Chronical haloperidol and clozapine treatment in rats: Differential RNA display analysis, behavioral studies and serum level determination

Pq.

Nan-Psychophamad

&Rid

Psychiat 1998. Vol. 22, pp. 1129-l 139 Copyright 0 1998 Elsetier Science Inc. Printed

ELSEVIER

in the USA. All rights reserved 0278.5%X6/98 $19.00 + .OO

PI1 SO278-5846(98)00065-7

CHRONICAL HALOPERIDOL AND CLOZAPINE TREATMENT IN RATS: DIFFERENTIAL RNA DISPLAY ANALYSIS, BEHAVIORAL STUDIES AND SERUM LEVEL DETERMINATION VIOLA FISCHER’, ULRICH SCHMITT’, HARALD WEIGMANN’. BORIS v. KELLER’, STEFAN REUSS’. CHRISTOPH HIEMKE’ and NORBERT DAHMEN’ Departments

of Psychiatry’

and Anatomy’, University

(Final form,

August

of Mainz, Mainz, Germany

1998)

Abstract Fischer, Viola, Ulrich Schmitt, Harald Weigmann, Boris v. Keller, Stefan Reuss, Christoph Hiemke and Norbert Dahmen: Chronical Haloperidol and Clozapine Treatment in Rats: Differential RNA Display Analysis, Behavioral Studies and Serum Level Determination. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1998, 2. pp. 1129-l 139. 0 1998 Elsevier Science Inc. 1. 2.

3.

4.

Adult, female rats were treated orally for 23 days with 1.6 mgikg haloperidol or 36 mg/kg clozapine per day, to study chronic effects of the two neuroleptics. At five time points during the neuroleptic treatment, animal behavior was recorded in an open field and locomotive activity was analysed. At the end of the experiment, rats were decapitated, blood samples were collected and serum concentrations of haloperidol and clozapine were determined by a radioreceptor or HPLC assay, respectively. RNA was isolated from each brain, without cerebellum, and subjected to differential RNA display. Mean serum concentrations were 8 ngiml for haloperidol and 21 ngiml for clozapine. Analysis of open field behavior showed that haloperidol and clozapine decreased the total distance moved and the velocity as measures of the overall activity, whereas the number of rearings and the number of entries into the center, reflecting risk assessment behavior, were differentially affected. Three neuroleptic-regulated gene fragment bands were identified in differential RNA display experiments. Two gene fragments of 281 bp and 266 bp were sequenced. We conclude that our study design that used behavioral, pharmacokinetic and molecular analysis increase the likelihood of finding relevant molecular events underlying the pharmacotherapeutic effects of neuroleptics in animal models.

Keywords:

behavioral

studies, chronical treatment, clozapine, differential

RNA display, haloperidol.

Introduction Neuroleptics

are a chemically

such as schizophrenia.

diverse group of compounds

used to treat various psychiatric

disorders

Based on their clinical side effects, they are divided into two groups, i.e. typical

and atypical neuroleptics.

Haloperidol

is the most common typical neuroleptic 1129

(Settle and Ayd, 1983;

1130

V. Fischer et aL

Janssen et al., 1959). Unwanted dyskinesia

extrapyramidal

can appear during the treatment

prototype

of an atypical neuroleptic

Kerwin, 1994). Antipsychotic

side effects such as dystonia, parkinsonism

with haloperidol.

The neuroleptic

that causes no extrapyramidal

medications

clozapine

are believed to exert at least a part of their therapeutic

has low affinity for dopamine D2 receptors in vitro (Bartholini,

receptor

related side effects.

represents

the

side effects (Weiner et al., 1996;

by blocking one or more of the five dopamine receptors that have been distinguished

with low D2 receptor occupancy

and tardive

effects

so far. Clozapine

1976) and clinical response is associated

(Pilowsky et al., 1992), a fact that may account for the absence of D2

After cloning

the D4 dopamine

receptor

it has become

apparent

that

clozapine binds to the D4 receptor with an affinity 10 times higher than to the D2 receptor (van To1 et al., 1991), differentiating

it from the typical antipsychotic

drugs. Haloperidol

binds with high affinity to

the D2 receptor but also has considerable

affinity to the D3 and D4 dopamine receptors. In vivo binding

studies in rat brain show that clozapine

in therapeutically

binding sites, whereas haloperidol and Andersen.

doses occupies

also Dl receptor

binds to D2-like receptors (Leysen et al., 1992; Nielsen

1992).

