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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:590 –596, 1999
Vol. 288, No. 2
Printed in U.S.A.
Atypical Neuroleptics Enhance Histamine Turnover in Brain Via
5-Hydroxytryptamine2A Receptor Blockade1
S. MORISSET, U. G. SAHM, E. TRAIFFORT, J. TARDIVEL-LACOMBE, J. M. ARRANG and J.-C. SCHWARTZ
Unité de Neurobiologie et Pharmacologie Moléculaire (U.109) de Institut National de la Santé et de la Recherche Médicale, Paris, France
Accepted for publication August 14, 1998
This paper is available online at http://www.jpet.org
During the last decade a number of novel antipsychotic
drugs were developed with the aim of obtaining agents displaying therapeutic advantages over the first drug generation, often designated “typical” antipsychotic drugs. These
include a lower propensity to elicit extrapyramidal side-effects, an improved therapeutic efficacy on negative and affective symptomatology of schizophrenia as well as on refractory forms of the disease. These drugs, often designated
“atypical” antipsychotics, a rather vague terminology, were
mostly modeled after clozapine, which seems to display such
advantages but is not devoid of toxic side-effects (for reviews
see Meltzer and Nash, 1991; Buchanan, 1995; Kinon and
Lieberman, 1996). In addition to dopamine receptors blocked
by all antipsychotics, clozapine potently blocks with Ki of 1 to
20 nM a variety of aminergic receptors including muscarinic,
a1 adrenergic, H1 histaminergic, and 5-hydroxytryptamine
(5-HT)2, 5-HT6, and 5-HT7 serotoninergic receptors. Although there are many individual differences among drugs
designated “atypical antipsychotics”, all these compounds
share some properties of clozapine, e.g., they display significant affinity toward several aminergic receptors and/or lower
Received for publication April 30, 1998.
1
This work was supported by grants from the Biomedical and Health
Research Program EEC BMH4 CT96 to 0204, the Direction des Recherches
Etudes et Techniques (DRET 92/045), and the Wellcome Trust.
This effect was 1) additive with that of a pure H3-receptor
antagonist, ciproxifan, 2) mimicked by a 5-HT2A receptor antagonist, ketanserin, 3) reversed by a 5-HT2A receptor agonist,
DOI, 4) not shared by antipsychotics with low affinity for the
5-HT2A receptor, i.e., haloperidol, sulpiride, raclopride, or remoxipride that, on the contrary, tended to reduce tele-methylhistamine levels. We conclude that in contrast to “typical”
antipsychotics, “atypical” antipsychotics stimulate histamine
neuron activity via blockade of the 5-HT2A receptor in vivo. This
effect does not appear to account for their reduced extrapyramidal side-effects but may underlie their pro-cognitive properties.
propensity to cause catalepsy in rodents as well as positive
effects on animal models of cognition (reviewed by Arnt and
Skarsfelt, 1998). To date the results of clinical studies have
confirmed the predictions of lower incidence of extrapyramidal side-effects after administration of these novel antipsychotic drugs at doses that demonstrate antipsychotic efficacy.
Controlled studies with some of these drugs, e.g., clozapine,
risperidone, or olanzapine, have shown them to decrease
negative symptomatology, whereas haloperidol is ineffective.
It is not entirely clear to which receptor subtype(s) blockade,
clozapine and these novel antipsychotic agents owe their
peculiar animal behavioral and human clinical properties.
Recently clozapine was shown to block the histamine H3
receptor as evidenced on the release of noradrenaline or
serotonin from brain slices and confirmed in radioligand
binding assays, whereas typical antipsychotics were ineffective in this respect (Kathmann et al., 1994; Alves-Rodrigues
et al., 1995).
In fact, the H3 receptor was initially described as an inhibitory autoreceptor through which histamine controls its own
synthesis in and release from tuberomammillary neurons in
brain (Arrang et al., 1987), so that it could be predicted that
its blockade by a drug like clozapine should enhance the
activity of these neurons in vivo.
