Ž .Brain Research 853 2000 377–380www.elsevier.comrlocaterbres

Short communication

Human midbrain dopamine neurons express serotonin 2A receptor: animmunohistochemical demonstration

Keiko Ikemoto a,), Akiyoshi Nishimura b, Nobuo Okado c, Masahiko Mikuni d, Katsuji Nishi b,Ikuko Nagatsu a

a Department of Anatomy, School of Medicine, Fujita Heath UniÕersity, Toyoake, Aichi 470-1192, Japanb Department of Legal Medicine, Shiga UniÕersity of Medical Science, Otsu 520-2192, Japan

c Department of Anatomy, Institute of Basic Medical Science, UniÕersity of Tsukuba, Tsukuba, Ibaraki 305-0018, Japand Department of Neuropsychiatry, Gumma UniÕersity School of Medicine, Maebashi 371-8511, Japan

Accepted 12 October 1999

Abstract

Ž . Ž .We demonstrated intense serotonin 5-HT 2A receptor immunoreactivity in the human ventral tegmental area VTA using by aŽ .recently raised antibody against 5-HT2A receptor. The substantia nigra SN neurons also showed 5-HT2A receptor immunoreactivity.

Ž .Double immunohistochemistry of 5-HT2A receptor and tyrosine hydroxylase TH revealed many neurons doubly labeled by 5-HT2Areceptor and TH in the VTA and SN. It is suggested that activity of human midbrain dopaminergic neurons might be strongly regulatedvia 5-HT2A receptors at the level of their originating nuclei. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Human; Substantia nigra; Ventral tegmental area; Oculomotor nucleus; Serotonin 2A receptor; Serotonin; Tyrosine hydroxylase; Dopamine;Immunohistochemistry

Ž . Ž .Serotonin 5-HT –dopamine DA interaction via 5-HT2A receptors has been implicated in etiology of psy-choses and therapeutic actions of antipsychoticsw x2,9,15,16,18 . Although there are numerous animal studiesthat show inhibitory actions of 5-HT on DA neuronsw x9,10,16,18 , little has been known in human materialsw x17 . Recently, 5-HT2A receptor immunoreactivity hasbeen reported in a small number of neurons of the rat

Ž . Ž .ventral tegmental area VTA and substantia nigra SNw x1,3 , the originating nuclei of mesolimbic or mesostriataldopaminergic neurons. These 5-HT2A receptor immunore-

Ž .active -ir neurons might be DA neurons. However, con-sidering some species differences in mammalian

w xmonoaminergic neuronal systems 4,5,7,11,13 , expression

) Corresponding author. Fax: q81-562-93-2649; e-mail:kikemoto@fujita-hu.ac.jp

of 5-HT2A receptors in the human homologous areasmight be different from that in the rat.

The present study was conducted to elucidate morpho-logical interaction between DA neurons and 5-HT2A re-ceptors in the VTA and SN in the human, with immunohis-tochemical method by using antibodies against 5-HT2A

w x Ž . w xreceptor 3 and tyrosine hydroxylase TH 12 .Human brains were obtained from four autopsied cases

Ž24–47 years old, post-mortem interval to fixation: 4–16.h in Department of Legal Medicine, Shiga University of

Medical Science, Japan. These cases died from naturalcauses and had no known clinically and pathologicallydetectable neurological and psychiatric diseases. Brainswere immediately sliced into 1 cm slabs and immersed in

Ž .the fresh fixative pH 7.4 containing 5% glutaraldehyde orŽ .4% paraformaldehyde in 0.1 M phosphate buffer PB at

48C for 5–72 h. The slices were then transferred to PBcontaining 15% sucrose and 0.1% sodium azide for storageat 48C. The brain sections were made using a cryostat in50 mm thick in coronal planes. The sections were treated

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 02237-4

( )K. Ikemoto et al.rBrain Research 853 2000 377–380378

with 40% methanol and 1% H O for 20 min to inhibit2 2w xendogenous peroxidase 4 .