To date, no comprehensive molecular

exclusively

effective

model

level. The time lag between

targets, the neurotransmitter

receptors,

exists

explaining

the immediate

the clinical interaction

efficacy

of neuroleptics

of neuroleptics

and the relief from psychotic

symptoms

with their primary suggests

that further

processes are relevant. Among the discussed processes modulatory effects on gene expression particular importance

or clozapine

administered

survey of genes influenced

in a pharmacologically

locomotive

treatment,

the animal behavior

activity. At the end of the treatment,

RNA display consequence

was used as a method

or clozapine.

was recorded

identify

relevant dose in

At five time points

in an open field to analyse the

serum concentrations

to systematically

by chronical treatment with

and behaviorally

the rat model. Rats were treated orally for 23 days with haloperidol during the neuroleptic

may be of

(Nestler, 1997).

This study was set out to initiate a systematic either haloperidol

on the

were monitored

changes

and differential

in gene expression

as a

of the chronical treatment.

Methods Studv Design For this study, nine adult, two month old female Sprague Dawley rats, ranging in weight from 200-230 g, were used. Animals were divided into three groups with n=3, i.e. a control, a haloperidol clozapine treated group. Rats were decapitated

after 23 days of treatment.

treated and a

Chronical haloperidol Chronic Administration

and clozapine treatment

in rats

1131

of Drugs

The animals were kept under 12 hr light/l2 hr dark cycle with food and water ad libitum. Drug treated animals received either 1.6 mg/kg haloperidol received

tap water.

concentration

Water was sweetened

or 36 mg/kg clozapine in all groups

of 3% (w:v) to avoid taste variations

in their drinking water. Controls

by the addition

between

of sucrose

to the final

the drugs. The drinking

volume

was

examined every morning and every third day the body weight was controlled. Behavioral Tests The three groups of rats were subjected to behavioral test sessions in a standard open field. The tests were carried out at pretreatment

day three (control level) and at days two, nine, 16, and 23 of the chronic

treatment. The following parameters

were recorded during the trials: total distance moved (cm), velocity

(cm/s), number of rearings (n) and number of entries into the center (n). The open field arena consisted of dark-gray plastic with the bottom painted in ochre. It was divided into 25 squares (Al to E5) by gray lines. The arena measured

100 x 100 x 35 cm3. Diffuse illumination

200 cm above the field. Rats were transported

was provided by two 200 W-lamps,

within their home cages to the test room 1 hr before

starting the tests to minimize the influence of transportation

stress. The test room temperature

was 22°C.

After each trial, the test arena was carefully cleaned. For all tests, the path of each rat was registered automatically computer

by a computerized

combined

image analysis system. The hardware consisted

with a videodigitizer

acquisition and analysis was EthoVision@

(Noldus Information

The scores from the different treatment as the change to the respective

and a CCD video camera.

of an IBM-type

The software

Technology,

AT

used for data

Utrecht, Netherlands).

days were pooled for each group. The results were expressed

untreated

status. Statistically

significant

differences

were revealed by

Student’s t-test. Serum Concentrations

of Haloneridol

The rats were decapitated Serum was prepared determined

after 23 days of treatment in the morning and blood samples were collected.

by centrifugation

by a radioligand

were determined

and Clozanine

at 3200 g for 5 min. Haloperidol

serum concentrations

binding assay as described by Rao (1986). Clozapine

by HPLC with on-line

column

included adsorption of clozapine and its metabolites

switching

(Weigmann

on a cyanopropyl

were

serum concentrations

et al., 1997). The method

(CN) coated clean-up column (10

urn; 20 mm x 4.6 mm I. D.), and separation on Cl8 ODS Hypersil reversed phase material (5 urn; 250 mm x 4.6 mm I. D.). Ultraviolet-detection

was performed

at 254 nm.