The role, if any, of histaminergic neurons in psychiatric
ABBREVIATIONS: HA, histamine; t-MeHA, tele-methylhistamine; (R)a-MeHA, (R)a-methylhistamine; HALO, haloperidol; CLZ, clozapine; OLZ,
olanzapine; KET, ketanserin; CPX, ciproxifan; 5-HT, 5-hydroxytryptamine.
590
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ABSTRACT
Clozapine and olanzapine behave as weak H3-receptor antagonists in vitro with Ki values around 1 and 50 mM, respectively.
Despite these modest apparent affinities, both compounds
given orally to mice, nearly doubled steady-state tele-methylhistamine levels in brain, with ED50 values as low as 1 and 3
mg/kg, respectively, an effect comparable to those of potent
H3-receptor antagonists. This effect corresponded to an enhancement of histamine turnover rate from 45 to 73 ng/g/h as
measured in the case of olanzapine using the pargyline test.
Other antipsychotics displaying, such as clozapine and olanzapine, high 5-hydroxytryptamine (5-HT)2A receptor antagonist
potency, i.e., risperidone, thioridazine, seroquel, and iloperidone, also enhanced markedly tele-methylhistamine levels.
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Atypical Neuroleptics and Histaminergic Neurons
diseases is not well understood (Schwartz et al., 1995) but a
relationship between histamine and schizophrenia is suggested by several pieces of evidence. In agreement, decreased
H1 receptor-mediated response to histamine is consistently
observed among schizophrenic patients (Rauscher et al.,
1980; Nakai et al., 1991). Levels of tele-methylhistamine
(t-MeHA), the major histamine metabolite in brain (Schwartz
et al., 1971, 1991) are significantly enhanced in the cerebrospinal fluid of schizophrenic patients (Prell et al., 1995).
Finally, a polymorphism within the H2 receptor gene was
recently reported to be associated with schizophrenia (Orange et al., 1996).
We have evaluated the changes in histamine neuron activity induced in mice by administration of a variety of antipsychotic drugs by measuring the levels of t-MeHA in several
brain regions.
[ I]Iodoproxyfan Binding Assay. The procedure was that
described by Ligneau et al. (1994). Aliquots of membrane suspension
from mouse cerebral cortex, striatum, or hypothalamus (20 mg of
protein) were incubated for 60 min at 25°C in 50 mM Na2HPO4/
KH2PO4 buffer, pH 6.8 with [125I]iodoproxyfan alone or together with
competing drugs. Specific binding was defined as that inhibited by 1
mM (R)a-MeHA. Incubations were performed in triplicate and
stopped by dilution with ice-cold medium, followed by rapid filtration
through glass microfiber filters (GF/B Whatman, Clifton, NJ) presoaked in 0.3% polyethyleneimine. Radioactivity trapped on the filters was measured with a LKB (Rockville, MD) gamma counter (82%
efficiency).
[3H]HA Release from Synaptosomes. Release experiments
with synaptosomes were performed according to Arrang et al. (1985)
with slight modifications (Garbarg et al., 1992). A crude synaptosomal preparation from mouse cerebral cortex was preincubated for 30
min with [3H]L-histidine (0.4 mM) at 37°C. Synaptosomes were then
washed and resuspended in fresh 2 mM K1-Krebs-Ringer’s medium.
After a 5-min preincubation in the presence of drugs, synaptosomes
were incubated for 2 min with 2 or 30 mM K1. When required,
R(a)-MeHA (1 mM), a specific H3 receptor agonist (Arrang et al.,
1987) was added to the medium. Incubations were ended by a rapid
centrifugation and [3H]HA levels in the supernatant were determined (Garbarg et al., 1983).
t-MeHA Levels in Brain. Male Swiss mice (18 –20 g) (Iffa-Credo,
L’Arbresle, France) were fasted for 12 h before drug administration.
