Experimental Brain Research

, Volume 99, Issue 1, pp 34–42 | Cite as

Mapping of the colocalization of calretinin and tyrosine hydroxylase in the rat substantia nigra and ventral tegmental area

  • Krystyna R. Isaacs
  • David M. Jacobowitz
Original Paper


The distribution of calretinin (CR), a calcium binding protein, was compared with that of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, throughout the rostrocaudal extent of the rat subsantia nigra (SN) and ventral tegmental area (VTA). After mapping the cells using double-labelling immunofluorescence, it was possible to distinguish three distinct cell types: cells immunoreactive for CR only, cells immunoreactive for TH only, and cells in which the two proteins were colocalized (CR+TH). Colocalized cells in rat brain sections comprised approximately 40–55% of the fluorescent labelled cells in the SN compacta, 30–40% in the VTA, and 55–80% in the SN lateralis. Colocalized cells in the SN reticulata were infrequent except in the more caudal sections where a majority of the TH-immunoreactive cells also contained CR. The percentage of CR cells that contained TH was approximately 80% in the SN compacta and averaged 65% in the VTA. Overall, the percentage of TH-immunoreactive cells which also contained CR was approximately 50% in the SN compacta and 45% in the VTA. These data reveal a significant degree of colocalization of CR in dopamine-producing cells of the SN and VTA and suggest the need for studies concerning the fate of these individual cell types following experimental manipulations.