V.

1132

Differential

RNA Display

After decapitation,

brains were immediately

each brain was isolated (Chomczynski

isothiocyanate-phenol-chloroform

and Sacci, 1987) with the modification

transcriptase

of an additional

(Promega,

Mannheim,

Germany).

(downstream

Oligo-dT

(Gene Ex Screen

Biomedicals, polyacrylamide

primers

with

two

additional

of the poly(A) tail. The second

with the same 3’primer and a second decamere arbitrary primer (upstream primer

Meckenheim,

of mRNA with MLV-

primers) (Gene Ex Screen primer Kit, Biometra,

Giittingen, Germany) were used to anchor the reaction to the beginning

primer)

method

DNAse clean up of the RNA

RNA display was the reverse transcription

in all possible combinations

step was PCR amplification

extraction

extraction.

The first step of the differential

nucleotides

removed and frozen in liquid nitrogen. Total RNA from

using the guanidinium

followed by phenol/chloroform

reverse

Fischer et al

Kit, Biometra)

Germany).

in presence

Amplification

gel. Gels were dried and exposed

of a-[“P]dATP,

300 Ci/mmol

(ICN

products were analysed on a nondenaturating for 1.5 hr to autoradiographic

6%

film (XAR2., Kodak,

Rochester, New York, U.S.A.). The differentially acetate, glycogen,

regulated bands were cut out of the gel. DNA was eluted, precipitated and ethanol and reamplified.

QIAEX II Gel Extraction

culture on agar supplemented selection.

samples were then purified using the

Kit (Quiagen, Hilden, Germany). The cDNA was cloned into the pCR-Script

SK(+) plasmid (Stratagene,

blue/white

The reamplified

with sodium

Heidelberg,

Germany).

with ampicilline

The positive clones were identified

(50 mg/l), X-Gal (80 mg/l) and IPTG (0.1 mM) by

Positive clones were grown overnight

plasmid DNA, inserts were PCR-amplified

in a Petri dish

in a multi well plate. After preparation

with primers specific to the insert-flanking

of

DNA stretches of

the plasmid. Inserts were then sequenced by a modified Sanger method (dye-dideoxy-termination). Data Analvsis The obtained sequences Biotechnology

were compared with the entries of the databases from the National Center for

Information

(NCBI),

e.g. European

Molecular

Biology

Laboratory

(EMBL),

NIH

Genetic Sequence Databases (GenBank) and DNA DataBank of Japan (DDBJ). The programs BLASTN and BLASTX

(Pearson

and Lipman,

1988) were used for the homology

comparison

between

the

queried and the database sequences.

Results Open Field Behavior Open field behavior of rats treated with haloperidol different parameters.

and clozapine

was analysed with respect to four

Total distance moved (cm) and velocity (cm/s) reflected the overall activity. The

Chronical haloperidol and clozapine treatment

1133

in rats

number of rearings (n) and of entries into the center (n) measured risk assessment. Rats treated with either haloperidol parameter

or clozapine

“distance moved” in comparison

showed a statistically

“velocity”, only the haloperidol

decrease (Fig. 1; t=2.93 p
“entries into the center” (t=4.26 p
“rearing?

by haloperidol

groups were compared

were significantly the difference

in the

t=2.57 ~~0.05; treated group

(t=6.46 p
but not by clozapine.

contrast, clozapine increased the number of rearings but this lacks statistical significance. treatment

reduction

to the control group (Fig. 1; haloperidol,

clozapine t=2.87 p
significant

In

When the two

with each other, the numbers of rearings and entries into the center

lower in the haloperidol

group (t=6.6 p
whereas

in distance moved and velocity did not reach statistical significance.

