Number of striatal D-neurons is reduced inautopsy brains of schizophrenics

Keiko Ikemotoa,b,c,*, Akiyoshi Nishimurad, Tatsuro Odae, Ikuko Nagatsub, Katsuji Nishic

aClinical Research Institute, National Minami Hanamaki Hospital, Hanamaki, Iwate, 025-0033, JapanbDepartment of Anatomy, Fujita Health University, School of Medicine, Toyoake, Aichi, 470-1192, Japan

cDepartment of Legal Medicine, Shiga University of Medical Science, Setatsukinowacho, Otsu, 520-2192, JapandDepartment of Legal Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan

eNational Shimofusa Sanatorium, Chiba, 226-0000, Japan

Abstract

The human striatum, especially its ventral part, the nucleus accumbens, contains numerous neurons immunoreactive for

aromatic l-amino acid decarboxylase (AADC, the second-step monoamine synthesizing enzyme, ¼ DDC: dopa decarboxy-

lase), but not for tyrosine hydroxylase (TH, the first-step catecholamine synthesizing enzyme) or tryptophan hydroxylase (TPH,

the first-step serotonin synthesizing enzyme) (Neurosci Lett 232 (1997) 111–114). These AADC (1)/TH (2)/TPH (2) neurons

are named as D-neurons (Jaeger CB, Ruggiero DA, Albert VR, Joh TH, Reis DJ. Immunocytochemical localization of

aromatic-l-amino acid decarboxylase. In: Bjorklund A, Hokfelt T, editors. Classical transmission in the CNS, Part I, Handbook

of chemical neuroanatomy, vol. 2. Amsterdam: Elsevier, 1984. pp. 387–418). The nucleus accumbens is one of the brain

regions that is involved in the pathogenesis of schizophrenia. We examined the distribution of striatal D-neurons using AADC

immunohistochemistry and postmortem brains obtained by legal and pathological autopsies (nine controls (27–75 years old)

and nine schizophrenics (32–78 years old), postmortem interval to fixation (PMI): 2–30 h). Because the number of AADC-

positive neurons per section had a tendency to reduce in the case with longer PMI, we analyzed specimens of five controls (27–

64 years old) and six schizophrenics (51–78 years old) in which the PMI was less than 8 h. The number of AADC-positive

neurons was reduced in the striatum of schizophrenics compared to that of controls. The reduction was significant in the nucleus

accumbens (P , 0:05, t-test). D-Neurons might be involved in the pathogenesis of schizophrenia. Further studies using sex-,

age- and PMI-matched controls are essential.

q 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Nucleus accumbens; Aromatic l-amino acid decarboxylase; Tyrosine hydroxylase; Schizophrenia; Immunohistochemistry; D-

Neuron

1. Introduction

Aromatic l-amino acid decarboxylase (AADC,

¼ DDC: dopa decarboxylase) is the second-step

monoamine synthesizing enzyme, which converts l-

dopa, 5-hydroxytryptophan (5-HTP) and exogenous

droxidopa (l-threo-DOPS) to dopamine (DA), seroto-

nin and noradrenaline, respectively, and is also the

rate-limiting synthesizing enzyme of trace amines

including tyramine and betaphenylethylamine [1].

Recently, we found that human striatum, especially

its ventral part, the nucleus accumbens (Acc),

contains many neurons immunoreactive for AADC,

but not for tyrosine hydroxylase (TH) or 5-HTP

Legal Medicine 5 (2003) S221–S224

1344-6223/03/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.

doi:10.1016/S1344-6223(02)00117-7

www.elsevier.com/locate/legalmed

* Corresponding author. Clinical Research Institute, National

Minami Hanamaki Hospital, 500 Suwa, Hanamaki, Iwate, 025-

0033, Japan. Tel.: 181-198-24-0511; fax: 181-198-24-1721.

E-mail address: ikemoto@shanamaki.hosp.go.jp (K. Ikemoto).

[2,3]. These AADC (1)/TH (2)/TPH (2) neurons are

named as D-neurons [4–6].

The Acc is thought to be an interface nucleus that

has functions to convert emotion to action [7], and the

nucleus receives DA projection from the midbrain

ventral tegmental area (A10) [8]. This mesolimbic

DA system has been thought to be involved in the

etiology of mental disorders including schizophrenia

[9,10]. Therefore, we suspected that these D-neurons

might be related to the pathogenesis of schizophrenia.

In the present study, we examined the distribution

of striatal D-neurons of schizophrenic patients using

AADC immunohistochemistry [2,11,12] and post-

mortem brains obtained by legal and pathological

autopsies.

2. Materials and methods

2.1. Tissue preparations

Human brains were obtained from 12 legal autopsy

cases (32–75 years old, postmortem interval to fixa-

tion (PMI): 4–30 h, control: nine, schizophrenia:

three) in the Department of Legal Medicine, Shiga

University of Medical Science, Japan, in compliance

with the ethical code of the Ethical Committee of the

Japanese Society of Legal Medicine, and nine patho-

logical autopsy cases (27–78 years old, PMI: 2–21 h,

schizophrenia: six, senile psychosis: three) of the

National Shimofusa Sanatorium, Japan with approval

of the Ethical Committee of the National Shimofusa

Sanatorium. A total of 18 brains, nine control brains

(27–75 years old, PMI: 4–30 h) and nine schizophre-

nic brains (32–78 years old, PMI: 2–21 h), were used.