Administrations were performed with drugs dissolved in 1% lactic
acid and diluted as required in 1% methylcellulose or 0.9% NaCl for
p.o. and i.p. administration, respectively. Rectal temperature of the
animals was measured and, when necessary, maintained at 37°C by
using a heating lamp. Animals were sacrificed by decapitation. The
brain was dissected out and the cerebral cortex, striatum, and hypothalamus were homogenized in 10 volumes (w/v) of ice-cold perchloric acid (0.4 M). The perchloric acid extracts were centrifuged
(4000g for 20 min) and the supernatant was stored at 220°C. tMeHA levels were determined by radioimmunoassay after derivatization of samples with benzoquinone as described previously (Garbarg et al., 1989b, 1992).
Assessment of Catalepsy. For assessment of catalepsy in an
all-or-none manner, mice received haloperidol (0.25 mg/kg, i.p.) and,
1 h later, each mouse was placed gently so that both front limbs
rested on top of a horizontal rod placed at a height of 5 cm above the
floor. When required, H3-receptor ligands were given 1 h before
haloperidol. An animal was considered to be in catalepsy if it remained with its hind legs on the floor and its front limbs on the rod
for more than 5 s.
For assessment of catalepsy duration, mice received haloperidol
(0.8 mg/kg, i.p.) and, 1 h later, were placed in the position described
before. When required, H3-receptor ligands were administered 1 h
before haloperidol. Scoring consisted in measuring the time during
which they kept the position. Scoring were made under unblinded
conditions.
Analysis of Data. Maximal effects, ED50, IC50, and pseudo Hill
coefficients (nH) were determined by nonlinear regression using an
iterative computer least-squares method and a one-site cooperative
model (Parker and Waud, 1971). Ki values of drugs at the H3 receptor
were calculated from their IC50 values, assuming a competitive antagonism and using the relationship Ki 5 IC50/(1 1 S/Kd) where S
and Kd represent, respectively, either the concentration and the
apparent dissociation constant of [125I]iodoproxyfan in binding experiments or the concentration and EC50 of histamine in release
experiments (Cheng and Prusoff, 1973). Protein contents were determined according to the method of Lowry et al. (1951), using bovine
serum albumin as the standard. Statistical evaluation of the results
was by Student’s t test.
Radiochemicals and Drugs. The drugs and their sources were as
follows: ciproxifan [cyclopropyl-(4-(3–1H-imidazol-4-yl)propyloxy)phenyl)ketone], nafadotride and (R)a-MeHA (Laboratoire Bioprojet, Paris,
France), risperidone and iloperidone (Hoechst-Roussel Pharmaceuticals, Somerville, NJ), clozapine and thioridazine (Sandoz, Basel, Switzerland), olanzapine (Eli Lilly and Co., Indianapolis, IN), seroquel (Zeneca, Wilmington, DE), haloperidol (Janssen Pharmaceutica, Beerse,
Belgium), raclopride and remoxipride (Astra, Läkemedel AB, Sweden),
ketanserin and (6)-DOI [(6)-2,5-dimethoxy-4-iodoamphetamine hydrochloride] (Research Biochemicals International, Natick, MA),
(2)sulpiride (Synthélabo, Paris, France). [125I]iodoproxyfan (2000 Ci/
mmol) and L-[2,5-3H]histidine (50 Ci/mmol) were purchased from Amersham (Amersham, UK). All drug weights are expressed as free base.
Results
Interaction of Clozapine and Olanzapine with the Histamine H3 Receptor. The specific binding of [125I]iodoproxyfan, a selective H3 receptor radioligand (Ligneau et al., 1994), to
membranes from mouse cerebral cortex, striatum, and hypothalamus was monophasically inhibited by clozapine and olanzapine in increasing concentrations with pseudo Hill coefficients (nH) of 0.9 6 0.1 and 0.9 6 0.2, respectively (Fig. 1 and
data not shown). Analysis of the displacement curve of the
binding obtained in the three brain regions studied yielded IC50
Fig. 1. Inhibition by clozapine and olanzapine of [125I]iodoproxyfan binding to membranes from mouse striatum. Membranes were incubated for
1 h at 25°C with [125I]iodoproxyfan (30 pM) and drugs in increasing
concentrations. Specific binding was defined as that displaced by 1 mM
(R)a-MeHA, corresponded to 71% of total and represented 72 6 3 fmol/mg
protein. Results are expressed as percentage of this value. Each point
represents the mean of results from two to three different experiments
with triplicate determinations.