Fig 1. Shown are the changes compared to untreated conditions of the mean values of four test sessions during the neuroleptic treatment with either haloperidol or clozapine. Statistical significance is marked by an circle (0) for the comparison both at ~~0.05 (Student’s t-test).

to controls and by asterisk (*)

for intertreatment

comparison

for

V. Fischer et al

1134

Serum Concentrations From rats treated decapitation.

chronically

with

haloperidol

The receptor assay used for haloperidol

of 8 ng/ml. Clozapine serum levels determined Differential

blood

was obtained

after

showed mean serum concentrations

by HPLC revealed mean serum levels of 21 ngiml.

regulated bands have been identified by differential

These bands were obtained with the downstream primer (5’GGA downstream

of Bauer et al., 1993, these primers were termed

primer DlO and upstream primer U5, respectively. display gel, subcloned

and sequenced.

by haloperidol

with independent

shows a representative

One of these bands was cut out of the

The band consisted of two regulated gene fragments,

as well as by clozapine. The two sequences

281 bp and 266 bp. Reproducibility

of the results

cDNA preparations

RNA display analysis,

primer (5’-TTT TTT TTT TTA G-3’) and the upstream

ACC AAT C-3’). In the nomenclature

both dowmegulated

experiment

quantification

trunkal

RNA Display

To date, three differentially

differential

or clozapine,

was confirmed

obtained had a length of

by five-fold

replication

of the

and PCR reactions from each of the animals. Figure 2

gel image.

Discussion Behavioral Changes Behavior of rats treated chronically The behavioral differential

with either haloperidol

or clozapine was analysed in an open field.

changes after treatment with both neuroleptics

effects were consistent

atypical neuroleptic.

with the classification

indicated pharmacological

of the two substances

In the light of the main focus, the molecular

receptor

antagonists

“distance

moved”

was in line with behavioral

(Paulus and Geyer, confirms

the results

equivalent decrease in ambulation

as an either typical or

analysis of altered gene expression,

the animal numbers and the group sizes could be kept small. Nevertheless, overall activity under haloperidol

efficacy. The

the observed reduction of the

effects reported

for multiple dopamine

1991; Olivier et al., 1990). The decrease

in the parameter

of van den Buuse and den Jong (1989) who reported

scores in the open field after haloperidol

an

treatment in two different rat

strains. The other two parameters,

“number of rearings” and “entries into the center” are related to explorative

behavior and anxiety. In these parameters,

haloperidol

treated animals showed a significant

decrease

Chrontcal haloperidol

and clozaptne treatment

in rats

compared to controls whereas clozapine treatment resulted in an insignificant rearings.

Such a trend

observation haloperidol. changes

towards

of Bruhwyler However,

in activity.

increased

explorative

et al. (1990) who reported

anxiety

In addition,

and exploration

behavior

increase in the number of

was in line with the open

an anxiolytic

related

1135

parameters

potential

of clozapine

are potentially

field

but not of

confounded

it should be noted that the open field test is not specific

by

for the

evaluation of anxiety. In summary, the behavioral induce pharmacon-specific

examination

showed that the chronical treatment

regime was capable to

behavioral changes relevant to the clinical efficacy

Fig. 2. Representative 6% polyacrylamide differential RNA display gel. Amplification was carried out with the primer combination downstream primer DlO; upstream primer U5. The arrows mark the three differentially regulated gene fragments. K : cDNA of control animal, H : cDNA of haloperidol treated animal, C : cDNA of clozapine treated animal, M; marker.

