Calbindin immunoreactivity in the hippocampal formation and neocortex of schizophrenics

Pmg. N-sydtophmmaml

(L Bid

F3&iat.

cOp&ht 0 Printed

1999, Vol. 23, pp. 40942 1999 Elsevicr

in the USA.

front matter

PII 60278-as46(99)OOW’&6

CALBIRDIR IIUMUROREACTIVITY INTHEmmOCAMPALFORIuATI0~ AND NEOCORTEX OF SCHIZOPHRENICS SHVJI IRITANP, NORIOMI KVROKP, KENJI IKEDA** andhYJIME KAZAMATSVRP

*Dept. of Psychiatry, Tokyo Metropolitan Matsuzawa Hospital Tokyo,JAPAN,**Dept. of Neuropathology, Tokyo Institute of Psychiatry Tokyo, JAPAN (Final form! March

1999)

Abstract Iritani Shuji, Noriomi Kuroki, Kenji Ikeda and Hajime Kazamatsuri: Calhindin Immunoreactivity in The Hippocampal Formation and Neocortex of Schizophrenics. Prog. Neuro-Psychophdrmacol. & Biol. Psychiat. 1999,2& pp. 409421.01999 Elsevier ScienceInc. l.The authors studied the morphology of CalbindinDZgK (CaBp) immunoreactive ceils and processes in the hippocampal formation and the prefrontal cortex of schizophrenics using the immunohistochemicai technique of avidin-biotin-complex method (ABC method), and the results were compared with those from normal human brains. 2.In the hippocampal formation area CA2 of schizophrenics, many CaBp-immunopositive cell bodies and fibers were disordered in their arrangement compared to normal control brains. 3.In the prefrontal cortex (Brodmann area 9) of schizophrenics, many immunopositive cell bodies were exhibited irregular axis arrangement and fiber disarray. 4. The altered distribution pattern of CaBp-immunopositive structures in the hippocampal formation and the prefrontal cortex might indicate the existence of GABA(gamma-aminobutyric acid)ergic dysfunction in the brain of schizophrenic patients. Keywords: calbindinD28K, hippocampus, immunohistochemistry, prefrontal cortex, schizophrenia. . . e avidin-biotin-complex method (ABC method), CalbindinD28K (CaBp), Diagnostic and Statistical manual of Mental Disorders. 4th ed.(DSM-IV), gamma-aminobutyric acid (GABA), 2% normal goat serum (NGS), phosphate buffered saline (PBS), T&-Cl buffered saline (TBS; pH 7.4, 0.9% NaCl), 0.3% Triton X-lOO(TX) ductim Since the concept of dementia praecox was defined by Kraepelin nearly a century ago, many investigators have considered schizophrenia to be an organic disease. Many neuropdthoiogical findings in schizophrenic brains have been reported, but no consistent features have been established for this condition. In the last 20 years, two major streams of neuropathological studies of schizophrenia have developed. One stream of studies focuses on limbic structures. These studies have shown that the ventricle volume is enlarged in schizophrenic brain and the volume of the limbic system is reduced, as shown by neuroimaging(Bachneff,1991).

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These studies strongly suggest that the cognitive dysfunction

of schizophrenia is due to the disorganization of the limbic system. The other stream is focuses on neurodevelopmental disorders of the schizophrenic brain. Studies of neuropathological abnormalities in schizophrenic brain have been recently increasing and Roberts et

410

s. hitani et Ql.

a1(1986,1987) reported that there were no neuronal degeneration Moreover, there are no reports demonstrating neuritic plaque in the schizophrenic schizophrenia

is

neuropathological

a

like a gliosis in schizophrenic

abnormal protein deposits such as neurofibrillary

brain. These

neurodevelopmental,

findings

not

have been interpreted

neurodegenerative,

disorder.

studies have concentrated on quantitative measurements

instead of the qualitative histological analysis in schizophrenic

brains. tangle or

as evidence Therefore,

that recent

of the neuronal construction

brains, especially in the hippocampus.