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Materials and Methods
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was further enhanced by coadministration of clozapine, olanzapine, or ketanserin, a preferential 5-HT2A-receptor antagonist (Fig. 6). A significant increase in t-MeHA level was also
observed 3 h after the administration of ketanserin alone (8
mg/kg, p.o.), as compared with control mice, in the cerebral
cortex and striatum (154 and 181%, respectively, Fig. 6) as
well as hypothalamus (142%, data not shown). The coadministration of ketanserin did not further enhance the effect of
clozapine in the cerebral cortex and striatum (Fig. 7). DOI, a
preferential 5-HT2A/2C-receptor agonist, did not change significantly t-MeHA level when used alone but strongly decreased the t-MeHA accumulation induced by clozapine (262
and 272% in the cerebral cortex and striatum, respectively)
(Fig. 7). Given at a dose of 5 mg/kg DOI reversed the effect of
clozapine by 58% (not shown). Prazosin (5 mg/kg, p.o.) an
a1-receptor antagonist did not modify t-MeHA levels.
Although it induced a slight and nonsignificant decrease in
t-MeHA level (Figs. 3 and 8), haloperidol, a D2-like receptor
antagonist, significantly inhibited by ;50% the t-MeHA accumulation induced by ciproxifan in the cerebral cortex, striatum (Fig. 8), and hypothalamus (not shown). This decreasing effect of haloperidol was not observed on the t-MeHA
accumulation induced by coadministration of ketanserin
(Fig. 8).
Effect of (R)a-MeHA and Ciproxifan on Catalepsy in
Mice. (R)a-MeHA or ciproxifan, tested in a maximally effective dosage (20 and 3 mg/kg, respectively) (Garbarg et al.,
1989a; Ligneau et al., 1998), neither produced any significant
catalepsy when administered alone nor modified haloperidolinduced catalepsy in mice (Table 3).
Discussion
TABLE 1
Potencies of clozapine and olanzapine as inhibitors of
[125I]Iodoproxyfan binding in various mouse brain regions
Ki Values (mM)
Region
Cerebral cortex
Striatum
Hypothalamus
Fig. 2. Effect of clozapine on the inhibition by exogenous HA of the
K1-induced [3H]HA release from synatosomes of mouse cerebral cortex.
Synaptosomes preincubated with L-[3H]histidine were incubated for 5
min in the presence of HA (1 mM) and clozapine in increasing concentrations, and subsequently depolarized for 2 min in the presence of 30 mM
K1 (final concentration). In the absence of added agents, [3H]HA release
induced by 30 mM K1 (expressed as percent of total [3H]HA over spontaneous efflux) represented 19 6 3%. Results are expressed as percentage
of this value. Each point represents the results from three independent
experiments with triplicate determinations.
Clozapine
Olanzapine
2.2 6 0.4
1.2 6 0.2
3.1 6 0.8
22 6 6
36 6 5
72 6 6
Our main finding is that clozapine, as well as a number of
other atypical antipsychotic drugs, activate cerebral histaminergic neurons, as judged from the enhanced level of
t-MeHA they induce in several brain areas.
This was not unexpected in the case of clozapine, a drug
previously shown to be unique among antipsychotics in that
it displays significant antagonist activity at the H3-heterore-
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values of 2 to 5 mM for clozapine and 30 to 100 mM for olanzapine (Fig. 1 and data not shown). Taking into account a Kd value
of 72 6 9 pM for [125I]iodoproxyfan, as determined from
saturation kinetics at equilibrium in the three regions (data not
shown), a calculated Ki value of 1 to 3 mM was found for
clozapine and of 20 to 70 mM for olanzapine (Table 1).
