Introduction

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Histamine neurons originate from the tuberomammillary nucleus in the hypothalamus and project in a highly divergent manner to many brain regions, namely, in the limbic system where the highest density of terminals is encountered (Schwartz et al., 1991, 1995; Onodera et al., 1994). Whereas the function of these neurons in physiological processes such as arousal, attention, or hormonal controls is well documented, very little was known about their possible involvement in brain disorders.

Recently, however, several pieces of evidence have started to suggest a possible involvement of brain histaminergic neurons in the pathophysiology of schizophrenia and/or the action of typical and atypical antipsychotic agents. Overdose of various H₁ receptor antagonists of the first generation was repeatedly reported to result in toxic psychoses with hallucinations resembling schizophrenia and the hallucinogenic potential of these drugs has even led to abuse (Sangalli, 1997). In several open studies famotidine, an H₂ receptor antagonist, was found to improve schizophrenic patients, a finding that remains to be confirmed in control studies.

Methamphetamine, a psychotogenic drug that induces an enhanced dopamine release in schizophrenic patients (Laruelle et al., 1996), was shown to enhance histamine release in dialysates of rat striatum. This response was completely blocked by haloperidol, an antagonist at dopamine D₂-like receptors (Ito et al., 1996a). Consistent with a positive influence of endogenous dopamine in the control of histamine neuron activity, we recently showed that typical antipsychotic agents, such as haloperidol, reduce the level of tele-methylhistamine (t-MeHA) in mouse brain (Morisset et al., 1999).

Furthermore, repeated administration of methamphetamine, which results in behavioral sensitization to dopamine agonists, a cardinal feature of schizophrenia, was accompanied by an enhanced histamine release in rat striatum. This effect was blocked by haloperidol, presumably reflecting an increased tonic dopaminergic influence on histaminergic neurons (Ito et al., 1996a). Consistent with this proposal, Prell et al. (1995) showed that the level of tele-methylhistamine, a major histamine metabolite, was significantly elevated in the cerebrospinal fluid of patients with chronic schizophrenia, either under neuroleptic treatment or not, suggesting that hyperactivity of dopaminergic transmission was associated with an enhanced activity of histaminergic neurons.

In contrast to typical neuroleptics, we recently reported that atypical antipsychotics, such as clozapine and olanzapine, enhance histamine neuron activity. This effect was attributable to blockade of 5-HT₂ receptors in vivo and suggested the existence of a balance between a positive and negative tonic influence of dopamine and serotonin, respectively, on histamine neuron activity (Morisset et al., 1999).

In the present work, we have further explored the influence of endogenous dopamine on histamine neuron activity. To this purpose we have 1) assessed a possible direct histamine-releasing activity of methamphetamine in vitro, 2) evaluated the effects of acute and repeated treatment with methamphetamine on tele-methylhistamine levels in several brain areas, and 3) assessed the role of D₂ and D₃ receptors in the response to methamphetamine. In addition, we have assessed the changes in the respective tonic influences of endogenous dopamine and serotonin in mice displaying behavioral sensitization to methamphetamine.

	Materials and Methods

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Animals. Male Swiss mice (18-20 g) (Iffa-Credo, L'Arbresle, France) were housed with free access to food and water in a room maintained at 21-22°C under a 12-h light/dark cycle with lights on from 7 AM. The D₃ receptor mutant mice were obtained as previously described (Accili et al., 1996). Subsequent generations were obtained by crossing heterozygote mice. DNA was prepared from a piece of their tail (3-5 mm), by using the DNAeasy kit (QIAGEN, Courtaboeuf, France) and amplified with the mixture of primers 5'-GCA GTG GTC ATG CCA GTT CAC TAT CAG-3' and 5'-CCT GTT GTG TTG AAA CCA AAG AGG AGA GG-3', amplifying exon 3 of the wild-type D₃ receptor, and 5'-TGG ATG TGG AAT GTG TGC TGC GAG-3' and 5'-GAA ACC AAA GAG AGG GCA GGA C-3', amplifying the PGK cassette of the mutated gene. Agarose gel electrophoresis allowed us to detect homozygote wild-type mice (a single band at 137 bp), homozygote mutated mice (a single band at 200 bp), and heterozygote mice (two bands at 137 and 200 bp). D₃ receptor mutant mice (/) and their wild-type (+/+) littermates were used for the experiments.

