaminergic amygdala innervation.
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Alonso G, Gaillet S (1991) Differences in the immunoreactivity to phenylethanolamine-N-methyltransferase in the central adrenergic neurons of four strains of rats. Cell Tissue Res 265:307–315
Armstrong DM, Ross CA, Pickel VM, Joh TH, Reis DJ (1982) Distribution of dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes. J Comp Neurol 212:173–187
Asan E (1992) Dopaminergic innervation of somatostatin containing neurons in the rat central amygdaloid nucleus. Eur J Neurosci Suppl 5:26
Bayer VE, Towle AC, Pickel VM (1991) Vesicular and cytoplasmic localization of neurotensin-like immunoreactivity (NTLI) in neurons postsynaptic to terminals containing NTLI and/or tyrosine hydroxylase in the rat central nucleus of the amygdala. J Neurosci Res 30:398–413
Beato DK, Burke WJ, Joh TH, Haring JH (1987) Cortical epinephrine projections demonstrated by retrograde tracing combined with tyrosine hydroxylase and phenylethanolamine N-methyltransferase immunocytochemistry. Soc Neurosci Abstr 13:1336
Berod A, Hartman BK, Pujol JF (1981) Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase. J Histochem Cytochem 29:844–850
Björklund A, Lindvall O (1984) Dopamine-containing systems in the CNS. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy, vol 2, part I. Elsevier, Amsterdam, pp 55–122
Bouthenet ML, Matres MP, Sales N, Schwartz JC (1987) A detailed mapping of dopamine D-2 receptors in rat central nervous system by autoradiography with [125]iodosulpride. Neuroscience 20:117–155
Buijs TM, Geffard M, Pool CW, Hoorneman EMD (1984) The dopaminergic innervation of the supraoptic and paraventricular nucleus. A light and electron microscopical study. Brain Res 323:65–72
Campbell MJ, Lewis DA, Foote SL, Morrison JH (1987) Distribution of choline acetyltransferase-, serotonin-, dopamine-β-hydroxylase-, tyrosine hydroxylase-immunoreactive fibers in monkey primary auditory cortex. J Comp Neurol 261:209–220
Cassell MD, Gray TS (1989) Morphology of peptide-immunoreactive neurons in the rat central nucleus of the amygdala. J Comp Neurol 281:320–333
Chang HT, Kuo H (1989) Adrenergic innervation of the substantia innominata: co-localization of phenylethanolamine N-methyltransferase and tyrosine hydroxylase immunoreactivities within the same axons. Brain Res 503:350–353
Chan-Palay V, Zaborsky L, Köhler C, Goldstein M, Palay SL (1984) Distribution of tyrosine-hydroxylase-immunoreactive neurons in the hypothalamus of rats. J Comp Neurol 227:467–496
Decavel C, Geffard M, Calas A (1987) Comparative study of dopamine- and noradrenaline-immunoreactive terminals in the paraventricular and supraoptic nuclei of the rat. Neurosci Lett 77:149–154
Ellis ME (1985) Amygdala norepinephrine involved in two separate long-term memory retrieval processes. Brain Res 342:191–195
Ellis ME, Kesner RP (1983) The noradrenergic system of the amygdala and aversive information processing. Behav Neurosci 97:399–415
Fallon JH, Ciofi P (1992) Distribution of monoamines within the amygdala. In: Aggleton JP (ed) The amygdala, Wiley-Liss, New York, pp 97–114
Fallon JH, Moore RY (1978) Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J Comp Neurol 180:545–580
Fallon JH, Koziell DA, Moore RY (1978) Catecholamine innervation of the basal forebrain. II. Amygdala, suprarhinal cortex and entorhinal cortex. J Comp Neurol 180:509–532
Fibiger HC (1991) The dopamine hypotheses of schizophrenia and mood disorders: contradictions and speculations. In: Willner P, Scheel-Krüger J (eds) The mesolimbic dopaminergic system: from motivation to action. Wiley, Chichester, pp 615–637
Gallagher M, Kapp BS, Musty RE, Driscoll PA (1977) Memory formation: evidence for a specific neurochemical system in the amygdala. Science 198:423–425
Geffard M, Buijs RM, Seguela P, Pool CW, LeMoal M (1984) First demonstration of highly specific and sensitive antibodies against dopamine. Brain Res 294:161–165