These quantitative studies of the hippocampal,formation

include i) meaurement

of tissue volume

(Bogerts et al., 1985; Bogerts et al., 199(I), ii) measurement of neuronal density (Benes et al., 1991; Benes et al., 1993), iii) neurons counting (Falkai and Bogerts, 1986; Heckers et al., lYYl), iv) observation of the arrangement

of pyramidal

cells (Bents

et al., 1991; Chun and Shatz, 1YXY; Kovelman

and Scheibel,

1984), v) measurement of pyramidal cell size (Benes et al., 1991; Zaidel et al.. 1997) and vi) examination of various focused neurons or proteins by staining including immunohistochemical al., 1991). Benes et al (1996) reported that the number of gamma-aminobutyric in dentate gyms, area CA4, area CA3, subiculum, and the presubiculum in schizophrenic

brains. But the number of chemical neuroanatomical

Neuropathological reported(Benes

abnormalities

of cerebral

et al., 1986; Akbarian

Neuropsychological

frontal

et al., 1993; Akbarian

studies have suggested

In this study, the authors demonstrated

clarified (Baimbridge

studies are limited. in schizophrenics

have also been

et al.. 1996: Selemon

the hypothesis

proteins

group,

pathogenesis

1982; Celio. 1990).

in the hippocampal

using the ABC immunohistochemical

technique,

with those from normal human brain. CaBp is a member

and is widely However,

distributed its precise

in the mammalian physiological

central nervous

function

et al., 1992). On e of its function is believed to be the regulation

excitability through altering concentrations

of calcium ions within neurons.

has not been of neuronal

Further, cortical GABAergic

neurons also contain CaBp in the cytoplasm, and this protein may act as a neuromodulator Moreover,

et al., 1995). may be related

that schizophrenia

the distribution of CaBp immunorcactivity

cortex of schizophrenics

and compared the findings from schizophrenics

system(Baimbridge,

were elevated

of the frontal cortex.

formation and the prefrontal

of calcium binding

of hippocampus

that cognitive impairment in schizophrenics

to the prefrontal cortex (Faux et al., 1988), supporting involves the dysfunction

cortex

staining (Arnold et

acid (GABA, ) receptors

(Celia, 1990).

CaBp colocalized in the cholinergic neurons of the nucleus basalis of Meynert in primates,

but this protein was absent in rats(Celio and Norman,

1985; Chang and Kuo, 1YYl). These studies

suggest that CaBp may be involved in the higher level neuronal functions like memory and/or learning. In the present study, the authors examined the neurons containing CaBp in the hippocampal and prefrontal cortex of the schizophrenic

formation

brains.

Three normal control brains and brains of 6 schizophrenic

patients were used. All caSes had been

CaJ3p containing admitted and treated in our hospital.

Brains without neuropsychiatric

control cases. Diagnosis of schizophrenia Association,

neurons in schizophrenics

411

disease were selected as normal

was conducted by applying the DSM-IV(American

1994). None of the cases had a history of alcohol abuse, convulsions

which may have the neuropathologically

Psychiatric

or encephalitis,

altered the central nervous system. The clinical characteristics of

all cases are shown in Table 1.

Table 1. Clinical

Characteristics Normal Case Norm.1 Case Norm.2 Case Norm.3 Mean SD

of Control

Age (years) 76

Sex

and Schizophrenic

Patients

Subjects.

PMI* (hours) 3

Cause of Death

M

Brain Weight(g) 1320

54

M

1440

8

Renal dysfunction

61

F

1360

18

Gallbladder cancer

63.7

1373

9.18

49.89

Schizophrenia

Age (years)

Sex

Brain Weight(g)

CaseSch.1

56

M

Stomach cancer

Duration of Illness (years)

PMI* (hours)

Cause of Death

1370

25

12

Pneumonia

7

Gastric ulcer

2

Acute heart failure

Case Sch.2

68

M

1290

31

Case Sch.3

56

F

1220

23

Case Sch.4

63

F

1100

30

2

Acute heart failure

Case Sch.5

43

M

1380

15

10

Pneumonia

Case Sch.6

63

M

1320

42

15

Bone cancer

Mean

5x.2

12h2

SD

8.28

96.00

*PMI: Postmortem interval

Also, all cases lacked neuropathological

changes associated with neurodegenerdtion

cerebral vascular disease, this was confirmed by several neuropathologists

like gliosis or/and

by microscopic

examination

of Bematoxylin Eosin stained or Kltiver-Barrera stained specimens. The bereaved families of all cases consented to this study. Procedure In all cases, autopsy were performed