1136

V. Fischer et aL

Serum Concentrations

of Haloperidol

Mean serum concentrations concentrations

and Clozanine

of haloperidol

in rats amounted to 8 ngiml. This was in the range of serum

of 5-12 ngiml found optimal for the therapy of schizophrenic

of clozapine in rats were 2 1 ng/ml. Clozapine

1992). Mean serum concentrations recommended

patients (van Putten et al.,

for the acute therapy

of schizophrenic

patients

serum concentrations

range between

350 and 450 ng/ml

(Hasegawa et al., 1993; Perry et al., 1991). However, one should be aware that clozapine is metabolised about 10 times faster in rats than in humans (Baldessarini pharmacological

effects

et al., 1993). Taken this and the observed

in the open field into consideration,

achieved were sufficiently

we concluded

that the serum levels

high to act on brain functions most propably related to psychic disorders.

Differential RNA Display Analysis The expression

of genes for dopamine

early transcription Meador-Woodruff

factors

receptors.

glutamate receptors,

have been shown to be modulated

neuropeptides

by neuroleptics

and immediate

(Marin et al., 1996;

et al., 1996: Waters et al., 1996; Mac Gibbon et al., 1994; Robertson

and Fibiger,

1992). This indicates that post receptor events bring about adaptive changes thought to be necessary for therapeutic effects. The clinical relevance of these distinct effects, however, is still a matter of debate. The method of differential

display enables a systematic

by drugs or other manipulations. based on the assumption mRNA molecules

The technique

was first described

that every cell expresses

can be reversely

transcribed

differential

RNA display

analysis

haloperidol

or clozapine

so far revealed

approach to identify genes that are regulated by Liang and Pardee (1992). It is

some 15,000 genes and that individual

and amplified

of brain RNA. obtained

by the polymerase

chain reaction.

from rats chronically

three differentially

regulated

species of Our

treated with either

bands. One of the bands was

further analysed and two gene fragments with a length of 28 1 bp and 266 bp were obtained. Both gene fragments

showed

fragments

were present

clozapine

treated rats. Therefore.

neuroleptics

the same regulatory

but markedly

they are suggested

or clozapine

downregulated

were conducted

some authors have expressed

In addition,

separately

the band chosen

treatment.

The

in the haloperidol

and

to be related to mechanisms concerned

RNA display (Liang et al., 1993). To minimize the possibility

clones. all experiments PCR reactions.

after either haloperidol

in the control animals

and their activity. However,

in differential

profile

common

about false positives

of obtaining false positive

with three different cDNA preparations

for subcloning

appeared

to both

to be regulated

in different in an on-off

fashion. The two regulated gene fragments showed no significant homology to entries in Gene Bank and EMBL data base so that we suppose that these sequences regions have not been cloned in rats so far.

represent genes whose 3’-prime untranslated

Chronical haloperidol

and clozapme treatment

1137

in rats

Conclusion In conclusion,

the study design that combined

may enable the systematic effects of neuroleptics

approach

behavioral,

to identify

pharmacokinetic

molecular

in an animal model. Our experiments

adaptive changes in the pharmacodynamic

events

and molecular

underlying

analysis,

pharmacotherapeutic

provide further evidence

for the role of

action of neuroleptics.

Acknowledgements The authors gratefully thank Professor blood plasma levels of haloperidol

Marie-Luise

Rao and Mrs. Gabriele Stroba for measuring

the

and clozapine, respectively

This study was supported by the Deutsche Forschungsgemeinschaft

(grant Da 37011-I)

References BALDESSARINI, R. J., CENTORRINO, F., FLOOD, J. G., VOLPICELLI, S. A., HUSTON-LYONS. D. and COHEN, B. M. (1993) Tissue Concentrations of Clozapine and its Metabolites in the Rat. Neuropsychopharmacol. 9: 117- 124. BARTHOLINI, G. (1976) Differential Effects of Neuroleptic Drugs on Dopamine Extrapyramidal and Limbic Systems. J. Pharm. Pharmacol. 3: 429-433.

