Short communication

Effects of zotepine on excitatory synaptic responses in the perforant

path–dentate gyrus pathway in chronically prepared rabbits

Takashi Kubota*, Itsuki Jibiki, Sonoko Kurokawa

Department of Neuropsychiatry, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan

Received 20 August 2002; received in revised form 11 September 2002; accepted 17 September 2002

Abstract

The effects of an atypical antipsychotic drug, zotepine, were examined on excitatory synaptic responses elicited in the dentate gyrus by

single electrical stimulation of the perforant path and the induction of long-term potentiation in this pathway in chronically prepared rabbits.

Doses of 1.0, 2.0 and 5.0 mg/kg of zotepine intraperitoneally injected had virtually no effect on the excitatory synaptic responses. However,

these doses of zotepine dose dependently suppressed the induction of long-term potentiation. According to our previous studies, these results

indicate that the effects of zotepine are different from those of the other atypical antipsychotic drugs, clozapine, but are rather similar to those

of a typical antipsychotic drug, haloperidol and the 5-HT-dopamine receptor antagonist, risperidone. Furthermore, the zotepine-induced

blockade of long-term potentiation induction may be associated with drug-induced cognitive dysfunction such as memory disturbance.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Atypical antipsychotic drug; Zotepine; Perforant path–dentate gyrus pathway; Long-term potentiation

1. Introduction

Zotepine is an antipsychotic drug, effective against both

negative (affective flattening, abulia and social withdrawal,

etc.) and positive (hallucination and delusion, etc.) symp-

toms of schizophrenia and with a low propensity to induce

extrapyramidal side-effects (Petit et al., 1996). Further,

zotepine is not only a 5-HT-dopamine receptor antagonist

with a higher affinity for 5-HT2A receptors than for

dopamine D2 receptors but also a multi-acting receptor-

targeted agent with affinity for various subtypes of neuro-

transmitter receptors (Sumiyoshi et al., 1995; Arnt and

Skarsfeldt, 1998).

Both zotepine and clozapine are considered to be atypical

antipsychotics with clinical and pharmacological profiles

similar to each other but different from those of typical

antipsychotics such as haloperidol.

We previously found that clozapine, 20 mg/kg intra-

peritoneally injected, potentiated the excitatory synaptic

responses elicited in the dentate gyrus by single electrical

stimulation of the perforant path in chronically prepared

rabbits. We called this phenomenon ‘clozapine-induced

potentiation’ (Kubota et al., 1996). Further, in a recent

study, we found that a noncompetitive N-methyl-D-aspartate

(NMDA) receptor antagonist, dizocilpine, at 1.0 mg/kg

completely prevented the clozapine-induced potentiation

(Kubota et al., 2000). This finding indicated that the

clozapine-induced potentiation was caused by NMDA

receptor activation. Such potentiation was not induced by

haloperidol injected intraperitoneally, not only at a high

dose, 0.8 mg/kg (Jibiki et al., 1993) but also even at low

doses of 0.1 or 0.4 mg/kg (Kubota et al., 2001). However,

haloperidol suppressed the induction of long-term potentia-

tion in the perforant path–dentate gyrus pathway in chroni-

cally prepared rabbits (Jibiki et al., 1993), whereas

clozapine had no inhibitory effect on long-term potentiation

induction (Kubota et al., 1996). Findings similar to ours

have been reported by several investigators (Arvanov et al.,

1997). Further, it has been reported that other types of

antipsychotics, pimozide, and the phenothiazine neuroleptic,

trifluoperazine, blocked the induction of long-term poten-

tiation as did haloperidol in our study, whereas sulpiride and

domperidone did not (Finn et al., 1980; Mody et al., 1984;

Frey et al., 1990).

It has been recently proposed that inactivation of the

glutamatergic excitatory system, especially the NMDA

receptor-mediated one, may contribute to the negative

0014-2999/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0014 -2999 (02 )02447 -0

* Corresponding author. Tel.: +81-76-286-2211; fax: +81-76-286-3341.