Geffard M, Patel S, Dulluc J, Rock AM (1986) Specific detection of noradrenaline in the rat brain using antibodies. Brain Res 363:395–400
Glavin GB, Murison R, Overmier JB, Pare WP, Bakke HK, Henke PG, Hernandez DE (1991) The neurobiology of stress ulcers. Brain Res Rev 16:301–343
Grima B, Lamoroux A, Boni C, Julien JF, Javoy-Agid F, Malet J (1987) A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics. Nature 326:707–711
Gugten J van der, Palkovits M, Wijnen HLJM, Versteeg DHG (1976) Regional distribution of adrenaline in rat brain. Brain Res 107:171–175
Gustafson EL, Greengard P (1990) Localization of DARPP-32 immunoreactive neurons in the bed nucleus of the stria terminalis and central nucleus of the amygdala: codistribution with axons containing tyrosine hydroxylase, vasoactive intestinal polypeptide, and calcitonin gene-related peptide. Exp Brain Res 79:447–458
Hökfelt T, Johansson O, Goldstein M (1984) Central catecholamine neurons as revealed by immunohistochemistry with special reference to adrenaline neurons. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy, vol 2, part 1. Elsevier, Amsterdam, pp 157–276
Hornby PJ, Piekut DT (1989) Opiocortin and catecholamine input to CRF-immunoreactive neurons in rat forebrain. Peptides 10:1139–1146
Jaeger CB, Ruggiero DA, Albert VR, Joh TH, Reis DJ (1984) Immunocytochemical localization of aromatic-L-aminoacid decarboxylase. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy vol 2, part 1. Elsevier, Amsterdam, pp 387–408
Joh TH, Reis DJ (1975) Different forms of tyrosine hydroxylase in central dopaminergic and noradrenergic neurons and sympathetic ganglia. Brain Res 85:146–151
Lavoie B, Smith Y, Parent A (1989) Dopaminergic innervation of the basal ganglia in the squirrel monkey as revealed by tyrosine hydroxylase immunohistochemistry. J Comp Neurol 289:36–52
Lenard L, Hahn Z (1982) Amygdalar noradrenergic and dopaminergic mechanisms in the regulation of hunger and thirst-motivated behaviour. Brain Res 233:115–132
Liang K, Juler RG, McGaugh JL (1986) Modulating effects of posttraining epinephrine on memory: involvement of the amygdala noradrenergic system. Brain Res 368:125–133
Lindvall O, Björklund A (1974) The organization of the ascending catecholamine neuron systems in the rat brain. Acta Physiol Scand Suppl 412:1–48
Liposits Z, Phelix C, Paull WK (1986a) Electron microscopic analysis of tyrosine hydroxylase, dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat. Histochemistry 84:105–120
Liposits Z, Sherman D, Phelix C, Paull WK (1986b) A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Histochemistry 85:95–106
McDonald AJ (1982) Cytoarchitecture of the central amygdaloid nucleus of the rat. J Comp Neurol 208:401–418
Milner TA, Bacon CE (1989) Ultrastructural localization of tyrosine hydroxylase-like immunoreactivity in the rat hippocampal formation. J Comp Neurol 281:479–495
Moore RY, Bloom FE (1979) Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu Rev Neurosci 2:113–168
Moore RY, Card P (1984) Noradrenaline-containing neuron systems. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy, vol 2, part 1. Elsevier, Amsterdam, pp 123–156
Nitecka L, Ben-Ari Y (1987) Distribution of GABA-like immunoreactivity in the rat amygdaloid complex. J Comp Neurol 266:45–55
Noack HJ, Lewis DA (1989) Antibodies directed against tyrosine hydroxylase differentially recognize noradrenergic axons in monkey neocortex. Brain Res 500:313–324
Palkovits M, Mezey E, Skirboli LR, Hökfelt T (1992) Adrenergic projections from the lower brainstem to the hypothalamic paraventricular nucleus, the lateral hypothalamic area and the central nucleus of the amygdala in rats. J Chem Neuroanat 5:407–415
Paxinos G, Watson C (1982) The rat brain in stereotaxic coordinates. Academic Press, Sydney
