do all of human midbrain tyrosine hydroxylase neurons synthesize dopamine?

4
Ž . Brain Research 805 1998 255–258 Short communication Do all of human midbrain tyrosine hydroxylase neurons synthesize dopamine? 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, Japan b 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 dual Ž . labeling immunohistochemical technique of TH and aromatic L-amino acid decarboxylase AADC . In the substantia nigra, besides many Ž w neurons 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 indicated that the number of midbrain TH neurons does not reflect the exact number of DA neurons. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Human; Substantia nigra; Ventral tegmental area; Aromatic L-amino acid decarboxylase; Tyrosine hydroxylase; Dopamine; Immunohistochem- istry Ž . Dopamine DA is related to extrapyramidal motor control and its lack in the central nervous system evokes parkinsonism. Its first step and rate-limiting enzyme is Ž . tyrosine hydroxylase TH . TH has been used as a marker w x of putative dopaminergic neurons 2,13 . The existence of this enzyme in the human midbrain neurons has been considered to be an evidence of DA synthesis in this area. On the other hand, midbrain neurons which contain L- w x DOPA as an endproduct has been also described 8,10 , suggesting that these neurons lack the second step DA- synthesizing enzyme, aromatic L-amino acid decarboxylase Ž . AADC . In fact, TH-positive but AADC-negative Ž Ž . Ž .. TH qrAADC y neurons has been reported in various w x mammalian brain including the humans 1,4,9 . These neurons are not catecholaminergic. The present study was conducted to examine whether all of the human midbrain TH 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 cases Ž . 24–64 years old, postmortem interval: 4–5 h in the Department of Legal Medicine, Shiga University of Medi- cal Science, Japan. These cases died from natural causes and had no known clinically and pathologically detectable neurological 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 0.1 Ž . M phosphate buffer PB at 48C for 48–72 h. The slices were then transferred to PB containing 15% sucrose and 0.1% sodium azide for storage at 48C. The brain sections were made using a cryostat in 50 mm thick in coronal planes. The sections were treated with 40% methanol and wx 1% H O for 20 min to inhibit endogenous peroxidase 3 . 2 2 The sections of the human brain were incubated in antibodies against TH or AADC diluted 1:10,000–70,000 in PBS containing 0.3% Triton X-100 at 48C for 1 week. Ž Then they were incubated in biotinylated rabbit IgG Vec- . tor Laboratory, 1:1000 for 12 h at 48C, and finally Ž avidin–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

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Page 1: Do all of human midbrain tyrosine hydroxylase neurons synthesize dopamine?

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

Page 2: Do all of human midbrain tyrosine hydroxylase neurons synthesize dopamine?

( )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.

Page 3: Do all of human midbrain tyrosine hydroxylase neurons synthesize dopamine?

( )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.

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