Determination of tele-Methylhistamine Levels in Brain. Drugs dissolved in saline solution (0.9% NaCl, w/v) were administered intraperitoneally. After treatment, animals were sacrificed by decapitation. The brain was dissected out and the cerebral cortex, caudate putamen, nucleus accumbens, or hypothalamus was homogenized in 10 volumes (w/v) of ice-cold perchloric acid (0.4 N). The clear supernatant was stored at 20°C immediately after centrifugation (4000g for 20 min). t-MeHA levels were determined using an enzimoimmunoassay derived from a radioimmunoassay described previously (Garbarg et al., 1989, 1992). Briefly, t-MeHA of the sample was derivatized with p-benzoquinone (BZQ) (2.8 mg/ml). The reaction was allowed to proceed at pH 7.9 for 3 h then 2 M glycine was added to eliminate the excess of BZQ. The derivatized extract was mixed with t-MeHA-BZQ-Leu-Tyr-acetylcholinesterase as a tracer and an antiserum raised in rabbits against t-MeHA conjugated with bovine serum albumin via p-benzoquinone in a plate (Nunc Immuno-Plate Maxi-Sorp Surface; Nunc, Roskilde, Denmark) pretreated with swine anti-rabbit IgG (Cayman Chemical, Ann Arbor, MI). After incubation for 16 h at 15°C, plates were washed and the substrate for acetylcholinesterase, Ellmann's reagent, was added. After 5 h, the optical density was measured with a Dynatech Mr 5000 at 405 nm. The limit of the detection was 5 pg of t-MeHA.

Locomotor Activity. Mice were introduced into a IMétronic actimeter (Pessac, France), consisting in individuals boxes (length = 20 cm, width = 10 cm, and height = 10 cm) placed in a quiet room. After a 30-min habituation period, locomotor activity was evaluated by numbering infrared crossed beams during the next 90-min period. Mice received saline or methamphetamine 30 min after placement into the actimeter. When required, ciproxifan was administered 2 h before methamphetamine treatment. When locomotor sensitization elicited by repeated treatment with methamphetamine was evaluated, animals were trained to the actimeter by being placed 1 h daily for three consecutive days before the beginning of methamphetamine treatment.

[³H]Histamine and [³H]Noradrenaline 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, striatum, or hypothalamus was preincubated for 30 min with L-[³H]histidine (0.4 µM) or [³H]noradrenaline (NA) (30 nM) at 37°C. Synaptosomes were then washed and resuspended in fresh 2 mM K⁺-Krebs-Ringer medium. Synaptosomes were then incubated for 3 min with or without methamphetamine at various concentrations in 2 mM Krebs-Ringer medium. Incubations were ended by a rapid centrifugation and [³H]amine levels in the supernatant were determined, either directly ([³H]NA) or after isolation by ion-exchange chromatography ([³H]HA) (Garbarg et al., 1983). As a control, synaptosomes were also depolarized for 3 min with 30 mM K⁺ (final concentration) before measurement of the [³H]HA released in the supernatant (Garbarg et al., 1992).

Analysis of Data. Dose-response curves were fitted with Prism program (GraphPad Software, San Diego, CA). This program was also used to draw curves. Statistical evaluation of the results was performed using one- or two-way ANOVA where appropriate. Comparisons between individual groups were conducted using the Student-Newman-Keuls procedure or the Mann-Whitney test.

Radiochemicals and Drugs. The drugs and their sources were as follows: ciproxifan and nafadotride (Laboratoire Bioprojet, Paris, France), haloperidol (Janssen Pharmaceutica, Beerse, Belgium), ketanserin (Sigma/RBI, Natick, MA) and methamphetamine (Sigma Chemical, St. Louis, MO). L-[2,5-³H]Histidine (50 Ci/mmol) and [³H]noradrenaline (48 Ci/mmol) were purchased from Amersham plc (Little Chalfont, Buckinghamshire, UK). All drug weights are expressed as free base.