Clozapine was examined for its ability to modulate HA
release from cortical synaptosomes labeled with L-[3H]histidine (Arrang et al., 1985). It failed to mimic the autoinhibitory effect of 1 mM exogenous HA but progressively and
completely reversed it at the presynaptic H3 receptor with an
IC50 value of 10 6 3 mM (Fig. 2). Taking into account an EC50
value of 0.06 mM for exogenous HA (Garbarg et al., 1992), an
apparent Ki value of 0.6 mM was calculated for clozapine
acting as an H3-receptor antagonist in this functional model.
Effects of Typical and Atypical Antipsychotic Drugs
on t-MeHA Levels. The effects of 10 antipsychotic drugs
belonging to various chemical classes and classified as either
typical or atypical agents were analyzed on t-MeHA level, an
index of HA neuronal activity in three mouse brain regions.
In cerebral cortex, striatum, and hypothalamus, acute administration of haloperidol, sulpiride, raclopride, or remoxipride tended to slightly decrease (by ;10 –30%) basal tMeHA levels, but this inhibitory effect was hardly significant
except in striatum (Fig. 3). In contrast, all other agents,
sometimes classified as atypical antipsychotics, strongly and
significantly enhanced t-MeHA level in the cerebral cortex,
striatum, and hypothalamus. In the first two regions, the
increase was of similar amplitude (about 180%) with all
these compounds and in the same range as that observed
after oral administration of the potent and selective HA
H3-receptor antagonist ciproxifan (about 1100%) (Ligneau et
al., 1998), whereas it was of a lower amplitude (about 140%)
in the hypothalamus (Fig. 3; Table 2). Clozapine, as well as
olanzapine, displayed a similar potency at increasing
t-MeHA levels in the three brain regions, with ED50 values of
;1 and ;3 mg/kg, respectively (Fig. 4 and Table 2). In the
striatum, t-MeHA accumulation induced by oral administration of olanzapine was clearly additive with that induced by
pargyline, a monoamine oxidase inhibitor and previously
shown to be an index of neuronal HA turnover (Schwartz et
al., 1991). In mice receiving pargyline, t-MeHA level increased linearly with time at a rate of 45 ng/g/h, which was
enhanced to 73 ng/g/h in mice receiving pargyline and olanzapine (Fig. 5).
Effects of Clozapine, Olanzapine, 5-HT2A Receptor
Ligands, and Haloperidol on t-MeHA Levels in the Absence or Presence of a H3-Receptor Antagonist. Oral
administration of ciproxifan alone in a maximally effective
dosage (3 mg/kg) (Ligneau et al., 1998) induced a doubling of
t-MeHA levels in the cerebral cortex, striatum, and hypothalamus (Figs. 3 and 6). This effect of the H3-receptor antagonist
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Atypical Neuroleptics and Histaminergic Neurons
593
TABLE 2
Effects of clozapine and olanzapine on t-MeHA levels in various mouse
brain regions
ED50 (mg/kg)
Hypothalamus
Striatum
Cerebral cortex
Maximal Effect
(% of Control)
Clozapine
Olanzapine
Clozapine
Olanzapine
1.5 6 0.6
0.9 6 0.2
1.6 6 0.8
3.5 6 1.6
3.0 6 0.6
2.9 6 0.9
148 6 4
182 6 3
182 6 6
157 6 3
210 6 4
191 6 8
Fig. 4. Changes in t-MeHA levels in mice receiving clozapine (E) or
olanzapine (F) in increasing dosages. Mice were sacrificed 3 h after oral
administration of the drug. Values are means 6 S.E.M. from 12 to 20
animals.
