Ž .Brain Research 805 1998 255–258
Short communication
Do all of human midbrain tyrosine hydroxylase neurons synthesizedopamine?
Keiko Ikemoto a,) , Ikuko Nagatsu a, Akiyoshi Nishimura b, Katsuji Nishi b, Ryohachi Arai a
a Department of Anatomy, Fujita Heath UniÕersity, School of Medicine, Toyoake, Aichi, 470-1192, Japanb Department of Legal Medicine, Shiga UniÕersity of Medical Science, Otsu, 520-2121, Japan
Accepted 16 June 1998
Abstract
Ž . Ž .We examined whether all of human midbrain tyrosine hydroxylase TH neurons substantially synthesize dopamine DA using dualŽ .labeling immunohistochemical technique of TH and aromatic L-amino acid decarboxylase AADC . In the substantia nigra, besides many
Ž wneurons doubly stained for TH and AADC, neurons stained only for TH and only for AADC D-neurons C.B. Jaeger, D.A. Ruggiero,V.R. Albert, T.H. Joh, D.J. Reis, Immunocytochemical localization of aromatic L-amino acid decarboxylase, in: A. Bjorklund, T. Hokfelt¨ ¨Ž .Eds. , Handbook of Chemical Neuroanatomy, Classical Transmitters in the CNS, Vol. 2, Part 1, Elsevier, Amsterdam, 1984, pp.
x.387–408. were identified. In the ventral tegmental area, dually labeled neurons and TH-only-positive neurons were found. It is indicatedthat the number of midbrain TH neurons does not reflect the exact number of DA neurons. q 1998 Elsevier Science B.V. All rightsreserved.
Keywords: Human; Substantia nigra; Ventral tegmental area; Aromatic L-amino acid decarboxylase; Tyrosine hydroxylase; Dopamine; Immunohistochem-istry
Ž .Dopamine DA is related to extrapyramidal motorcontrol and its lack in the central nervous system evokesparkinsonism. Its first step and rate-limiting enzyme is
Ž .tyrosine hydroxylase TH . TH has been used as a markerw xof putative dopaminergic neurons 2,13 . The existence of
this enzyme in the human midbrain neurons has beenconsidered to be an evidence of DA synthesis in this area.On the other hand, midbrain neurons which contain L-
w xDOPA as an endproduct has been also described 8,10 ,suggesting that these neurons lack the second step DA-synthesizing enzyme, aromatic L-amino acid decarboxylaseŽ .AADC . In fact, TH-positive but AADC-negativeŽ Ž . Ž ..TH q rAADC y neurons has been reported in various
w xmammalian brain including the humans 1,4,9 . Theseneurons are not catecholaminergic. The present study wasconducted to examine whether all of the human midbrainTH neurons contain AADC using dual labeling immuno-
) Corresponding author. Fax: q81-562-93-2649; E-mail:[email protected]
histochemical technique and a confocal laser-scanning mi-croscope.
Human brains were obtained from four autopsied casesŽ .24–64 years old, postmortem interval: 4–5 h in theDepartment of Legal Medicine, Shiga University of Medi-cal Science, Japan. These cases died from natural causesand had no known clinically and pathologically detectableneurological and psychiatric disease. Brains were immedi-ately sliced into 1 cm slabs and immersed in the fresh
Ž .fixative pH 7.4 containing 4% paraformaldehyde in 0.1Ž .M phosphate buffer PB at 48C for 48–72 h. The slices
were then transferred to PB containing 15% sucrose and0.1% sodium azide for storage at 48C. The brain sectionswere made using a cryostat in 50 mm thick in coronalplanes. The sections were treated with 40% methanol and
w x1% H O for 20 min to inhibit endogenous peroxidase 3 .2 2
The sections of the human brain were incubated inantibodies against TH or AADC diluted 1:10,000–70,000in PBS containing 0.3% Triton X-100 at 48C for 1 week.
ŽThen they were incubated in biotinylated rabbit IgG Vec-.tor Laboratory, 1:1000 for 12 h at 48C, and finally
Žavidin–biotin–peroxidase complex Vector Laboratory,
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 98 00661-1
( )K. Ikemoto et al.rBrain Research 805 1998 255–258256
.1:1000 for 1 h at room temperature. Peroxidase activityŽ .was then revealed with 50 mM Tris–HCl buffer pH 7.6
containing 0.0003% H O , 0.01% 3,3X-diaminobenzidine-2 2Ž .4HCl DAB and 1% nickel ammonium sulfate. Details of
the production, characterization, and specificity of theAADC and TH antisera have been described elsewherew x11,12 .