Key words

Fluorescence immunohistochemistry Calcium-binding protein Dopamine Neuroprotection Rat 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arai R, Winsky L, Aral M, Jacobowitz D (1991) Immunohistochemical localization of calretinin in the rat hindbrain. J Comp Neurol 310:21–44Google Scholar
  2. Bankiewicz KS, Plunkett RJ, Jacobowitz DM, Porrino L, di Porzio U, London WT, Kopin IJ, Oldfield EH (1990) The effect of fetal mesencephalon implants on primate MPTP-induced parkinsonism: histochemical and behavioral studies. J Neurosurg 72:231–244Google Scholar
  3. Bankiewicz KS, Plunkett RJ, Jacobowitz DM, Kopin IJ, Oldfield EH (1991) Fetal nondopaminergic neural implants in parkinsonian primates: histochemical and behavioral studies. J Neurosurg 74:97–104Google Scholar
  4. Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DJ, Kopin IJ (1983) A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Natl Acad Sci USA 80:4546–4550Google Scholar
  5. Campbell KJ, Takada M (1989) Bilateral tectal projection of single nigrostriatal dopamine cells in the rat. Neuroscience 33:311–321Google Scholar
  6. Chronister RB, Waldins JS, Aldo LD, Marco LA (1988) Interconnections between SN reticulata and medullary reticular formation. Brain Res Bull 21:313–317Google Scholar
  7. Clavier RM, Atmadja S, Fibiger HC (1976) Nigrothalamic projections in the rat is demonstrated by orthograde and retrograde tracing techniques. Brain Res Bull 1:379–384Google Scholar
  8. Fallon JH, Loughlin SE (1985) Substantia nigra. In: Paxinos G (eds) The rat nervous system, vol I. Academic, Sydney, pp 353374Google Scholar
  9. 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 180:545–580Google Scholar
  10. Gerfen CR, Baimbridge KG, Miller JJ (1985) The neostriatal mosaic: compartmental distribution of calcium binding protein and parvalbumin in the basal ganglia of the rat and monkey. Proc Natl Acad Sci USA 82:8780–8784Google Scholar
  11. Gerfen CR, Herkenham M, Thibault J (1987a) The neostriatal mosaic. II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J Neurosci 7:3915–3934Google Scholar
  12. Gerfen C, Baimbridge K, Thibault J (1987b) The neostriatal mosaic. III. Biochemical and developmental dissociation of patch-matrix mesostrial systems. J Neurosci 7:3935–3944Google Scholar
  13. German DC, Manaye KF, Sonsalla PK, Brooks BA (1992) Midbrain dopaminergic cell loss in Parkinson's disease and MPTP-induced parkinsonism: sparing of calbindin-D28K-containing cells. Ann NY Acad Sci 648:42–62Google Scholar
  14. Hallman H, Lange J, Olson L, Strömberg I, Jonsson G (1985) Neurochemical and histochemical characterization of neurotoxic effects of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine on brain catecholamine neurones in the mouse. J Neurochem 44:117–127Google Scholar
  15. Hof P, Morrison JH (1991) Neocortical neuronal subpopulations labeled by a monoclonal antibody to calbindin exhibit differential vulnerability in Alzheimer's disease. Exp Neurol 111:293–301Google Scholar
  16. Hökfelt T, Johansson O, Fuxe K, Goldstein M, Park D (1976) Immunohistochemical studies on the localization and distribution of monoamine neuron systems in the rat brain. I. Tyrosine hydroxylase in the mes- and diencephalon. Med Biol 54:427–453Google Scholar
  17. Hökfelt T, Johansson O, Goldstein M (1984a) Chemical anatomy of the brain. Science 225:1326–1334Google Scholar
  18. Hökfelt T, Martensson R, Björklund A, Kleinau S, Goldstein M (1984b) Distributional maps of tyrosine hydroxylase-immunoreactive neurons in the rat brain. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy, vol 2. Eisevier, Amsterdam pp 55–115Google Scholar
  19. Iacopino AM, Christakos S (1990) Specific reduction of calciumbinding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases. Proc Natl Acad Sci USA 87:4078–4082Google Scholar
  20. Ichimiya Y, Emson PC, Mountjoy CQ, Lawson DEM, Heizmann CW (1988) Eoss of calbindin-28K immunoreactive neurones from the cortex in Alzheimer-type dementia. Brain Res 475:156–159Google Scholar
  21. Jacobowitz D, Winsky E (1991) Immunocytochemical localization of calretinin in the forebrain of the rat. J Comp Neurol 304:198–218Google Scholar
  22. Jacobowitz DJ, Burns RS, Chuang C, Chiueh CC, Kopin I (1984) N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes destruction of the nigrostriatal but not the mesolimbic dopamine system in the monkey. Psychopharmacol Bull 20:416–422Google Scholar
  23. Juraska JM, Wilson CJ, Groves PM (1977) The substantia nigra of the rat: a Golgi study. J Comp Neurol 172:585–600Google Scholar
  24. Kobayashi K, Emson PC, Mountjoy CQ, Thornton SN, Eawson DEM, Mann DMA (1990) Cerebral cortical calbindin D28K and parvalbumin neurones in Down's syndrome. Neurosci Lett 113:17–22Google Scholar
  25. Eavoie B, Parent A (1991) Dopaminergic neurons expressing calbindin in normal and parkinsonian monkeys. Neuroreport 2:601–604Google Scholar
  26. Moriizumi T, Leduc-Cross B, Wu J-Y, Hattori T (1992) Separate neuronal populations of the rat substantia nigra pars lateralis with distinct projection sites and transmitter phenotypes. Neuroscience 43:711–720Google Scholar
  27. Parmentier M, Lefort A (1991) Structure of the human brain calcium-binding protein calretinin and its expression in bacteria. Eur J Biochem 196:79–85Google Scholar
  28. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd edn. Academic, SydneyGoogle Scholar
  29. Pickel VM, Joh TH, Field PM, Becker CG, Reis DJ (1975) Cellular localization of tyrosine hydroxylase by immunohistochemistry. J Histochem Cytochem 23:1–12Google Scholar
  30. Résibois A, Rogers JH (1992) Calretinin in rat brain: an immunohistochemical study. Neuroscience 46:101–134Google Scholar
  31. Ribak CE, Vaughn JE, Roberts E (1980) GABAergic nerve terminals decrease in the substantia nigra following hemitransections of the striatonigral and pallidonigral pathways. Brain Res 192:413–420Google Scholar
  32. Rogers JH (1987) Calretinin: a gene for a novel calcium binding protein expressed principally in neurons. J Cell Biol 105:1343–1353Google Scholar
  33. Rogers JH (1992) Immunohistochemical markers in rat brain: colocalization of calretinin and calbindin-D28K with tyrosine hydroxylase. Brain Res 587:203–210Google Scholar
  34. Rogers JH, Résibois A (1992) Calretinin and calbindin D28K in rat brain: patterns of partial colocalization. Neuroscience 51:843–865Google Scholar
  35. Seto-Ohshima A, Emson PC, Eawson E, Mountjoy CQ, Carrasco EH (1988) Eoss of matrix calcium-binding protein-containing neurons in Huntington's disease. Eancet 1(8597):1252–1256Google Scholar
  36. Sun CJ, Johannessen JN, Gessner W, Namura I, Singhaniyom W, Brossi A, Chiueh CC (1988) Neurotoxic damage to the nigrostriatal system in rats following intranigral administration of MPDP+ and MPP+. J Neural Transm 74:75–86Google Scholar
  37. Sutherland MK, Wong E, Somerville MJ, Yoong EKK, Bergeron C, Parmentier M, McEachlan DR (1993) Reduction of calbindin-28k mRNA levels in Alzheimer as compared to Huntington hippocampus. Mol Brain Res 18:32–42Google Scholar
  38. Trump BF, Berezesky IK (1992) The role of cytosolic Ca2+ in cell injury, necrosis and apoptosis. Curr Opin Cell Biol 4:227–232Google Scholar
  39. Winsky E, Nakata H, Martin B, Jacobowitz D (1989) Isolation, partial amino acid sequence and immunohistochemical localization of a brain-specific calcium binding protein. Proc Natl Acad Sci USA 86:10139–10143Google Scholar
  40. Yamada T, McGeer P, Baimbridge K, McGeer E (1990) Relative sparing in Parkinson's disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain Res 526:303–307Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Krystyna R. Isaacs
    • 1
  • David M. Jacobowitz
    • 1
  1. 1.Laboratory of Clinical Sciences, National Institute of Mental HealthBethesdaUSA

Personalised recommendations