ceptor controlling [3H]noradrenaline release in brain slices
as well as in radioligand binding tests (Kathmann et al.,
1994; Alves-Rodrigues et al., 1995). In this study, we have
confirmed this activity, showing that the drug reverses the
action of histamine at the H3-autoreceptor, controlling
[3H]HA release from synaptosomes (Arrang et al., 1985; Garbarg et al., 1992) and inhibits [125I]iodoproxyfan binding to
cerebral membranes (Ligneau et al., 1994). The apparent Ki
value (about 1 mM) of clozapine at H3 receptors regulating
HA release is in the same range as that reported at H3 receptors
regulating noradrenaline (Kathmann et al., 1994) or serotonin
(Alves-Rodrigues et al., 1995) release. These similar Ki values
at H3 receptors of a nonimidazole compound further argue
against the existence of several H3 receptor subtypes previously
suggested by functional studies (Clapham and Kilpatrick, 1992;
Leurs et al., 1996; Schlicker et al., 1996). The clozapine-related
atypical antipsychotic drug olanzapine also inhibited [125I]iodoproxyfan binding at the H3 receptor but with a very modest
potency, its apparent Ki value being of about 50 mM.
Many previous studies, using thioperamide and a variety
of other antagonists, have shown that H3-receptor blockade
in vivo activates histaminergic neuron activity (Schwartz et
al., 1991, 1995) and enhances [3H]histamine synthesis (Arrang et al., 1987), endogenous HA release (Itoh et al., 1991;
Mochizuki et al., 1991), and levels of t-MeHA, a major metabolite in brain (Garbarg et al., 1989a,b; Oishi et al., 1989).
These effects all reflect the tonic inhibition of histaminergic
neurons that endogenous HA exerts via H3-autoreceptors
and no other tonic inhibitory mechanism controlling the activity of these neurons has been reported. In addition, clozapine administration resulted in an enhancement of t-MeHA
levels of about 100%, i.e., in the same range as that elicited
by H3-receptor antagonists such as thioperamide (Garbarg et
al., 1989a) or ciproxifan (Ligneau et al., 1998 and present
data). Also, as in the case of H3-receptor antagonists
(Schwartz et al., 1991), the effect of clozapine on steady-state
t-MeHA levels resulted from an enhanced HA turnover rate,
shown to be nearly doubled according to its evaluation via
measurement of pargyline-induced t-MeHA accumulation
(see Results).
Nevertheless, despite these various observations, several
findings led us to the conclusion that this effect could not be
ascribed to blockade of H3 receptors. First, in vitro, clozapine
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Fig. 3. Effects of typical and atypical antipsychotics on t-MeHA levels in three brain regions. Mice were sacrificed 3 h after the oral administration
of vehicle, haloperidol (1 mg/kg), ciproxifan (3 mg/kg), raclopride, remoxipride, risperidone (5 mg/kg), iloperidone (20 mg/kg), thioridazine, seroquel,
clozapine, olanzapine (30 mg/kg), or sulpiride (100 mg/kg). Results from at least 15 mice are expressed as percent change as compared with control
t-MeHA levels (59 6 2, 112 6 4, and 272 6 6 ng/g in the cerebral cortex, striatum and hypothalamus respectively). *P , .05, **P , .01, ***P , .001
as compared with controls.
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Morisset et al.
Vol. 288
Fig. 5. Time course of the changes in striatal t-MeHA levels in
mice treated with pargyline and/or olanzapine. Mice were sacrificed at various times after the administration of olanzapine (30
mg/kg, p.o.) and/or pargyline (65 mg/kg, i.p.). Values are means 6
S.E.M. from 12 to 18 animals. *P , .01, **P , .001 as compared
with controls; §P , .01 as compared with pargyline alone.
Fig. 7. Effect of ketanserin and
DOI, a preferential 5-HT2A-receptor antagonist and agonist,
respectively on the t-MeHA accumulation induced by clozapine. Clozapine was given
orally at a dose of 30 or 6 mg/kg
when coadministered with ketanserin (KET, 8 mg/kg, p.o.) or
DOI (20 mg/kg, s.c.), respectively. Values are means 6
S.E.M. of data from 8 to 12 animals. *P , .01 as compared
with controls; §P , .05 as compared with clozapine alone.
was 100 to 1000 times less potent at the H3-receptor than
thioperamide, both in binding and release experiments.