	Results

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Effect of Methamphetamine on Spontaneous Efflux of [³H]HA from Synaptosomes. Synaptosomes of mouse cerebral cortex were used to study the effect of methamphetamine on the release of newly synthesized [³H]histamine. The spontaneous efflux of [³H]HA (at 2 mM K⁺) represented 15 ± 2% of the total [³H]HA in cerebral cortex synaptosomes and was not significantly modified after incubation for 3 min with methamphetamine at concentrations up to 30 µM (Fig. 1). In contrast, the spontaneous efflux of [³H]NA was enhanced by methamphetamine in a concentration-dependent manner (EC₅₀ = 0.3 µM), with a maximal increase of ~80% (Fig. 1). Whereas a highly significant [³H]HA release was evoked over spontaneous efflux by 30 mM K⁺, methamphetamine (1-10 µM) did not significantly modify the spontaneous efflux of [³H]HA from synaptosomes of cerebral cortex, striatum, or hypothalamus (Table 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of methamphetamine on [3H]HA or [3H]NA release from synaptosomes of mouse cerebral cortex. The spontaneous efflux represented 205 ± 2 and 9927 ± 97 cpm/incubation for [3H]HA and [3H]NA release, respectively. The data are expressed as a percentage of these values and are means of four determinations from two separate experiments.

Effect of Single Administration of Methamphetamine (MET) on t-MeHA Levels and Locomotor Activity. MET enhanced t-MeHA levels in striatum (Fig. 2; Table 2), cerebral cortex, and hypothalamus (Table 2) in a dose-dependent manner. In the first two regions, the increase was of similar amplitude (about +150%), whereas it was of lower amplitude (about +80%) in the hypothalamus. MET displayed a similar potency at increasing t-MeHA levels in the three brain regions, with ED₅₀ values of ~1 mg/kg (Table 2). In striatum, the increase in t-MeHA levels induced by MET (2 mg/kg) was similar in the caudate putamen and nucleus accumbens (~+20% and ~+80% at 30 min and 3 h after administration, respectively) (Table 3).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Changes in striatal t-MeHA levels and locomotor activity in mice receiving MET in increasing dosages. After a 30-min habituation period in the actimeter, mice received MET at the indicated dosage, and locomotor activity was recorded for 90 min. Another group of mice received MET in increasing dosage and was sacrificed 3 h later for measurement of t-MeHA levels. Values are means ± S.E.M. from six to eight animals. , p < 0.05; , p < 0.001 versus saline controls in Student-Newman-Keuls test.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Temporal pattern of locomotor activity and concomitant changes in t-MeHA levels after a single injection of MET (2 mg/kg i.p.). Mice received MET (2 mg/kg i.p) at various times and were all sacrificed between 11 AM and 3 PM. t-MeHA levels are expressed in percentage of increase compared with levels in corresponding controls (139 ± 18 ng/g). Values are means ± S.E.M. from 6 to 18 animals. , p < 0.05; , p < 0.01; , p < 0.001 versus saline controls in Student-Newman-Keuls test.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of ciproxifan, an H3 receptor antagonist/inverse agonist, on the hyperlocomotor activity of methamphetamine. Ciproxifan (CPX) (3 mg/kg i.p.) was administered 2 h before MET (2 mg/kg i.p.). Each point represents the mean ± S.E.M. of the values from six to eight animals. a, two-way ANOVA revealed a significant difference between the two groups of mice (pretreated or not with ciproxifan) (treatment: F1,252 = 27.25, p < 0.0001; time: F1,252 = 4.42, p < 0.0001; interaction: F1,252 = 0.7, N.S.).