E-mail address: t-kubota@kanazawa-med.ac.jp (T. Kubota).

www.elsevier.com/locate/ejphar

European Journal of Pharmacology 453 (2002) 245–250

symptoms or cognitive dysfunction (attention and memory

disturbances, and executive dysfunction, etc.) seen in schiz-

ophrenia, based on evidence such as represented by so-

called ‘PCP psychosis’, in which schizophrenia-like psy-

chotic symptoms are caused by intake of the noncompetitive

NMDA receptor antagonist, phencyclidine (PCP) in humans

(Olney and Farber, 1995). Further, it is well known that

long-term potentiation is also an NMDA receptor-mediated

event and may be related to mechanisms underlying mem-

ory function (Jibiki et al., 1993). Possibly, the NMDA

receptor-mediated potentiation by clozapine of excitatory

neurotransmission is related to its atypical clinical profile, it

being effective against the negative symptoms or cognitive

dysfunction in schizophrenia, whereas haloperidol-induced

blockade of long-term potentiation induction conversely

implies that the drug may produce or exacerbate such

symptoms.

In a recent study, we found that risperidone, an atypical

antipsychotic with the pharmacological profile of a repre-

sentative 5-HT-dopamine receptor antagonist, showed no

effects on the excitatory synaptic responses elicited in the

dentate gyrus by single electrical stimulations and sup-

pressed the induction of long-term potentiation, as does

haloperidol (Kubota et al., 2001). These results indicate that

individual atypical antipsychotics are different in their

neurophysiological actions from each other.

In the present study, for the purpose of examining the

actions of atypical antipsychotics on the glutamatergic

excitatory system, we investigated whether zotepine shows

clozapine-like effects on the excitatory synaptic responses

elicited in the dentate gyrus by single electrical stimulation

of the perforant path and the induction of long-term poten-

tiation in this pathway.

2. Materials and methods

2.1. Animal model

Chronic experiments were carried out with 20 adult male

rabbits weighing 2.5–3.5 kg. Each surgical procedure was

conducted under intraperitoneal pentobarbital sodium anes-

thesia (20–30 mg/kg). A tungsten microelectrode for

recordings (tip diameter: 1–2 Am, resistance: 1–5 kV)

and a concentric stimulating electrode for laminar analysis

(0.4 mm in diameter) were attached to a holder with the tips

aligned 1 mm apart. The tungsten microelectrode was

connected to a memory oscilloscope (Nihon Kohden:

VC10, bandpath: 0.08–3000 Hz) through a preamplifier.

After unilateral craniotomy, these electrodes were inserted

from the pial surface at the P4 and L6 position on Ridge’s

map to the dentate gyrus, using an oil hydraulic microdrive

(Narishige), with laminar analysis every 50 or 100 Am, as in

previous studies (Jibiki et al., 1993; Kubota et al., 1994,

1996). Next, another concentric stimulating electrode was

inserted from the pial surface at the P4 and L1 position to

the perforant path ipsilateral to the dentate gyrus, while

observing the maximal responses elicited in the dentate

gyrus by single shocks at a constant intensity delivered

through the stimulating electrode. After a 10-day post-

surgical recovery period, chronic experiments were per-

formed as below. Animal care and use procedures were in

accordance with approved protocols of the Animal Research

Committee of Kanazawa Medical University.

Zotepine was obtained from Fujisawa Pharmceutica

(Japan).

2.2. Experiment 1

In 5/20 rabbits, control experiments were performed to

examine the magnitude of the excitatory synaptic responses

in the dentate gyrus. The threshold intensities of single

shocks to the perforant path for inducing population spikes

in the dentate gyrus were initially examined. The intensities

just above the threshold were determined as those of single

shocks to elicit control responses, which consisted of a small

population spike with an amplitude of less than 0.5 mV

preceded by the leading edge population excitatory post-

synaptic potential (EPSPs) of a low positive wave, and the

subsequent slow component (Kubota et al., 1994). Then, the

baseline responses was recorded for 30 min with single

stimuli at a fixed intensity (monopolar square pulse of 0.2–

0.5 ms duration, 400–800 AA, 30 s stimulus intervals).