However, its in vivo effect surprisingly occurred with an
ED50 value in the same milligram per kilogram range (Fig. 4;
Table 2) as that previously reported for thioperamide in the
same test (Garbarg et al., 1989a). Even more strikingly, an
effect of similar amplitude and occurring with a similarly low
ED50 value (;3 mg/kg) was obtained for olanzapine which
displays a 50-fold lower potency than clozapine at the H3
receptor in vitro and the same increase in t-MeHA levels
could be observed with various other atypical antipsychotics
that display low, if any, affinity at this receptor (Schlicker
and Marr, 1996). Second, the effects of clozapine and olanza-
pine on t-MeHA levels were additive with those of ciproxifan
used in a maximally effective dose. Moreover, despite its
higher affinity at the H3-receptor, clozapine at the highest
dose tested (100 mg/kg) did not further enhance t-MeHA
level, strongly suggesting a low degree of H3-receptor occupation.
If it was not mediated by the H3 receptor, what could be the
mechanisms through which clozapine and other atypical antipsychotics activate histaminergic neurons?
A characteristic property shared by all these antipsychotic
compounds is their relatively low affinity for the D2 receptor
and high affinity for the 5-HT2A/2C receptor, leading to a
higher 5-HT2A/D2 affinity ratio as compared with typical
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Fig. 6. The increase in t-MeHA
level induced by clozapine
(CLZ, 30 mg/kg), olanzapine
(OLZ, 30 mg/kg), or ketanserin
(KET, 8 mg/kg) is additive with
that induced by ciproxifan
(CPX, 3 mg/kg), a H3-receptor
antagonist. Mice were sacrificed 3 h after oral administration of vehicle or drug. Values
are means 6 S.E.M. from 12 to
18 animals. *P , .01, **P ,
.001 as compared with controls;
§P , .05, §§P , .01, as compared with ciproxifan alone.
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Atypical Neuroleptics and Histaminergic Neurons
595
Fig. 8. Effect of haloperidol
(HALO, 1 mg/kg, p.o.), on
t-MeHA accumulation induced
by ciproxifan (3 mg/kg, p.o.) or
by ketanserin (8 mg/kg, p.o.).
Mice were sacrificed 3 h after
the administration of haloperidol alone or in combination
with ciproxifan or ketanserin.
Values are means 6 S.E.M. of
data from 10 to 16 animals.
*P , .01; **P , .001 as compared with controls; §P , .05,
§§P , .01 as compared with
ciproxifan alone.
Drugs
All-or-none scoring (no. of cataleptic mice)
Haloperidol (0.25)
(R)a-MeHA (20)
Haloperidol 1 (R)a-MeHA
Haloperidol (0.25)
Ciproxifan (3)
Haloperidol 1 ciproxifan
Duration of catalepsy (in sec, n 5 20–40)
Haloperidol (0.8)
Haloperidol (0.8) 1 (R)a-MeHA (20)
Haloperidol (0.8) 1 ciproxifan (3)
Catalepsy
6/10
0/10
5/10
14/22
2/18
15/22
121 6 10
143 6 14
128 6 16
Drugs were administered intraperitoneally at the indicated dosage (mg/kg). H3receptor ligands were administered 2 h and haloperidol 1 h before assessment of
catalepsy.
neuroleptics (Meltzer and Nash, 1991). Accordingly, these
compounds elicit a predominant 5-HT2A receptor occupancy
in vivo accompanied by a moderate D2-receptor occupancy, a
balance proposed to underlie their atypical profile (Brunello
et al., 1995; Schotte et al., 1996; Busatto and Kerwin, 1997).
