Psychopharmacology (2009) 205:177187 DOI 10.

1007/s00213-009-1528-1

ORIGINAL INVESTIGATION

Anxiolytic-like profiles of histamine H3 receptor agonists in animal models of anxiety: a comparative study with antidepressants and benzodiazepine anxiolytic
Fumikazu Yokoyama & Miki Yamauchi & Masayo Oyama & Kunihiro Okuma & Kaname Onozawa & Takako Nagayama & Rie Shinei & Makoto Ishikawa & Yasuo Sato & Nobukazu Kakui

Received: 15 June 2008 / Accepted: 22 March 2009 / Published online: 9 April 2009 # Springer-Verlag 2009

Abstract Rationale Histamine H3 receptor functions as a presynaptic auto- and hetero-receptor on histaminergic and nonhistaminergic neurons in the brain regulating the synaptic release of numerous neurotransmitters. Therefore, the ligands for this receptor have been proposed to be of therapeutic interest for the treatment of various neuropsychiatric disorders. At present, however, the psychopharmacological profiles of H3 ligands, particularly H3 agonists, have not been extensively studied. Objective The present study investigated the anxiolytic-like profiles of H3-selective agonists in a variety of classical (benzodiazepine-sensitive) and atypical (antidepressanteffective) animal models of anxiety. Comparator drugs used were diazepam and both fluvoxamine and desipramine in the former and latter models, respectively. Results H3 agonist R--methylhistamine and immepip were inactive in rat elevated plus maze test and Vogel type conflict test where diazepam (5 mg/kg) produced significant anxiolytic-like effects. Meanwhile, these H3 agonists (10 30 mg/kg) significantly reduced isolation-induced vocalizations in guinea pig pups and isolation-induced aggressive behavior in mouse residentintruder test. Moreover, in rat conditioned fear stress test, R--methylhistamine (30 mg/kg) and immepip (10 mg/kg) significantly decreased freezing time, which were completely reversed by concomitant

treatment with H3 antagonist, thioperamide (10 mg/kg). In contrast to the limited efficacy obtained with desipramine (30 mg/kg), fluvoxamine (2060 mg/kg) exhibited anxiolyticlike effects in all the latter three atypical models. Conclusions These data suggest that the H3 agonists may have anxiolytic-like effects similar to those of selective serotonin reuptake inhibitors but not benzodiazepine anxiolytics and represent a novel strategy for the treatment of some anxiety disorders in which selective serotonin reuptake inhibitors are prescribed. Keywords Histamine H3 receptor . R--Methylhistamine . Immepip . Selective serotonin reuptake inhibitor . Elevated plus maze test . Vogel type conflict test . Isolation-induced vocalization test . Residentintruder test . Conditioned fear stress test . Anxiolytic-like effects

Introduction Since Schwartz (1975) found the existence of histaminergic neurons, increasing evidence supports a role for histamine as a neurotransmitter and neuromodulator in various brain functions, including cognition, emotion, stress, and feeding (Leurs et al. 2005). In four different subtypes of G proteincoupled histamine receptors (H1H4), H3 receptor was discovered by Arrang et al. (1983) using cortical slice cultures, where auto-regulation of histamine release was demonstrated. This receptor subtype, as a presynaptic autoreceptor, suppresses the synthesis and release of histamine in the central nervous system (CNS; Arrang et al. 1985, 1987). It is also shown to behave as a presynaptic heteroreceptor, modulating the release of several important neurotransmitters such as serotonin (5-HT; Schlicker et al. 1988), noradrenaline

F. Yokoyama : M. Yamauchi : M. Oyama : K. Okuma : K. Onozawa : T. Nagayama : R. Shinei : M. Ishikawa : Y. Sato : N. Kakui (*) Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan e-mail: nobukazu_kakui@meiji.co.jp