as soon as possible after death. Brains were cut into small

412

S. Iritani et al.

blocks(5mm thick) for immersion fixation. Brains were fixed in the Zamboni solution(Zamboni Marino, 1967) for two nights(CC),

and De

then immersed in 20% sucrose-O. 1M Phosphate Buffer solution for

at least 3 days or more(4”C). Thirty micron-thick

sections were made of the fixed brain slices using a

freezing microtome or cryostat. Sections were collected, rinsed and stored in phosphate

buffered saline

(PBS), O.g%NaCl in 0.1M PB(pH 7.4), for at least 3 days and for up to two weeks prior to the next immunohistochemical

staining procedures.

Brain sections were rinsed in O.lM Tris-Cl buffered saline (TBS; pH 7.4, 0.9% NaCl), containing 0.3% Triton X-100 (TX) and 2% normal goat serum(NGS), sections

were incubated

1) in a commercially

(Sigma,; dilutions of l/1000-l/2000 medium containing temperature(45

biotinylated

min),and

available monoclonal

in 2% NGS-0.3% anti-rabbit

for 30-60 min. at room temperature. The

IgG (Vector:

then 3) in medium

anti-CalbindinD28K

TX-O. 1M THS solution), diluted l/100

containing

avidin-biotin

antibody

overnight (4°C) 2) in

in NGS-TX-TBS) peroxidase

at room

complex

[ABC

method(Hsu et al., 1981)] for 45 min. After each incubation step, the sections were rinsed in NGS-TXTBS solution.

Finally, the sections were rinsed in TBS twice for 10 min reacted with 0.05%,

diaminobenzine-HCl

in 0.05M Tris-Cl buffer (pH7.6)

3,3’

two or three times, and mounted onto gelatin

coated slides. After drying, the sections were dehydrated and coverslipped

with Permount,and

examined

with a light microscope. Control studies were conducted immunocytochemical

using the same immunostaining

labeling the diluted antiCalbindinD28K

pg/ml of Calcium-binding

procedure,

was preabsorbed

except that prior to with 10 pg/ml or 50

protein(Vitamin D-induced) (Sigma).

In case of normal human controls,

CaBp positive cells exhibited well ordered

run in a uniform direction in the CA2 area of the hippocampus.

ardyS

and their fibers

Positive cells in the CA2 area were

mostly pyramidal cells with nearly identical cell body size. Cells orientation was also nearly uniform. On the other hand, in all cases of schizophrenia,

CaBp-positive

neurons exhibited disoriented arrays. CaBp-

positive neurons were scarce and their shapes were not uniform compared to normal control neurons. The density of CaBp positive cells in the CA2 region of all schizophrenic than that of normal controls. Many of these cells in schizophrenic coinciding with their disarray, and their processes

cases was significantly

lower

brains had long or short processes

were orientated at random directions (Fig 1, Fig 2a,

Fig 2b).

In the prefrontal cortex (Brodmann III-IV

of three schizophrenic

area 9) of the schizophrenic

cases, CaBp-positive

cases were disarrayed compared to normal controls.

neurons in layer In normal control

cases, these positive cells were in well ordered arrays and their processes oriented perpendicular to the

CaBp containing

neurons

413

in schizophrenics

_

&Observed

Fig 1. Photomictogrqhs of the CA2 region of the hippocampal fotmrrt’on in u normal control(A) wrd II schizophrenia brain(B). Noted that CaBp positive cells were rare wld not unifktm in shqe itt schizophrenia brains, compared to he twrmal controls.(Bar=50 0 m) surface of the cortex. On the other hand, in the case of schizophrenia, arrayed

and their processes

schizophrenic

were

oriented

almost

completely

these positive cells were randomly randomly.

cases were running in somewhat arbitrary directions (Fig 3, Fiig 4).