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Effect of haloperidol (HAL, 1 mg/kg i.p.) and nafadotride (NAF, 2 mg/kg i.p.) alone or with MET (2 mg/kg i.p.) on striatal t-MeHA levels. Mice were sacrificed 3 h after intraperitoneal administration of vehicle or drugs. Values are means ± S.E.M. from 12 animals. , p < 0.05; , p < 0.001 versus saline control; §, p < 0.001 versus methamphetamine alone in Student-Newman-Keuls test.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Effect of methamphetamine on t-MeHA levels of control mice (+/+) and D3 receptor-deficient mice (/). Mice were sacrificed 3 h after the injection of vehicle or methamphetamine (MET 2 mg/kg i.p.). t-MeHA levels are expressed in nanograms per gram of tissue. Means ± S.E.M. of values from 12 animals. Two-way ANOVA revealed a significant effect of methamphetamine in control (+/+) and D3 receptor-deficient mice (/) (cerebral cortex: F1,28 = 170.6, p < 0.0001; striatum: F1,28 = 40.56, p < 0.0001; and hypothalamus: F1,28 = 49.66, p < 0.0001). There were no main effect of genotype and no genotype × treatment interaction in the three regions. , p < 0.01; , p < 0.001 versus the corresponding control in Student-Newman-Keuls test.

Effect of Repeated Administration of Methamphetamine on t-MeHA Levels and Locomotor Activity. In mice pretreated with two daily injections of saline or MET (2 mg/kg), the hyperlocomotor effect of MET was progressively enhanced after the fifth and ninth injection compared with the first injection, whereas the responsiveness was not modified after repeated administration of saline (Fig. 7A).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   A, effects of repeated methamphetamine pretreatments on locomotor activity. Mice received methamphetamine (2 mg/kg i.p.) twice daily for 5 days. Locomotor activity was evaluated after the first (, ), fifth (, ), or ninth (, ) injection of methamphetamine (2 mg/kg, filled symbols) or saline solution (open symbols). Means ± S.E.M. of values from 8 to 10 animals. B, comparison of the effect of a challenge dose of methamphetamine (0.5 or 2 mg/kg i.p.) on striatal t-MeHA levels in mice pretreated twice daily for 4 days with saline solution or methamphetamine (2 mg/kg). Means ± S.E.M. of values from 5 to 10 animals. , p < 0.01; , p < 0.001 versus corresponding saline controls; §, p < 0.05 versus saline control of mice pretreated with saline in Student-Newman-Keuls test.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Comparison of the effects of ketanserin (KET, 8 mg/kg i.p.), haloperidol (HAL, 1 mg/kg i.p.), and ciproxifan (CPX, 3 mg/kg i.p.) on striatal t-MeHA levels in mice pretreated twice daily for 5 days with saline (SAL) solution (controls) or methamphetamine (2 mg/kg) (sensitized) as evaluated after an 11-day interruption of treatment. Means ± S.E.M. of values from 12 mice. , p < 0.05; , p < 0.01; , p < 0.001 versus corresponding saline controls; §, p < 0.01 versus saline control of mice pretreated with saline in Student-Newman-Keuls test.

	Discussion

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Our results show that methamphetamine affects histaminergic neuron activity in brain both acutely and in a long-term manner. A single administration of methamphetamine enhances t-MeHA levels markedly in all brain regions analyzed. This response occurs with ED₅₀ values of about 1 mg/kg, in the same range as those required to elicit the hyperlocomotor response in the same animals, whereas at doses above 4 mg/kg the locomotion is reduced in relationship with the appearance of stereotypies (Hirabayashi and Alam, 1981). The enhanced t-MeHA level very likely reflects an enhanced activity of all histaminergic neurons, which is consistent with increased histamine levels in microdialysates of rat striatum (Ito et al., 1996a). There is no evidence for selective projections of histaminergic subpopulations to particular brain regions (Köhler et al., 1985). However, the maximal effect of methamphetamine on t-MeHA levels was higher in the cerebral cortex and striatum than in the hypothalamus. Its influence on histamine levels was also stronger in the cortex and striatum than in the diencephalon (Ito et al., 1996b). These findings may reveal some functional heterogeneity among histaminergic neurons, as suggested from the regulation of histamine release by presynaptic galanin receptors (Arrang et al., 1991).