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(Schlicker et al. 1994), dopamine (Schlicker et al. 1993), and acetylcholine (Clapham and Kilpatrick 1992). In consideration of the multiple roles of these neurotransmitters and the widespread existence of H3 receptor in the CNS, the specific ligands for this receptor could be expected to have therapeutic potential in the treatment of various CNS disorders. With the latest advances in pharmacological investigations, the putative therapeutic application of H3 antagonists has been discussed in a recent review (Leurs et al. 2005). There are strong indications that H3 antagonists can be used to correct sleep disorder, cognitive disorder, or attention deficit hyperactivity disorder (Witkin and Nelson 2004). Oppositely, there is quite limited information available regarding the psychopharmacological profiles of H3 agonists, in particular their therapeutic values for anxiety disorders. A previous study reported that R-methylhistamine at the single dose did not affect time spent in open arms in elevated plus maze test (Perez-Garcia et al. 1999). Their study, however, seems to require further pharmacological elucidation for dose response analysis (efficacy at the higher doses of R--methylhistamine) and evaluation of another H3 agonist. Anxiety disorders are categorized by their diagnostic criteria into obsessive-compulsive disorder, panic disorder, social phobia, generalized anxiety disorder, and posttraumatic stress disorder. It is generally accepted that the clinical effectiveness of benzodiazepines is limited to generalized anxiety disorder, social phobia, and panic disorder (Borsini et al. 2002). Meanwhile, antidepressants such as selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs), and serotonin noradrenaline reuptake inhibitors (SNRIs) have been shown to be effective in patients with a wider spectrum of anxiety disorders (Kent et al. 1998; McLeod et al. 1990). Thus, as is well known, the antidepressant drugs are clinically beneficial for not only depressive but also anxiety disorders. Here, the critical issue to be considered is that the most commonly used classical animal models of anxiety, such as the elevated plus maze test and the Vogel type conflict test, which are sensitive to benzodiazepines, but not antidepressants (Griebel et al. 1997; Petersen and Lassen 1981). This discrepancy clearly serves to indicate the necessity for preclinical pharmacological studies, which reflect the efficacy of clinically more useful drugs. From these viewpoints, the following atypical anxiety models are a matter worthy of further preclinical examinations. The efficacy of various types of antidepressants has been demonstrated in the isolation-induced vocalization in guinea pig pups and the residentintruder test in mice (Borsini et al. 2002; Millan et al. 2001). The anxiolytic-like action of SSRIs but not TCAs has also been found in the rat conditioned fear stress test (Hashimoto et al. 1996).

Accordingly, these atypical models may have great potential to predict clinical outcome of antidepressants or some related compounds in anxiety disorders. The purpose of the present study was to characterize the anxiolytic-like profiles of H3 agonists with a variety of animal models, including classical and atypical ones. Thus, we first assessed the effects of R--methylhistamine and immepip in comparison to the benzodiazepine anxiolytic, diazepam, in the classical anxiety models (elevated plus maze test and Vogel type conflict test). And, second, we assessed the effects of the two H3 agonists in three atypical animal models (guinea pig isolation-induced vocalization test, mouse residentintruder test, and rat conditioned fear stress test) in comparison to the other reference drugs, fluvoxamine (serotonergic antidepressant) and desipramine (noradrenergic antidepressant). With our current observations, the similarity or difference of the anxiolytic-like profiles among H3 agonists and the existing drugs and the pathophysiological and therapeutic insights of H3 receptor agonism will be discussed.

Materials and methods Animals Male Wistar rats (for Vogel type conflict test; 120200 g, Clea Japan, Tokyo, Japan: for conditioned fear stress test; 200250 g, Japan SLC, Hamamatsu, Japan) and male SpragueDawley rats (for elevated plus maze test, 150 180 g, Charles River Japan, Atsugi, Japan) housed in groups of five to six upon arrival were used. Male ICR mice (4048 g, Japan SLC) were housed ten to 12 per cage and used for residentintruder test and locomotion measurements. Pregnant guinea pigs were obtained from Japan SLC and housed in individual cages until parturition and thereafter with their pups throughout the study. All these animals were maintained under a 12-h light/dark cycle (light on 0700 hours, light off 1900 hours) in a temperatureand humidity-controlled room. Chow and water were available ad libitum except during experiment. Animal care was performed according to the protocols reviewed by the Ethical Committee for Animal Experiment in Meiji Seika Pharmaceutical Research Center. Drugs R--methylhistamine dihydrochloride, thioperamide maleate, desipramine hydrochloride, immepip dihydrobromide (TOCRIS, Bristol, UK), and fluvoxamine maleate (Meiji Seika, Tokyo, Japan) were dissolved in sterile saline and injected intraperitoneally (i.p.) in a volume of 10 ml/kg (mice), 1 ml/kg (rats), or 2 ml/kg (guinea pig pups).