Next, vehicle solution [dimethylsulfoxide (0.5 ml)] was

administered as a single injection intraperitoneally. Soon

thereafter, single stimuli at a fixed intensity, the same as

used for the control recording, were given to the perforant

path for 60 min to observe the response changes in the

dentate gyrus. Next, a tetanic stimulation to induce long-

term potentiation was delivered to the perforant path. It is

well known that long-term potentiation is easily produced in

the perforant path–dentate gyrus pathway, as shown in

previous studies (Jibiki et al., 1993; Kubota et al., 1994,

1996). The tetanic stimulation was repeated three times at 3-

min intervals. The stimulus parameters were monopolar

square pulses of 0.2–0.4 ms duration, 200–600 AA, 60

Hz and 1 s in total duration. Soon thereafter, single shocks at

the fixed intensity were delivered again for about 30 min to

observe the response in the dentate gyrus.

2.3. Experiment 2

In 15 rabbits, experiments were performed to examine

whether changes in the excitatory synaptic responses and

long-term potentiation were induced after zotepine admin-

istration. The baseline recording was initially performed as

in experiment 1, after which zotepine dissolved in dime-

thylsulfoxide (0.5 ml) was administered as a single injection

intraperitoneally. The doses of zotepine were 1.0, 2.0 and

5.0 mg/kg, using five rabbits for each group. These doses

were considered to be low, moderate and high ones as used

clinically. Soon after the risperidone injection, single shocks

T. Kubota et al. / European Journal of Pharmacology 453 (2002) 245–250246

at the fixed intensity were again given to the perforant path

for 60 min to observe the response changes, as in experi-

ment 1. Next, long-term potentiation to induce tetanic

stimulation was delivered to the perforant path as in experi-

ment 1. Soon thereafter, single shocks at the fixed intensity

were delivered again for about 30 min.

2.4. Data analysis

In each experiment, four sets of responses were averaged

using a DAT 1100 (Nihon Kohden) and recorded with an X–

Y recorder. To analyze the response changes, the amplitude of

the population spike and the slope of the population EPSP

were measured as in previous studies (Jibiki et al., 1993;

Kubota et al., 1994, 1996). Both the population spike

amplitudes and the EPSP slopes in the 60 responses were

averaged for the whole experimental time of 120 min and

analyzed by repeated measure analysis of variance (ANOVA)

to examine whether there were significant differences

between the four groups, i.e. the control group of experiment

1 and the zotepine 1.0, 2.0 and 5.0 mg/kg dose groups of

experiment 2. Then, the respective values for the 15

responses averaged over 30 min in each of the four sessions,

i.e. baseline, the first half and latter half of the 60-min

observation period after vehicle or zotepine injection, and

after tetanus were analyzed by one-way ANOVA followed by

Scheffe’s multiple comparison, in which it was examined

whether there were significant differences between the four

sessions in each group and whether there were significant

differences between the four groups in each session.

3. Results

3.1. Visual evaluation

3.1.1. Experiment 1

In all five rabbits in experiment 1, the baseline responses

were virtually unaltered during the baseline recordings.

After vehicle injection, the responses showed no changes

during the 60-min observation period after the administra-

tion of vehicle with regard to both the population spikes and

the EPSP slopes. The responses were markedly potentiated

soon after the tetanic stimulation, with regard to population

spikes and EPSP slope, showing the induction of long-term

potentiation (Figs. 1A and 2, Control).

3.1.2. Experiment 2

In the zotepine 1.0 mg/kg dose group, the responses were

virtually unaltered during the baseline recordings and during

the 60-min observation period after zotepine administration.

After tetanic stimulation, the responses to single shocks

were potentiated as compared with the previous responses

for both population spike amplitudes and EPSP slopes,

seemingly showing the induction of long-term potentiation

in all five rabbits (Fig. 2, Zotepine 1.0 mg/kg).

In the zotepine 2.0 mg/kg dose group, the responses

were unaltered during the baseline recordings and during

the 60-min observation period after zotepine administration.