For dual labeling of TH and AADC, the sections werew xincubated in a mixture of rabbit anti-AADC antibody 12
Ž . Ždiluted 1:10,000 and mouse anti-TH antibody Incstar,.LNCI, diluted 1:1000 for 1 week, followed by incubation
in a mixtures of secondary antibodies; fluorescein-con-Ž .jugated donkey anti-rabbit IgG Chemicon; AP182F, 1:200
for AADC and Cy3-conjugated donkey anti-mouse IgGŽ .Chemicon; AP192C, 1:200 for TH. The specimens wereexamined under a confocal laser-scanning microscopeŽ . w xLSM410; Zeiss 4 .
A microphotograph of a TH-stained section through thehuman midbrain was shown in Fig. 1. TH-positive neuronswere distributed throughout the ventral tegmental areaŽ . Ž .VTA and the substantia nigra SN . In the SN, besides
Ž . Ž . ŽTH q rAADC q neurons Fig. 2A, shown in yellow or. Ž . Ž . Žorange , TH q rAADC y neurons Fig. 2A, red, arrow. Ž . Ž . Žheads and TH y rAADC q neurons Fig. 2A, green,.arrows were found under a laser-scanning microscope.
The three types were fusiform, bipolar or multipolar inŽ .shape, are medium- to large-sized 20–35 mm in diameter ,
and had long processes. In the VTA, most neurons werelabeled for TH and AADC in the center of the nucleusŽ . Ž . Ž .Fig. 2B, yellow or orange . TH q rAADC y neuronsŽ .Fig. 2B, red, arrow heads are intermingled in the dually
Ž . Ž .labeled neurons. The number of TH q rAADC y neu-rons were high in the ventral region of the VTA and dorsal
Ž .margin of the VTA Fig. 2B .The present study demonstrated that a part of TH-posi-
tive neurons in the human midbrain does not containAADC, suggesting that they do not synthesize DA. Such
Ž . Ž .neurons may be L-DOPA neurons or TH q rL-DOPA yw x Ž . Ž .neurons 8 . A significant number of TH q rAADC y
neurons in the VTA is in accordance with the report whichshows the existence of the VTA neurons containing L-
w xDOPA as an endproduct 10 .The present study also showed the presence of
Ž . Ž . Ž w x.AADC q rTH y neurons D-neurons 5 in the humanw xSN. D-neurons has been described in the rat VTA 6 .
There are species difference. In another mammal, labora-tory shrew study, D-neurons have been shown to synthe-
w xsize DA after administration of exogenous L-DOPA 7 .The human SN D-neurons might synthesize DA whenL-DOPA is given.
ŽFig. 1. Photomicrograph of a TH-stained section of the human midbrain. Photographs of a dually-labeled section of TH and AADC in the SN area. Ž .indicated by ‘A’ and VTA area indicated by ‘B’ were shown in Fig. 2A and B, respectively. Abbreviations: SNC, substantia nigra pars compacta; SNR,
substantia nigra pars reticulata; VTA, ventral tegmental area; PBP, parabrachial pigmented nucleus; CLi, central linear nucleus; IP, interpeduncularnucleus; cp, cerebral peduncle; scp, superior cerebellar peduncle.
( )K. Ikemoto et al.rBrain Research 805 1998 255–258 257
Ž . Ž . Ž .Fig. 2. Double-immunostaining of TH and AADC of a section through the human midbrain. A The SN. TH q rAADC q neurons are shown in yellowŽ . Ž . Ž . Ž . Ž . Ž . Ž . Ž . Ž .or orange, TH q rAADC y neurons in red arrow heads , and TH y rAADC q neurons in green arrows . B The VTA. TH q rAADC y
Ž .neurons arrows are shown in red. Barss100 mm.
Further studies should be conducted to elucidate thefunctions of the human midbrain neurons which containmonoamine-synthesizing enzymes.
Acknowledgements
This study was supported by Grant-in-Aid for ScientificResearch on Priority Areas, Ministry of Education, Sci-
ence, Sports and Culture of Japan to K.I., I.N., A.N. andR.A.
References
w x1 P. Gaspar, B. Berger, A. Febvret, A. Vigny, M. Krieger-Poulet, C.Borri-Voltattorni, Tyrosine hydroxylase-immunoreactive neurons inthe human cerebral cortex; a novel catecholaminergic group?, Neu-
Ž .rosci. Lett. 80 1987 257–262.