This response to methamphetamine does not reflect a direct effect of the drug upon histaminergic neurons because it did not release [³H]histamine from synaptosomes. In contrast, its expected effect on [³H]noradrenaline release was observed in the same preparations. Releasing effects of amphetamines on catecholamines and indolamines result mainly from their interaction with the plasma membrane transporters rather than with the vesicular monoamine transporter 2 (Giros et al., 1996). Vesicular monoamine transporter 2 transports histamine and is expressed in histaminergic neurons (Peter et al., 1995), whereas no active uptake of histamine (HA) could be evidenced at the plasma membrane (Schwartz et al., 1991). This difference between histamine and other monoamine neurons accounts for the lack of direct histamine-releasing effect of methamphetamine that we evidence herein.

Our data show that the enhancing effect of methamphetamine on t-MeHA levels results from the stimulation of histaminergic neurons by endogenous dopamine activating selectively D₂ receptors. In agreement, this effect was completely blocked by haloperidol, a D₂/D₃ receptor antagonist, but remained unchanged either after administration of nafadotride used at a dose inducing a selective blockade of the D₃ receptor (Sautel et al., 1995), or in the brain of mice lacking functional D₃ receptors. In agreement, endogenous dopamine released by methamphetamine enhances striatal histamine release by interacting with D₂-like receptors (Ito et al., 1996a). Moreover, we have shown that haloperidol as well as other "typical" neuroleptics (Morisset et al., 1999) decrease basal t-MeHA levels by ~20%, thereby revealing a tonic activation of histaminergic neurons by endogenous dopamine under basal conditions. Using [¹²⁵I]iodosulpride as a ligand, D₂-like receptor binding sites have been evidenced by autoradiography at the level of the tuberomammillary nucleus (Bouthenet et al., 1987), an area in which D₃ receptors could not be detected (J. Diaz, personal communication). Therefore, endogenous dopamine may directly activate histamine neurons by interacting with D₂ receptors located upon their perikarya or dendrites, as also supported by retrograde transport studies showing that some of the dopamine-containing axons emanating from the ventral tegmental area or substantia nigra project to the tuberomammillary nucleus (Ericson et al., 1989). D₂ receptors located on histaminergic nerve endings do not seem to be involved because apomorphine fails to significantly affect histamine release from slices of rat cerebral cortex (Schwartz et al., 1990). Although it cannot be entirely ruled out, some involvement of endogenous serotonin in the enhancing effect of methamphetamine on t-MeHA levels seems unlikely. Serotonin was reported to induce histamine release after local perfusion in the rat hypothalamus (Laitinen et al., 1995) and to increase the firing rate of tuberomammillary neurons in vitro (Eriksson et al., 2001). However, these local stimulations may play a minor part in the overall in vivo regulation of histamine neurons by endogenous serotonin because we recently showed that histamine neuron activity is under tonic inhibition by endogenous serotonin via 5-HT₂ receptors (Morisset et al., 1999).

Although our findings leave little doubt that the changes in t-MeHA levels induced by methamphetamine are due to an increase in dopamine release, microdialysis studies have shown that maximal extracellular dopamine concentrations are generally achieved within 30 min after drug administration then decline to basal levels within 3 h (Kuczenski and Segal, 1999a). In contrast, the changes in t-MeHA levels were maximal at 3 h and still highly significant 9 h after drug administration, demonstrating a dissociation in the time course of the two methamphetamine-induced responses. A dissociation was also evidenced in the temporal profiles of dopamine release and stereotypies induced by a single administration of amphetamine and has been explained by the rapid development of a hypersensitivity of dopamine receptors, allowing their activation by low concentrations of dopamine (Kuczenski and Segal, 1999b). The activation of supersensitive D₂ receptors by endogenous dopamine might also result in the strong and long-lasting changes in t-MeHA levels induced by methamphetamine.

The hyperlocomotor effect observed after drug administration did not parallel either the increase in striatal t-MeHA levels, suggesting that histaminergic neurons are not directly involved in the locomotor response to methamphetamine. Even more, the comparison of the two temporal profiles suggests that histaminergic neurons are involved in a compensatory manner in the modulation of this response, inasmuch as t-MeHA levels were also increased in the nucleus accumbens, known to be associated with the regulation of locomotor function (Svensson et al., 1995). Consistent with this proposal, enhanced release of endogenous histamine attenuates stimulant-induced locomotor activity (Itoh et al., 1984; Clapham and Kilpatrick, 1994; Ito et al., 1997), and ciproxifan, an H₃ receptor inverse agonist known to enhance histamine neuron activity (Morisset et al., 2000a), strongly decrease methamphetamine-induced hyperlocomotion.