Turnover

in the

BAUER, P., MULLER, H., REICH, J., RIEDL, H., AHRENKIEL, V., WARTHOE, P. and STRAUSS. M. (1993) Identification of Differentially Expressed mRNA Species by an Improved Display Technique (DDRT-PCR). Nut. Acid. Res. a: 4272-4280. BRUHWYLER, J., CHLEIDE, E., LIEGEOIS, Anxiolytic Potential of Sulpiride, Clozapine Biochem. Behav. 36: 57-61.

J. F., DELARGE, J. and MERCIER, M. (1990) and Derivates in the Open-Field Test. Pharmacol.

CHOMCZYNSKI, P. and SACCHI, N. (1987) Single-Step of RNA Isolation Thiocyanate-Phenol-Chloroform Extraction. Ana. Biochem. 162: 156-159.

by Acid Guanidinium

HASEGAWA, M., GUTIERREZ-ESTEINOU, R., WAY, L. and MELTZER, H. Y. (1993) Relationship Between Clinical Efficacy and Clozapine Concentrations in Plasma in Schizophrenia: Effect of Smoking. J. Clin. Psychopharmacol. 12: 383-390. JANSSEN, P. A. J., VAN DE WESTERINGH, C., JAGENAU. A. H. M., DEMOEN, P. A. J., HERMANS, B. K. F., VAN DAELE, G. H. P., SCHELLEKENS, K. H. L., VAN DER EYCKEN, C. A. M. and NIEMEGEERS, C. J. E. (1959) Chemistry and Pharmacology of CNS Depressants Related to 4-(4-Hydroxy-4-Phenylpiperidino)-Butyrophenone. Part 1 - Synthesis Acid Screening Data in Mice. J. Med. Pharm. Chem. L: 281-297. KERWfN, R. W. (1994) The New Atypical Antipsychotics.

British J. Psychiat. 164: 141-148.

LEYSEN, J. E., JANSSEN, P., GOMMEREN, W., WYNANTS, J., PAUWELS. P. J. and JANSSEN, P. A. J. (1992) In Vitro and In Vivo Binding and Effects on Monoamine Turnover in Rat Brain Regions of the Novel Antipsychotics Risperidone and Ocaperidone. Mol. Pharmacol. 4-l: 494-508. LIANG, P. and PARDEE, A. B. (1992) Differential Display of Eukaryotic Messenger of the Polymerase Chain Reaction. Science 257: 967-97 1.

RNA by Means

1138

V. Fischer et al.

LIANG, P., AVERBOUKH, L. and PARDEE, A. B. (1993) Distribution and Cloning of Eukaryotic mRNAs by Means of Differential Dislay: Refinements and Optimization. Nut. Acid. Res. 2-l: 32693275. MAC GIBBON, G. A., LAWLOR, P. A., BRAVO, R. and DRAGUNOW, M. (1994) Clozapine and Haloperidol Produce a Differential Pattern of Immediate Early Gene Expression in Rat CaudatePutamen, Nucleus Accumbens, Lateral Septum and Islands of Calleja. Mol. Brain Res. 23: 21-32. MARIN, C., ENGBER, T. M., BONASTRE, M., CHASE, T. N. and TOLOSA, T. (1996) Effect of Long-Term Haloperidol Treatment on Striatal Neuropeptides: Relation to Stereotyped Behavior, Brain Res. 73: 57-62. MEADOR-WOODRUFF. J. H., KING, R. E., DAMASK. S. P. and BOVENKERK, K. A. (1996) Differential Regulation of Hippocampal AMPA and Kainate Receptor Subunit Expression by Haloperidol and Clozapine. Mol. Psychiat. L: 41-53. NESTLER, E. J. (1997) Images in Neuroscience. Transcription Factors. Am. J. Psychiat. 154: 146.

Molecular

Biology,

IV.