The former had died from natural causes and had no

known clinically and pathologically detectable neuro-

logical and psychiatric diseases.

Brains were immediately sliced into 1 cm slabs and

immersed in the fresh fixative at 4 8C for 48–72 h.

Tissue preparations were performed based on

previous studies [2,11,12]. Cryostat 50 mm sections

were made in coronal planes through the levels of the

Acc and midbrain.

2.2. Immunocytochemistry

For AADC immunocytochemistry, two primary

antibodies (Ref. [14] and Eugene Tech) diluted

1:10,000–30,000 in 0.1 M phosphate-buffered saline

containing 0.3% Triton X-100 were used to confirm

the reliability of the results. The free floating method

and the ABC-DAB method were applied. Details of

immunocytochemical procedures for AADC or TH

were also described previously [13,14]. At least

three sections were stained for each antibody. The

number of AADC-positive neurons per section was

counted under a light microscopy. An atlas of Mai

et al. was used to identify the anatomical territories

[15].

The present study was approved by the Ethical

Committee of the Clinical Research Institute,

National Minami Hanamaki Hospital, Japan.

3. Results

In sections through the midbrain, DA neurons in the

substantia nigra and ventral tegmental area showed

immunoreactivity for AADC as well as TH. Some

brains in which the PMI was more than 15 h showed

weak AADC stainability.

The number of AADC-positive neurons in the stria-

tum per section had a tendency to decrease in the case

with longer PMI. Therefore, we analyzed specimens

of five controls (27–64 years old) and six schizophre-

nics (51–78 years old) in which the PMI was less than

8 h.

The distribution density of AADC-positive neurons

differed between individuals. In most cases, regard-

K. Ikemoto et al. / Legal Medicine 5 (2003) S221–S224S222

Fig. 1. AADC-immunoreactive neurons in the nucleus accumbens

(Acc). They are fusiform, bipolar or multipolar in shape, and have

thick dendritic arbor (s). Bars: 25 mm.

less of whether they were controls or schizophrenics,

the density of AADC-immunoreactive neurons was

higher in the Acc than in the caudate nucleus (Ca)

and putamen (Pu) (Fig. 1). In some cases, the density

of AADC-positive neurons in the dorsal area of the Pu

and Acc did not differ.

Generally, the brains of schizophrenic patients had

a smaller number of AADC-positive neurons in the

Ca, Pu and Acc. In the Acc, the number of AADC-

positive neurons in schizophrenic patients was signif-

icantly lower than that in controls (P , 0:05, t-test)

(Fig. 2).

4. Discussion

The significance of human striatal D-neurons, and

the functions of D-neurons in the Acc, the projection

field of the mesolimbic DA system, are as yet unclear

[16]. The Acc receives fiber projections of excitatory

amino acid from the hippocampus, anterior cingulate

cortex, amygdala and thalamus, and the nucleus

projects to ventral pallidum and midbrain [8]. The

Acc is thought to be an interface that converts emotion

to action [7]. It is also thought to relate to the etiology

of schizophrenia [9,10].

In the present study, the number of D-neurons in the

striatum, especially in the Acc, was reduced in

autopsy brains of schizophrenic patients. Although

the number of examined cases was small, the reduc-

tion of D-neurons in schizophrenic patients was clear

(Fig. 2). Does this reduction really represent pathog-

nomonic procedures of schizophrenia? At first, influ-

ences of antipsychotic drugs should be considered.

One schizophrenic patient who did not receive medi-

cation for 1 month before death also showed a reduc-

tion of the number of striatal D-neurons. In our

experiments, chronic administration of haloperidol

(1 mg/1 kg per day) for 2 months did not apparently

reduce the number of AADC-positive neurons in the

rat striatum (Ikemoto et al., unpublished data) [3]. To

the best of our knowledge, there is no evidence show-

ing that antipsychotics reduce the number of striatal

D-neurons. Further studies should be conducted by

using postmortem brains of schizophrenic patients

who have never taken antipsychotic medication. It is

also essential to examine the interaction between the

number of D-neurons and the duration of illness, and

to compare the results using age-, sex- and PMI-

matched controls.

AADC is not only the second-step synthesizing

enzyme for monoamines, but the rate-limiting synthe-

sizing enzyme for trace amines including tyramine

and betaphenylethylamine [1]. The present results

may indicate the possible involvement of trace amines

in human mental functions and the etiology of schizo-

phrenia [17].

Acknowledgements

This study was supported by Grants-in-Aid for

Scientific Research of the Japan Society for the

Promotion of Science (C1-10680713, C1-12680740),

Fujita Health University and Sumitomo Pharmaceuti-

cal Corporation.

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