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Diazepam (Wako Pure Chemical, Osaka, Japan) was suspended in 0.5% (v/v) carboxymethylcellulose (SigmaAldrich) solution and injected i.p. to animals. In oral administration tests, fluvoxamine and desipramine were dissolved in distilled water. All of these preparations were made immediately before their injection on the day of experiment. The dosing regimen of H3 agonists (Cannon and Hough 2005; Malmberg-Aiello et al. 1994) and other drugs (Li et al. 2001; Molewijk et al. 1996; Sanchez and Hyttel 1994; Wesolowska et al. 2006) used in this study was based on previous reports and our pilot studies (data not shown). Apparatus and procedure Elevated plus maze test The test was performed according to the method previously reported (Pellow et al. 1985). Fifteen minutes after the i.p. administration of R--methylhistamine (10 and 30 mg/kg) and immepip (10 and 30 mg/kg) or 30 min after the administration of diazepam (5 mg/kg), each rat was placed on the center of platform (1010 cm) facing one of the closed arms (5010 cm) with walls (40 cm height) and allowed to explore within the maze for 5 min. The behavior of rats was monitored by CCD camera located above the maze and analyzed by a computer system (Target System, Neuroscience, Tokyo, Japan). The assessment of anxiolyticlike activity was made with the measurement of the time spent in open arms. Vogel type conflict test A modified conflict test originally reported by Vogel et al. (1971) was employed to measure the anti-conflict activity. An operant chamber (312531 cm, Muromachi Kikai, Tokyo, Japan) equipped with a spout for water supply was used. In the training session, the rats were deprived of water for 24 to 31 h and then individually placed in the chamber to be trained to lick the spout for water with no electric shock. On the following day, they were again deprived of water for 24 to 31 h, and the number of spout licking response of each rat was counted for 10 min as a prevalue using an automated licking counter. The rats were randomly and evenly assigned to each of seven experimental groups to avoid any significant difference among groups. The next day, they were again deprived of water for 24 to 31 h and underwent test session. During the measurement, an electric shock of 2 mA for 0.2 s from grids connected to a shock generator (Model SGS-002, Muromachi Kikai) was given to the animals for every three licking responses. At 15 min after the administration of R--methylhistamine (10 and 30 mg/kg) and immepip (10 and 30 mg/kg) or 30 min after

diazepam (5 mg/kg), the number of licking responses in the animals was counted for 10 min. Isolation-induced vocalization test The test was conducted according to the previous report (Molewijk et al. 1996). The test plastic cage (2316.5 12 cm) equipped with illumination and microphone on the inside was placed in sound-proof boxes, and this box was closed during the test. From 7 days of age, guinea pig pups entered a pretest in which their vocalizations were recorded by mini disc recorder for 5 min. Only animals that exhibited more than 600 counts of vocalization were used for drug evaluation. On the next day, R--methylhistamine (3, 10, and 30 mg/kg) and immepip (3, 10, and 30 mg/kg) were injected i.p., and the pups were immediately returned to the home cage and remained there with their mother and litter mates for 15 min before testing. Fluvoxamine (30 mg/kg) and desipramine (30 mg/kg) were administered i.p. 30 min prior to the test. Then, the pups were moved into the test plastic cage, and their vocalizations were recorded for 5 min. The counts of vocalization were measured by observers blinded to the experimental groups of the animals. Residentintruder test The test was performed as described by Sanchez and Hyttel (1994). After an acclimation period, the resident mice were kept isolated for 6 weeks in the metal cage (3222 18 cm). All of these mice underwent a pretest for the detection of their levels of aggressiveness. In this pretest, an intruder mouse housed in groups was placed in a resident home cage, and duration of attacks by the resident on the intruder mouse was measured for 10 min by observer blind to the treatment conditions. The attack was defined as biting or as an attempt to bite the intruder mouse. Only mice with total attack time of longer than 10 s were included in drug administration test and randomly assigned to two or three groups based on their pretest values. In the test session, isolated mice were administered i.p. with R-methylhistamine (10 and 30 mg/kg), immepip (10 and 30 mg/kg), or vehicle 15 min prior to the test. Fluvoxamine (20 and 40 mg/kg) and desipramine (10 and 30 mg/kg) were given i.p 30 min before testing. Then, each of the intruder mice was confronted with each of the resident mice in the resident home cage, and the total attack time was measured for 10 min. Conditioned fear stress test In this procedure, rats were conditioned to anticipate a foot shock. On the first day, they were individually subjected to inescapable electric foot shocks for a total of 5 min (1 mA