Positive

fibcrs

in the

S. Iritani et al

414

7r

i

Fig 2a Camera&id of the CaBp positive products in the CA2 region of al! cases of the twrmul cotrtroi (Case Norm.1 -3; A-C). (Bar=50 LLm)

Discussion

s ln Schizo~

‘a

In this study, the authors investigated the morphological and fibers in the hippocampus with those of normal controls.

characteristics

and the prefrontal cortex of schizophrenic As shown

in Fig 2b, the positive

of CaBp-containing

neurons

hippocampus were less dense and more disarrayed than control specimens. accord with previous studies of the hippocampal formation of schizophrenics and Shatq1989;

Jeste and Lohr,1989).

distribution of CaBp-containing

neurons

patients and compared to them in the area CA2 of the

These findings are in good (Benes et al., 1991; Chun

Moreover, in this study, we also observed changes in neuronal

neurons.

to be involevcd in intercircuit neuronal

Cortical GABAergic neurons contain CaBp, which is thought connections

as a neuromodulater(Celio,

1990). The calcium-

binding proteins, CaBp, calretinin, and parvalbumin are present in separate subpopulations

of local

C&p containing

415

neurons in schizophrenics

D

F

J

Fig 2b. Cameralucid of the CaBppositiveproducts in the CA2 region of all cues of schizophrenia (Cue Sch. l-6; D-I). Noted that CaBp positive cell were less dense and their shapes were not uniform in schizophrenia cases compared to normal controls (A-C). Positive cells in the schizophrenia were sometimes disarrayed and had a long or short apical dendrite. Thepositive fibers were mndamly oriented compared to lwrmal controls.(Bar=_YOu m)

circuit neurons(CondC et a1.,1994), which together constitute 90% of GABAergic neurons(Gahbott Bacon, 1996). So, changes in CaBp-containing GABAergic neuronal dysfunction

neurons in schizophrenia

occurs in schizophrenic

brains(Benes

supports

et al., 1993).

the hypothesis

and that

416

S. Iritani ei al.

Fig 3. Photomicrograpks of luyer III-IV in tkeprefrontul cortex (Brodmrum ureu Y) of tlormd control(A) cold schizophrenia brains(B, C). Positive cells in the sckizophrerliu bruin sometime.s were di.surruyed or/and disoriented and distributed at rwtdom compured to rlormul control cuses. Normol ~mtrol.s were uniform itt their arrangemalts and orientationof theirfibers. (Bar = 100 :/ m) According to previous containing neurons

studies in the prefrontal

is significant

increased,

containing neuron densities (Tsung-Ung

cortex in schizophrenia,

the density of the CaBp-

but ollretinin (Daviss and L,ewis,lYY5) and parvalbumin-

Woo et al,.lYY7)are not different from normal controls. In this

CaBp containing

.

.

417

neurons in schizophrenics

>

J Fig 4. Cameralucid of lyer III-IV of the prefrontal cortex of r~~rmalcontrol(A), atul schizophrer~ic brains(Case Schl3;B, C, 0). Positive cells in schizophrenic brains were sometimes disarryed and distributed at random compared to normal control cases. Normal corltrol.~were ukform in their arrangements and orientationof their fibers. (Bar=100 11m) study, the density of CaBp-containing schizophrenia,

neurons was reduced in the CA2 region of the hippocampus

but not altered in the prefrontal cortex. Morphological

abnormalities

of

in CaBp positive

neurons and their fibers were observed in the prefrontal cortex. Some of the inconsistencies

between the previous studies and this study are probably the result of case

selection or small sample size. In any case, we believed that dysfunctional the hippocampal the reduction schizophrenia

of neurons.

GAE3Aergic neuron

dysfunction

The morphological

and may have several neuropathological

changes of CaBp-positive

without neuropathological

neurons in the hippocampus

findings such as neurodegeneration

this study might be the cause of the neurodevelopmental schizophrenia,

and these findings support the hypothesis

neurodevelopmental

abnormalities.

changes and/or

might be related to the symptoms

such as deterioration of cognition and/or learning. However, schizophrenia

syndrome of different etiologic backgrounds

schizophrenics

GABAergic neurons exist in

formation and the prefrontal cortex accompanied by the morphological

dysfunction

of

is considered a

features.

and prefrontal

cortex of

or gliosis of brain tissue in

of the central nervous system of

that the pathogenesis

of schizophrenia

is due to

418

S. Iritani et al.

tlons of CaBo m Psvchiatric Disease Calcium binding proteins may function as a buffer and participate in the transportation

of calcium ions

as well as regulate the function of enzymes within central nervous system neurons(Baimbridge Miller,1984;

Jande

et al.,

1981; Kijhr et al.,1991).

distributed in the central nervous protein sub-group,

system.