Identifying

DNA

NIELSEN, E. B. and Andersen P. H. (1992) Dopamine Receptor Occupancy in Vivo: Behavioral Correlates Using NNC-112, NNC-687 and NNC-756. New Selective Dopamine Dl Receptor Antagonists. Eur. J. Pharmacol. 219: 35-44. OLIVIER, B., MOS, J. and RASMUSSEN. D. (1990) Behavioural Eltoprazine. Drug Metabol. Drug Interact. 8: 3 l-83. PEARSON, W. R. and LIPMAN D. J. (1988) Improved Proc. Natl. Acad. Sci. USA a: 2444-2448.

Pharmacology

Tools for Biological

of the Serenic,

Sequence

Comparison.

PAULUS. M. P. and GEYER, M. A. (199 1) A Scaling Approach to Find Order Parameters Quantifying the Effects of Dopaminergic Agents on IJnconditioned Motor Activity in Rats. Prog. Neuropsychopharmacol. Biol. Psychiat. 15: 903-919. PERRY, P. J., MILLER, D. D., ARNDT, S. V. and CADORE?‘. R. J. (1991) Norclozapine Plasma Concentrations and Clinical Response of Treatment-Refractory Patients. Am. J. Psychiat. 148: 23 l-235.

Clozapine and Schizophrenic

PILOWSKY, L. S., COSTA, D. C., ELL, P. J., MURRAY. R. M., VERHOEFF, N. P. and KERWIN. R. W. (1992) Clozapine, Single Photon Emission Tomography, and the D2 Dopamine Receptor Blockade Hypothesis of Schizophrenia. Lancet 340: 199-202. RAO, M. L. (1986) Modification of the Radioreceptor Assay Technique for Estimation Neuroleptic Drug Levels Leads to Improved Precision and Sensitivity. Psychopharmacol. 553.

of Serum 90: 54%

ROBERTSON, G. S. and FIBIGER, H. C. (1992) Neuroleptics Increase c-fos Expression Forebrain: Contrasting Effects of Haloperidol and Clozapine. Neurosci. 46: 3 15-328. SETTLE, E. C. Jr. and AYD F. J. Jr. (1983) Haloperidol: Psychiat. 44: 440-448.

A Quarter Century of Experience.

in the J. Clin.

VAN DEN BUUSE, M. and DE JONG, W. (1989) Differential Effects of Dopaminergic Drugs on Open-Field Behavior of Spontaneously Hypersensitive Rats and Normotensive Wistar-Kyoto Rats. J. Pharmacol. Exp. Ther. 248: 1189-I 196. VAN PIJTTEN, T., MARDER, Levels and Clinical Response:

S. R.. MINTZ. J. and POLAND, R. E. (1992) Haloperidol Plasma A Therapeutic Window Relationship. Am. J. Psychiat. 149: 500-505.

Chronical haloperidol and clozapine treatment in rats

1139

VAN TOL, H. H., BRUNZOW, J. R., GUAN, H. C., SUNAHARA, R. K., SEEMAN, P., NIZNIK, H. B. and CIVELLI, 0. (1991) Cloning of the Gene for a Human Dopamine D4 Receptor with High Affinity for the Antipsychotic Clozapine. Nature 350: 610-614. WATERS, S. M., KONOKOY, C. S. and DAVIS, T. P. (1996) Haloperidol and Apomorphine Differentially Affect Neuropeptidase Activity. J. Pharmacol. Exp. Ther. 277: 113-120. WEIGMANN, H., BIERBRAUER, J., HARTTER, S. and HIEMKE, C. (1997) Automated Determination of Clozapine and Major Metabolites in Serum and Urine. Therap. Drug Monit. 19: 480-488. WEINER, I., SHADACH, E., TARRASCH, R., KIDRON, R. and FELDON, J. (1996) The Latent Inhibition Model of Schizophrenia: Further Validation Using the Atypical Neuroleptic, Clozapine. Biol. Psychiat. 40: 834-843.

Inquiries and reprint requests should be addressed to: Prof. Dr. Christoph Hiemke Department of Psychiatry University of Mainz Untere ZahlbacherstraBe 8 55 13 1 Mainz, Germany