( )K. Ikemoto et al.rBrain Research 805 1998 255–258258
w x2 T. Hokfelt, R. Martensson, A. Bjorklund, S. Kleinau, M. Goldstein,¨ ¨Distributional maps of tyrosine hydroxylase-immunoreactive neu-
Ž .rons in the rat brain, in: A. Bjorklund, T. Hokfelt Eds. , Handbook¨ ¨of Chemical Neuroanatomy, Classical Transmitters in the CNS, Vol.2, Part 1, Elsevier, Amsterdam, 1984, pp. 277–379.
w x3 K. Ikemoto, K. Kitahama, A. Jouvet, R. Arai, A. Nishimura, K.Nishi, I. Nagatsu, Demonstration of L-DOPA decarboxylating neu-
Ž .rons specific to human striatum, Neurosci. Lett. 262 1997 111–114.w x4 K. Ikemoto, I. Nagatsu, K. Kitahama, A. Jouvet, A. Nishimura, K.
Nishi, T. Maeda, R. Arai, A dopamine-synthesizing cell groupdemonstrated in the human basal forebrain by dual labeling immuno-histochemical technique of tyrosine hydroxylase and aromatic L-
Ž .amino acid decarboxylase, Neurosci. Lett. 243 1998 129–132.w x5 C.B. Jaeger, D.A. Ruggiero, V.R. Albert, T.H. Joh, D.J. Reis,
Immunocytochemical localization of aromatic L-amino acid decar-Ž .boxylase, in: A. Bjorklund, T. Hokfelt Eds. , Handbook of Chemi-¨ ¨
cal Neuroanatomy, Classical Transmitters in the CNS, Vol. 2, Part 1,Elsevier, Amsterdam, 1984, pp. 387–408.
w x6 C.B. Jaeger, D.A. Ruggiero, V.R. Albert, D.H. Park, T.H. Joh, D.J.Reis, Aromatic L-amino acid decarboxylase in the rat brain: im-munocytochemical localization in neurons of the brainstem, Neuro-
Ž .science 11 1984 691–713.w x7 N. Karasawa, R. Arai, G. Isomura, T. Nagatsu, I. Nagatsu, Chemical
features of monoaminergic and non-monoaminergic neurons in theŽ .brain of laboratory shrew Suncus murinus are changed by systemic
Ž .administration of monoamine precursors, Neurosci. Res. 24 199567–74.
w x8 K. Kitahama, M. Geffard, H. Okamura, I. Nagatsu, N. Mons, M.Jouvet, Dopamine- and DOPA-immunoreactive neurons in the catforebrain with reference to tyrosine hydroxylase-immunohistoche-
Ž .mistry, Brain Res. 518 1990 83–94.w x9 K. Kitahama, K. Ikemoto, A. Jouvet, I. Nagatsu, M. Geffard, H.
Okamura, J. Pearson, Dopamine synthesizing enzymes in paraven-Žtricular hypothalamic neurons of the human and monkey Macaca
. Ž .fuscata , Neurosci. Lett. 243 1998 1–4.w x10 K. Komori, S. Uesaka, H. Yamaoka, K. Fujita, K. Yamaoka, H.
Naitoh, M. Kuroda, N. Karasawa, T. Ito, Y. Kasahara, I. Nagatsu,Identification of L-DOPA-immunoreactivity in some neurons in thehuman mesencephalic region; a novel DOPA neuron group?, Neu-
Ž .rosci. Lett. 157 1993 13–16.w x11 I. Nagatsu, Y. Kondo, S. Inagaki, N. Karasawa, T. Kato, T. Nagatsu,
Immunofluorescent studies on tyrosine hydroxylase: Application forŽ .its axoplasmatic transport, Acta Histochem. Cytochem. 10 1977
494–499.w x12 I. Nagatsu, M. Sakai, M. Yoshida, T. Nagatsu, Aromatic L-amino
acid decarboxylase-immunoreactive neurons in and around the cere-brospinal fluid-contacting neurons of the central canal do not containdopamine or serotonin in the mouse and rat spinal cord, Brain Res.
Ž .475 1988 91–102.w x13 J. Pearson, M. Goldstein, K. Markey, L. Brandeis, Human brainstem
catecholamine neuronal anatomy as indicated by immunocytochem-Ž .istry with antibodies to tyrosine hydroxylase, Neuroscience 8 1983
3–32.