It was therefore of interest to assess the effect of 5-HT2Areceptor ligands on HA neuron activity. Ketanserin, a preferential 5-HT2A receptor antagonist, mimicked the enhancing effect of atypical antipsychotics on t-MeHA levels in
mouse cerebral cortex, striatum, and hypothalamus and, like
that of the latters, its effect was additive with that induced by
ciproxifan (Fig. 6). DOI, a 5-HT2A/2C-receptor agonist, did not
modify t-MeHA level but strongly reversed the effect of clozapine. Our findings therefore show that endogenous serotonin tonically inhibits HA neurons via 5-HT2A receptors, an
effect blocked by clozapine. These 5-HT2A receptors could be
located on HA neurons themselves, on interneurons or
nearby axon terminals impinging on the formers. This inhibitory effect of serotonin was apparently never described before but the 5-HT2A receptor displays inhibitory effects on the
spontaneous activity of locus ceruleus noradrenergic neurons
(Rasmussen and Aghajanian, 1988). In agreement with the
blockade of 5-HT2A receptors by atypical neuroleptics, the
effect of clozapine was not additive with that of ketanserin
and blockade of a1 adrenergic receptors, previously proposed also to contribute to the atypical profile of clozapine (Baldessarini et al. 1992), did not modify t-MeHA
levels. This strongly suggests that the activation of histamin-
ergic neurons by clozapine (and other novel antipsychotics) is entirely attributable to 5-HT2A receptor blockade and that, even at the highest clozapine dosage, H3-receptor blockade does not contribute.
Interestingly, antipsychotics devoid of significant affinity
for the 5-HT2A receptor, such as haloperidol or the benzamide
derivatives, not only failed to enhance t-MeHA levels but, on
the contrary, tended to decrease it. This inhibitory effect was
particularly clear in striatum (Fig. 3) or, in other regions,
when t-MeHA levels had been enhanced by treatment with
ciproxifan (Fig. 8 and data not shown). It is consistent with
the findings that endogenous dopamine released by administration of amphetamine enhances striatal HA release by
interacting with D2-like receptors (Ito et al., 1996). It is
interesting to underline that the tendency of antipsychotic
drugs to inhibit HA neuron activity via blockade of D2-like
receptors is dramatically reversed by ketanserin or with
atypical compounds displaying significant affinity for the
5-HT2A receptor.
Two main therapeutical advantages of atypical over typical
antipsychotics seem to derive from blockade of the 5-HT2A
receptor they induce, in addition to that of D2-like (D2 and
D3) receptors, that may potentially be related to enhanced
HA neuron activity. The first one is a reduced propensity to
elicit motor side-effects, exemplified by a reduced cataleptogenic activity in rodents (Meltzer and Nash, 1991; Arnt and
Skarsfeldt, 1998). Although a role for the H3 receptor present
on striatonigral neurons can be evoked (Garcia et al., 1997),
this property of clozapine and atypical antipsychotic drugs
cannot be ascribed to enhanced HA neuron activity inasmuch
as neither ciproxifan nor a H3-receptor agonist did modify
haloperidol-induced catalepsy (Table 3).
The second advantage of atypical antipsychotics is their
arousing and pro-cognitive effects resulting in a significant efficacy against negative symptomatology (Kinon and Lieberman,
1996). The positive functional role attributed to HA neurons in
processes such as wakefulness, attention, and cognition
(Schwartz et al., 1991, 1995) allows to propose that this property of atypical antipsychotics could be related to their unique
ability to activate HA neurons. In agreement, activation of
these neurons by H3-receptor blockade is accompanied in cats
and/or rodents with increase of vigilance, and fast cortical
rhythms as well as improved attention and learning ability (Lin
et al. 1990; Meguro et al., 1995; Miyazaki et al., 1995). Hence
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TABLE 3
Effects of a histamine H3-receptor agonist or antagonist on haloperidolinduced catalepsy
596
Morisset et al.
our observation that the positive effect of atypical antipsychotic
drugs on HA neuron activity can be further enhanced by a
H3-receptor antagonist, suggests the potential use of this class
of drugs in schizophrenia.
Acknowledgments
We are grateful to A. Galtier for processing this manuscript. We
also thank X. Ligneau for technical advice.
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