The sections of the human brains were incubated inŽantibodies against N-terminal of 5-HT2A receptor pro-

. w xvided by Dr. N. Okado, Japan 3 diluted 1: 200–400 orw xTH 12 diluted 1:10,000–70,000 in PBS containing 0.3%

Triton X-100 at 48C for 1 week. Then they were incubatedin secondary antibodies for 12 h at room temperature;

Žbiotinylated anti-chicken IgG Vector, Burlingame, CA,.USA; 1:1000 was used for the 5-HT2A receptor antibody,

Ž .biotinylated anti-rabbit IgG Vector; 1:1000 for the THantibody. Finally, they were incubated in avidin–biotin–

Ž .peroxidase complex Vector; 1:1000 for 1 h at roomtemperature. Peroxidase activity was then revealed with 50

Ž .mM Tris–HCl buffer pH 7.6 containing 0.0003% H O ,2 2Ž .0.01% 3,3-diaminobenzidine DAB –4 HCl and 1% nickel

Ž .ammonium sulfate DAB–nickel method . Details of theproduction, characterization, and specificity of the 5-HT2Areceptor and TH antisera have been described elsewherew x3,12 . To confirm the specificity of 5-HT2A receptorantiserum, an absorption test was performed. A synthe-sized peptide, corresponding to amino acids 14–33 in the

w xN-terminal extracellular region of rat 5-HT2A receptor 3was used as an antigen of the absorption test. An atlas of

w x w xNieuwenhuys et al. 14 and our previous study 6 wereused to determine the anatomical territories.

For dual labeling of 5-HT2A receptor and TH, thesections were incubated in a mixture of 5-HT2A receptor

w x Ž . w x Žantibody 3 diluted 1:200 and TH antibody 12 diluted.1:10,000 for 1 week. A half of the sections were incu-

bated in biotinylated anti-chicken IgG, followed by incuba-Ž .tion in avidin–biotin–peroxidase complex Vector; 1:1000

for 1 h at room temperature, and peroxidase activity wasŽ .revealed with 50 mM Tris–HCl buffer pH 7.6 containing

0.0015% H O and 0.05% DAB. Then, the sections were2 2

incubated in biotinylated anti-rabbit IgG, followed by incu-Žbation in avidin–biotin–peroxidase complex Vector;

.1:1000 , and peroxidase activity was revealed by DAB–nickel method. The other half of the sections were incu-bated in ultrasmall gold conjugate goat anti-rabbit IgGŽAurion, GAR GP-US, Wageningen, Netherlands; diluted

.1: 100 at 48C overnight, and TH immunoreactivity wasŽvisualized by a silver intensification kit IntenSEe Silver

Enhancement Kit, Amersham, RPN 491, Bucking-.hamshire, England . The sections were incubated in bio-

tinylated anti-chicken IgG, then in avidin–biotin–per-Ž .oxidase complex Vector; 1:1000 , and DAB–nickel

method was applied.

The absorption test resulted in no immunoreactivity instandard immunohistochemical procedures, which con-firmed the specificity of the 5-HT2A receptor antiserum.

In the VTA, intense 5-HT2A receptor immunoreactivityŽwas observed in neuromelanin-pigmented neurons Fig.

.1A,D . In the SN, neuromelanin-pigmented neurons alsoŽ .showed 5-HT2A receptor immunoreactivity Fig. 1B,E .