CaBp,

a calcium binding

The detailed physiological

function

protein,

and

is widely

of a calcium binding

the so-called EF-hand proteins, is unknown and the characteristic differences

among

the subclass of these proteins remains unclear. In previous biological studies of mental disorders,

it was reported that the concentration of calcium ion

in blood was altered in the patients with bipolar affective disorder, and calcium ion antagonists effective for the treatment of depression(Heizmann

and Braun, 1992). Also it was found that calcium

antagonist may be effective for the treatment of negative symptoms Changes in distribution

of CaBp have been reported

Nishiyama et al., 1993), Huntington Christakos,1990).

For example,

disease(Kiyama

may be

of schizophrenia

in Alzheimer’s

(Price,

disease(Ichimiya

lYX7).

et al., 1988;

et al., 199(J), and Parkinson disease(Iacopino

and

the number of CaBp positive neurons are reduced in the cortex of

Alzheimer patients as demonstrated

by immunocytochemical

ct al., 1988). These

techniques(Ichimiya

facts indicate that the pathology of calcium ion and/or calcium binding protein group arc rekited to mental disorders. poisons

CaBp has also been found to be involeved such

as

ischemia(Freund

h4TPT( l-metyl-4-phenyl-1,2,3,

et al., 1990), or convulsion

density of CaBp-containing

Gtetrahydropyridin)(

of neuron to neuronal

Lavoic

like a epileptic seizure(Sloviter

in GABAergic

neurons

of schizophrenia

Parcnt.1991).

shown in this stud)

and supports the hypothesis

is altered in schizophrenia.

hippocampal formation of schizophrenic

and

et al., 1YYl). The rcduccd

neurons in the hippocampal formation in schizophrenia

may be at least partly involved in the pathogenesis gene expression

with the vulnerability

brains may be more vulnerable than normal controls,

in the development

that

The neurons in area CA2 of the

vulnerability

may be involved

of schizophrenia.

Further

investigations

physiological

function of the EF-hand proteins like CaBp may be necdcd to understand

and this of the

ncuropsychiatric

diseases. Pharmacoloev

and Clinical Feature

The brains of patients used in this study had been subjected to long-term neuroleptic which may have altered the dynamism

of calcium ion movement,

calcium ion metabolism might change the expression

i.e., it is possible

of CaBp in neurons.

medication.

that changes in

All patients examined in this

study had been admitted to the psychiatric hospital for periods greater more than 15 years and presented severe negative symptoms

of schizophrenia,

group to a clinical medication. schizophrenic

so the patients in this study might represent

The results of this study may indicate the actual condition

patient group. Investigations

a resistant of only this

of the distribution of CaBp in other clinical types of patients

are needed. In any case, the present observations

suggest that both morphological

changes and alterations in the

CaBp containing neurotransmission

419

neurons in schizophrenics

mechanism exist in schizophrenia

The chemical-neuroandtomicaI

this study will hopefully be useful for discovering the etiology of schizophrenic

It is believed that CaBp immunoreactive interneurons

in the human of

schizophrenic

immunoreactivity

in the hippocampal

method. In the hippocampal

neurons mostly belong to a subpopulation

cerebral cortex.

pdthophysiology

GABAergic

disorder.

In

this

dysfunction study,

has been

the

authors

formation and neocortex of schizophrenic

formation of schizophrenia,

fibers of ared CA2 were altered in their arrangements. bodies had irregularly arrangements

approaches used in

disorder.

of GABAergic

implicated

in the

investigated

CaBp

brains using the ABC

some CaBp-immunopositive

cell bodies and

In the frontal cortex, some immunopositive

cell

of their axis and had disarrayed fibers. These observations

may

indicate GABAergic neuron dysfunction

in schizophrenic

disorder.

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Inquiries and reprint requests should be addressed to:

DrShuji

Iritani

Department of Psychiatry Tokyo Metropolitan Matsuzawa Hospital 2-l - 1 Kamikitazawa Setagayaku Tokyo 156-0057 JAPAN Tel +81-3-3303-7211,

Faxt81-3-3329-7586

E-mail; [email protected]

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