Histamine neuron activity exhibits a circadian rhythm and is the highest during arousal, that is, during the dark phase in rodents (Schwartz et al., 1991, 1995). In agreement, t-MeHA levels are maximal in the brain during this dark phase (Morisset et al., 2000b). In the present study, methamphetamine was administered during the light phase, that is, when histamine neuron activity is the lowest. The long-lasting decrease in basal t-MeHA levels observed 15 h after drug administration might reflect a disruption in the normal circadian pattern of histaminergic neuron activity by the psychomotor stimulant drug.

Repeated exposure to amphetamines is well known to produce behavioral sensitization, characterized by an augmented locomotor response to a subsequent challenge injection. In agreement with previous studies (Hirabayashi and Alam, 1981; Kolta et al., 1985), the intensity of the sensitized behavioral response in our mice was higher at extended withdrawal periods than shortly after the cessation of drug treatment. The effect of a challenge injection of methamphetamine on striatal t-MeHA levels remained unchanged in sensitized animals after the treatment and at any withdrawal times, suggesting that histaminergic neurons are not directly involved in the initiation or expression of the locomotor response. However, basal t-MeHA levels were enhanced in various brain regions of sensitized mice, showing that repeated administration of methamphetamine induced a long-lasting enhancement of histaminergic neuron activity in the whole brain, which is consistent with the increase in histamine release observed in the striatum of sensitized rats (Ito et al., 1996a). Like the response to acute administration, this effect of chronic treatment with methamphetamine on t-MeHA levels was blocked by haloperidol, strongly suggesting that it resulted from a sensitized release of dopamine from dopaminergic afferents, leading to a higher degree of activation of D₂ receptors. Although no single neuronal system is likely to be responsible for behavioral sensitization, the increased releasability of dopamine in the nucleus accumbens and striatum of animals sensitized to amphetamine is well documented (Kalivas and Stewart, 1991; Pierce and Kalivas, 1997). Moreover, the role of an enhanced dopamine tone in the enhanced histamine neuron activity observed during sensitization is confirmed by the time course of changes in basal t-MeHA levels: levels were clearly enhanced immediately after the cessation of the drug treatment and after a long withdrawal period (11 days), but not significantly modified after a shorter withdrawal (2 days), a pattern that parallels sensitized release of dopamine (Paulson and Robinson, 1996; Pierce and Kalivas, 1997).

Our results also indicated that the balance between tonic dopamine and serotonin influences on histamine neuron activity was modified in sensitized animals. As previously described (Morisset et al., 1999), endogenous dopamine (via D₂ receptors) and serotonin (via 5-HT₂ receptors) exerted opposite tonic influences in control mice. However, the serotonin influence, of a much larger amplitude than the dopamine influence in controls, was abolished in sensitized animals, as revealed by the disappearance of t-MeHA-enhancing response to ketanserin. Amphetamines also interact with the 5-HT transporter (Hoffman et al., 1991) but the involvement of 5-HT neurons in behavioral sensitization seems unclear (Pierce and Kalivas, 1997).

Behavioral sensitization to psychostimulants may represent an animal model of psychostimulant-induced and idiopathic psychosis (Pierce and Kalivas, 1997). It is interesting that enhanced t-MeHA levels in cerebrospinal fluid of schizophrenic patients were reported in one study (Prell et al., 1995), indicating that histamine neuron hyperactivity might be present in both the human disease and the rodent model. The apparent compensatory nature of this hyperactivity, its reduction by antipsychotics, and the high density of histaminergic terminals in limbic brain areas suggest that brain histamine receptors may represent novel targets for the therapeutics of psychotic disorders, a hypothesis that remains to be explored for H₂ and H₃ receptors.