The 5-HT2A-ir neurons in the VTA and SN were round,ovoid, bipolar or multipolar in shape and 20–35 mm indiameter. These 5-HT2A receptor-ir neurons possessed one

Ž .to two processes and well-stained nuclei Fig. 1D,E . Thenucleoli of these neurons showed weak immunoreactivityŽ .Fig. 1D,E . In the marginal area of the SN, 5-HT2Areceptor-ir neurons with no neuromelanin pigment wereoccasionally seen. In the neurons of the oculomotor nu-

Ž .cleus ON , intense 5-HT2A receptor immunoreactivityŽ .was observed Fig. 1C . In these 5-HT2A receptor-ir neu-

rons possessed some processes and well-stained nucleiŽ .Fig. 1F . 5-HT2A receptor immunoreactivity was espe-cially intense in the marginal regions of some cell bodies

Ž .of 5-HT2A receptor-ir neurons Fig. 1D,F arrow heads .In double immunohistochemistry for 5-HT2A receptor

and TH, many neurons were doubly stained for 5-HT2AŽ .receptor and TH in the VTA and SN Fig. 1G,H . In the

marginal area of the SN, some neurons were singly stainedŽ .for 5-HT2A receptor data not shown . Such 5-HT2A

receptor-only-ir neurons lacked neuromelanin pigment.The present study showed 5-HT2A receptor-ir neurons

in neuromelanin-pigmented neurons, putative DA neurons,in the human VTA and SN. The fact that 5-HT2A receptorand TH were doubly immunostained in the human VTAand SN is another evidence that human midbrain dopamin-ergic neurons express 5-HT2A receptors. In the rat VTAand SN, 5-HT2A receptor-ir neurons have been described,

w xhowever, the quantity is small 1,3 . In the human homolo-gous area, the density of 5-HT2A receptor-ir neuronsseems to be far higher than that in the rat. The speciesdifference is evident.

On the other hand, the existence of a number of 5-HT2Areceptor-ir neurons is likely to be common in the ON both

w xin the rat 1,3,19 and human.In the present results, 5-HT2A receptor immunoreactiv-

ity was more intense in the neurons in the VTA than thoseŽ .in the SN compare Fig. 1D with E . The VTA is an

originating nucleus of the mesolimbic DA system, whichrelates to motivation, attention and reward. The rat studieshave been shown that 5-HT2A receptors in the midbrainand dorsal raphe nucleus are synthesized in the neuronal

Fig. 1. Photomicrographs of immunostained brain sections obtained by an autopsy of a 35-year-old female. In this case, post-mortem period to fixation wasŽ .16 h, and fixation was performed with 5% glutaraldehyde in 0.1 M PB for 5 h. A–F Single-staining of 5-HT2A receptor in a section through the

Ž . Ž . Ž . Ž .midbrain. A,D The VTA. B,E The SN. C,F The ON. In the VTA and SN, many neuromelanin-pigmented neurons brown show 5-HT2A receptorŽ . Ž . Ž .immunoreactivity purple . The marginal regions of some 5-HT2A receptor-ir neurons are well-stained D,F arrow heads . G,H . Double-staining ofŽ . Ž . Ž . Ž .5-HT2A receptor brown and TH purple . TH-staining was intensified by nickel. G The VTA. H The SN. Many neurons are doubly stained for

5-HT2A receptor and TH. Abbreviations: VTA, ventral tegmental area; SN, substantia nigra; ON, oculomotor nucleus, mlf; medial longitudinal fasciculus.

( )K. Ikemoto et al.rBrain Research 853 2000 377–380 379

cell bodies, and are likely to be mainly transported retro-gradely to the dendrites rather than anterogradely to the

w xaxons 1,8 . Taken together with the present results, it isindicated that human mesolimbic DA systems might be

( )K. Ikemoto et al.rBrain Research 853 2000 377–380380

powerfully regulated by 5-HT via 5-HT2A receptors at thelevel of the midbrain originating nuclei.

Acknowledgements

This study was supported by Grant-in-Aid for ScientificResearch on Priority Areas, Ministry of Education, Sci-

Ž .ence, Sports and Culture of Japan to K.I. C-1 10680713 ,Ž . Ž .A.N., M.M. B 11470200 , K.N. C-2 11670413 and I.N.,

and Fujita Health University in Japan.

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