After tetanic stimulation, the responses to single shocks

were slightly potentiated as compared with the previous

responses for both population spike amplitudes and EPSP

slopes (Fig. 2, Zotepine 2.0 mg/kg).

In the zotepine 5.0 mg/kg dose group, the responses were

virtually unaltered throughout the experimental period, i.e.

during the baseline recordings, after risperidone injection

and tetanic stimulation (Fig. 2, Zotepine 5.0 mg/kg and

Fig. 1B).

These results indicated that zotepine had virtually no

effect on the perforant path–dentate gyrus responses elicited

by single shocks before tetanic stimulation, but dose

dependently suppressed the induction of long-term poten-

tiation after tetanic stimulation.

3.2. Statistical analysis

Repeated measures ANOVA showed significant differ-

ences among the four groups with regard to both the

population spike amplitude and EPSP slopes in the 60

responses averaged over the whole experimental period of

120 min [main time effect, F(59,944) = 23.164, P < 0.001 in

population spike and F(59,944) = 36.041, P < 0.001 in EPSP,

group� time course, F(177,944) = 5.678, P < 0.001 in pop-

ulation spike and F(177,944) = 4.959, P < 0.001 in EPSP].

One-way ANOVA, which examined whether the 15

responses averaged over the 30 min in each session differed

significantly between the four groups, showed significant

Fig. 1. (A) Typical averaged responses evoked in the dentate gyrus by

single shocks at fixed intensity to the perforant path in each session in a

single rabbit in experiment 1. Arrow: the single shock, Asterisk: population

spike. Dotted and solid lines in ‘BASELINE RECORDING’ express how to

measure the population spike amplitude from the tangent across the onset

and offset of the spike to the peak of the spike and population EPSP slope,

respectively. (B) Typical averaged responses in each session in a rabbit in

experiment 2 (ZOTEPINE 5.0 mg/kg, i.p., injection). The same intensity of

single shocks and symbols as in (A).

T. Kubota et al. / European Journal of Pharmacology 453 (2002) 245–250 247

differences among the four groups with regard to both the

population spike amplitude and EPSP slopes only in the last

session, i.e. after tetanus [main group effect, F(3,16) = 3.970,

P= 0.027 in population spike and F(3,16) = 3.484, P= 0.041

in EPSP in after tetanus]. The subsequent Scheffe’s multiple

comparison for the last session showed significant differ-

ences only between the control and zotepine 5.0 mg/kg dose

groups (P= 0.035 in population spike and P= 0.048 in EPSP,

respectively).

Next, one-way ANOVA, which examined whether the 15

responses averaged over the 30 min differed significantly

between the four sessions in each group, showed significant

differences among the four sessions for both the population

spike amplitudes and EPSP slopes only in the control group

Fig. 2. Serial changes of Mean and SE values of the population spike (PS) amplitudes and EPSP slopes in the 60 averaged responses, i.e. 240 real responses

during 120 min in four sessions, namely, baseline, the first half and latter half of the 60-min observation period after vehicle or zotepine, and after tetanus in

each experimental group consisting of five rabbits.

T. Kubota et al. / European Journal of Pharmacology 453 (2002) 245–250248

[main time course effect, F(3,16) = 8.933, P= 0.001 in

population spike and F(3,16) = 5.097, P= 0.011 in EPSP].

The subsequent Scheffe’s multiple comparison in the control

group showed significant differences only between baseline

and after tetanus with regard to both the population spike

amplitudes and the EPSP slopes (P= 0.006 in population

spike, P= 0.045 in EPSP).

These results indicated that the suppression of long-term

potentiation induction after tetanic stimulation in the zote-

pine 5.0 mg/kg dose group was statistically verified in the

comparison between sessions as well as in the comparison

with the control groups, whereas that in the zotepine 1.0

and 2.0 mg/kg groups was verified in the comparison

between sessions but not in the comparison with the

control group. Results suggested that zoptepine dose-

dependently suppressed the induction of long-term poten-

tiation, and that the suppression was most striking in the

zotepine 5.0 mg/kg dose group, and further that the

potentiation of responses to single shocks after tetanus in

the zotepine 1.0 and 2.0 mg/kg dose groups was modest

(Fig. 2, comparison between Control and Zotepine 1.0, 2.0

and 5.0 mg/kg).