Pickel VM, Joh TH, Field PM, Becker CG, Reis DJ (1975) Cellular localization of tyrosine hydroxylase by immunohistochemistry. J Histochem Cytochem 23:1–12
Price JL, Russchen FT, Amaral DG (1987) The limbic region. II: The amygdaloid complex. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy, vol 5, part I. Elsevier, Amsterdam, pp 279–388
Ray A, Henke PG, Sullivan RM (1987) The central amygdala and immobilization stress-induced gastric pathology in rats: neurotensin and dopamine. Brain Res 409:398–402
Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208–212
Reynolds GP (1983) Increased concentrations and lateral asymmetry of amygdala dopamine in schizophrenia. Nature 305:527–529
Roberts GW, Woodhams PL, Polak JM, Crow TJ (1982) Distribution of neuropeptides in the limbic system of the rat: the amygdaloid complex. Neuroscience 7:99–131
Roberts GW, Ferrier IN, Lee Y, Crow TJ, Johnstone EC, Ownes DG, Bacarese-Hamilton AJ, McGregor G, O'Shaughnessey D, Polak J, Bloom SE (1983) Peptides, the limbic lobe and schizophrenia. Brain Res 288:199–211
Ross CA, Ruggiero DA, Meeley MP, Park DH, Joh TH, Reis DJ (1984) A new group of neurons in hypothalamus containing phenylethanolamine N-methyltransferase (PNMT) but not tyrosine hydroxylase. Brain Res 306:349–353
Ruggiero DA, Ross CA, Anwar M, Park DH, Joh TH, Reis DJ (1985) Distribution of neurons containing phenylethanolamine N-methyltransferase in medulla and hypothalamus of rat. J Comp Neurol 239:127–154
Sadikot AF, Parent A (1990) The monoaminergic innervation of the amygdala in the squirrel monkey: an immunohistochemical study. Neuroscience 36:431–447
Samson Y, Wu JJ, Friedman AH, Davis JN (1990) Catecholaminergic innervation of the hippocampus of the cynomolgus monkey. J Comp Neurol 298:250–263
Sawchenko PE, Swanson LW (1982) The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat. Brain Res Rev 4:275–325
Seeman P, Ulpian C, Bergeron C, Riederer P, Jellinger K, Gabriel E, Reynolds GP (1984) Bimodal distribution of dopamine receptor densities in brains of schizophrenics. Science 225:728–731
Smiley JF, Williams SM, Szigeti K, Goldman-Rakic PS (1992) Light and electron microscopic characterization of dopamine-immunoreactive axons in human cerebral cortex. J Comp Neurol 321:325–335
Steinbusch HVM, Verhofstad AAJ, Penke B, Varga J, Joosten HWJ (1981) Immunohistochemical characterization of monoamine-containing neurons in the central nervous system by antibodies to serotonin and noradrenaline. A study in the rat and the lamprey. Acta Histochem Suppl 24:107–122
Ungerstedt U (1971) Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol Scand Suppl 367:1–48
Weiner DM, Levey AI, Sunahara RK, Niznik HB, O'Dowd BF, Seeman P, Brann MR (1991) D1 and D2 dopamine receptor mRNA in rat brain. Proc Natl Acad Sci USA 88:1859–1863
Wong DF, Wagner HN, Tune LE, Dannals RF, Pearlson GD, Links JM, Tamminga CA, Boussole EP, Ravert HT, Wilson AA, Toung JKT, Malat J, Williams JA, O'Tuama LA, Snyder SH, Kuhar MJ, Gjedde A (1986) Positron emission tomography reveals elevated D2 dopamine receptors in drug-naive schizophrenics. Science 234:1558–1562
Wray S, Hoffman GE (1983) Organization and interrelationship of neuropeptides in the central amygdaloid nucleus of the rat. Peptides 4:525–541
Zaborsky L, Heimer L (1989) Combinations of tracer techniques, especially HRP and PHA-L, with transmitter identification for correlated light and electron microscopic studies. In: Heimer L, Zaborsky L (eds) Neuroanatomical tract tracing methods 2. Plenum Press, New York, pp 49–96
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Asan, E. Comparative single and double immunolabelling with antisera against catecholamine biosynthetic enzymes: criteria for the identification of dopaminergic, noradrenergic and adrenergic structures in selected rat brain areas. Histochemistry 99, 427–442 (1993). https://doi.org/10.1007/BF00274095
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DOI: https://doi.org/10.1007/BF00274095