4. Discussion

In the present study, we found that zotepine had no effect

on the excitatory synaptic responses elicited in the dentate

gyrus by single electrical stimulation and suppressed the

induction of long-term potentiation. These results indicate

that the effects of zotepine are different from those of the

other atypical antipsychotic, clozapine, and are rather sim-

ilar to those of the typical antipsychotic drug, haloperidol,

and those of the other atypical antipsychotic (Jibiki et al.,

1993; Kubota et al., 1996, 2001).

It has been already mentioned that clozapine-induced

potentiation may be an NMDA receptor-mediated event.

However, it is well known that antipsychotic drugs, includ-

ing clozapine, have only a low affinity for binding sites of

glutamate receptors, whereas they show a high affinity for

those of monoamine receptors such as the dopamine, 5-HT

receptors and adrenoceptors (Arnt and Skarsfeldt, 1998;

Kapur and Remington, 2001). Therefore, it is unlikely that

clozapine-induced potentiation results from a direct inter-

action with glutamate receptors. Instead, it is likely to be

due to an indirect effect through monoamine receptors. It

has been reported that both clozapine and zotepine increase

the presynaptic release of noradrenaline and activate the

noradrenergic system in the central nervous system as a

result of their high affinity for the noradrenaline transporter

and their property as a2-adrenoreceptor antagonists (Rowley

et al., 1998; Green et al., 1993). In this respect, noradrena-

line may not exert an indirect effect on NMDA receptor

activation in clozapine-induced potentiation. It has been

reported that clozapine increases glutamate release in the

dentate gyrus and may activate the NMDA receptor (Arva-

nov et al., 1997). Like clozapine, zotepine may not activate

a glutamate system.

Clinically, like clozapine, zotepine is known to be

associated with a higher prevalence of seizures than other

neuroleptics (Kapur and Remington, 2001). The proconvul-

sant action of clozapine may be explained by the activation

of the glutamate system including the NMDA receptor,

while that of zotepine may not, suggesting a different

proconvulsive mechanism. In the present study, zotepine

dose dependently suppressed long-term potentiation. This

suppression may be caused by zotepine-induced inactivation

of calmodulin, an action shared with haloperidol (Jibiki et

al., 1993). This suppression has been observed with risper-

idone, too (Kubota et al., 2001). It has been demonstrated

that various types of neuroleptic compounds are potent

antagonists of calmodulin-activated enzymatic events (Jibiki

et al., 1993). Zotepine, too, may bind to calmodulin and act

as a potent inhibitor.

It has been reported that zotepine clinically shows anti-

depressant effects and effectiveness against negative symp-

toms or cognitive deficits of schizophrenia due to its

elevation of cortical noradrenaline levels (Rowley et al.,

1998). But, the present zotepine-induced blockade of long-

term potentiation induction may conversely imply that

zotepine at clinical doses is potent enough to produce or

exacerbate the negative symptoms and cognitive deficits.

In conclusion, zotepine had little effect on the excitatory

synaptic responses in the dentate gyrus induced by single

electrical stimulation of the perforant path but prevented the

induction of long-term potentiation in this pathway in a

dose-dependent manner. These results indicate that the

effects of zotepine are different from those of the other

atypical antipsychotic drug, clozapine, but are rather similar

to those of a typical antipsychotic drug, haloperidol, and the

5-HT-dopamine receptor antagonist, risperidone.

References

Arnt, J., Skarsfeldt, T., 1998. Do novel antipsychotics have similar phar-

macological characteristics? A review of the evidence. Neuropsycho-

pharmacology 18, 10–63.

Arvanov, V.L., Liang, X., Schwartz, J., Grossman, S., Wang, R.Y., 1997.

Clozapine and haloperidol modulate NMDA and non-NMDA receptor-

mediated neurotransmission in rat prefrontal cortical neurons in vitro. J.

Pharmacol. Exp. Ther. 283, 226–234.

Finn, R.C., Browning, M., Lynch, G., 1980. Trifluoperazine inhibits hippo-

campal long-term potentiation and the phosphorylation of a 40.000

dalton. Neurosci. Lett. 19, 103–108.

Frey, U., Schroeder, H., Matthies, H., 1990. Dopaminergic antagonists

prevent long-term maintenance of posttetanic LTP in the CA1 region

of rat hippocampal slices. Brain Res. 522, 69–75.

Green, A.I., Alam, M.Y., Sobieraj, J.T., Pappalardo, K.M., Waternaux, C.,

Salzman, C., Schatberg, A.F., Schildkraut, J.J., 1993. Clozapine re-

sponse and plasma catecholamines and their metabolites. Psychiatry

Res. 46, 139–149.

Jibiki, I., Wakita, S., Kubota, T., Kurokawa, K., Fukushima, T., Yamaguchi,

N., 1993. Haloperidol-induced blockade of induction of long-term po-

tentiation in perforant path–dentate gyrus pathway in chronically pre-

pared rabbits. Pharmacol. Biochem. Behav. 46, 847–852.

T. Kubota et al. / European Journal of Pharmacology 453 (2002) 245–250 249

Kapur, S., Remington, G., 2001. Atypical antipsychotics: new directions

and new challenges in the treatment of schizophrenia. Annu. Rev. Med.

52, 503–517.

Kubota, T., Jibiki, I., Fukushima, T., Kurokawa, K., Yamaguchi, N., 1994.

Carbamazepine-induced blockade of induction of long-term potentia-

tion in the perforant path–dentate gyrus pathway in chronically pre-

pared rabbits. Neurosci. Lett. 170, 171–174.

Kubota, T., Jibiki, I., Kishizawa, S., Kurokawa, K., 1996. Clozapine-induced

potentiation of synaptic responses in the perforant path–dentate gyrus

pathway in chronically prepared rabbits. Neurosci. Lett. 211, 21–24.

Kubota, T., Jibiki, I., Enokido, F., Nakagawa, H., Watanabe, K., 2000.

Effects of MK-801 on clozapine-induced potentiation of excitatory syn-

aptic responses in the perforant path–dentate gyrus pathway in chroni-

cally prepared rabbits. Eur. J. Pharmacol. 395, 37–42.

Kubota, T., Jibiki, I., Kurokawa, S., 2001. Effects of risperidone, an

atypical antipsychotic drugs, on excitatory synaptic responses in the

perforant path–dentate gyrus pathway in chronically prepared rabbits.

Pharmacol. Biochem. Behav. 70, 237–242.

Mody, I., Baimbridge, K.G., Miller, J.J., 1984. Blockade of tetanic- and

calcium-induced long-term potentiation in the hippocampal slice prep-

aration by neuroleptics. Neuropharmacology 23, 625–631.

Olney, J.W., Farber, N.B., 1995. Glutamate receptor dysfunction and schiz-

ophrenia. Arch. Gen. Psychiatry 52, 998–1007.

Petit, M., Raniwalla, J., Tweed, J., Leutenegger, E., Dollfus, S., Kelly, F.,

1996. A comparison of an atypical and typical antipsychotic, zotepine

versus haloperodol in patients with acute exacerbation of schizophrenia:

a parallel-group double-blind trial. Psychopharmacol. Bull. 32, 81–87.

Rowley, H.L., Kilpatrick, I.C., Needham, P.L., Heal, D.J., 1998. Elevation

of extracellular cortical noradrenaline may contribute to the antidepres-

sant activity of zotepine: an in vivo microdialysis study in freely mov-

ing rats. Neuropharmacology 37, 937–944.

Sumiyoshi, T., Suzuki, K., Sakamoto, H., Yamaguchi, N., Mori, H., Shiba,

K., Yokogawa, K., 1995. Atypicality of several antipsychotics on the

basis of in vivo dopamine-D2 and serotonin-5HT2 receptor occupancy.

Neuropsychopharmacology 12, 57–64.

T. Kubota et al. / European Journal of Pharmacology 453 (2002) 245–250250