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of scrambled shock, shock duration of 30 s5, and an intershock interval of 30 s) in an operant chamber (3125 31 cm, Muromachi Kikai) with a stainless grid floor. A scrambled current shock was delivered via a shock generator (Model SGS-002). The test session was performed about 24 h after the exposure to electric foot shock. The rats were again placed in the same box and observed for 5 min in the shock chamber with no current applied to the floor of the chamber. Freezing was defined as the absence of all observable movement of the skeleton and the vibrissae, except for those related to respiration, and measured by the observer who was blind to the experimental groups of the animals. In acute treatment tests, R-methylhistamine (10 and 30 mg/kg) and immepip (3 and 10 mg/kg) were injected i.p. 15 min before the observation period. Fluvoxamine (30 and 60 mg/kg) and desipramine (30 and 60 mg/kg) were administered orally 60 min before testing. As our preliminary studies strongly suggested that SSRIs need to be repetitively injected for the expression of significant anxiolytic actions, oral but not i.p. route was adopted to minimize stress that the animals may receive at the time of injections. In acute co-administration tests, H3 agonists and thioperamide (10 mg/kg) were given i.p. 15 min before testing. In repeated treatment tests, fluvoxamine (30 and 60 mg/kg) and desipramine (30 and 60 mg/kg) were orally administered once a day for 5 days. On day 1, these drugs were given 60 min after the electric foot shock, and later on days 24, the administration was conducted between 1300 and 1700 hours. On the day of the experiment (day 5), the drugs were given 60 min before testing. Locomotion measurements Effects of H3 receptor ligands, fluvoxamine, and desipramine on spontaneous locomotion were evaluated at the same time intervals as in the mouse residentintruder test. Immediately after the injection of R--methylhistamine (15, 30, and 60 mg/kg), immepip (10, 30, and 100 mg/kg), thioperamide (10 and 20 mg/kg), fluvoxamine (20 and 40 mg/kg), and desipramine (10 and 30 mg/kg), mice were individually placed in an open top plastic activity cage (172412 cm). Locomotion measurement system (DAS System Multidigital Counter, Neuroscience) consisted of an infrared sensor positioned just above the cage and the application software (Multidigital 32 Port Count System). After 15 min (for H3 ligands) or 30 min (for reference drugs), the locomotor activity was measured for 10 min and analyzed by the software. Statistics All results were expressed as meanSEM. Statistical analysis was performed with the SAS statistical package

(SAS Institute Japan, Tokyo, Japan). Statistical significance of the differences among multiple groups was tested by one-way analysis of variance followed by Dunnetts multiple comparison test. A two-tailed Students t test was used to evaluate the difference between two experimental groups. Differences were considered significant at p <0.05.

Results We first conducted efficacy studies in classical anxiety models. In the elevated plus maze test, the reference drug diazepam (5 mg/kg) almost doubled a time spent in the open arms (88.48.5 s vs. 42.917.7 s in vehicle group, p <0.05; Fig. 1). On the other hand, R--methylhistamine and immepip exhibited no significant anxiolytic-like effects at either of the tested doses (10 and 30 mg/kg; Fig. 1). In the Vogel type conflict test, the number of licking responses during the exposure to electric shock was prominently increased by diazepam at 5 mg/kg (85.7 23.0 vs. 23.72.8 in vehicle group, p <0.05; Fig. 2). In sharp contrast, neither R--methylhistamine nor immepip (1030 mg/kg) produced any statistically significant increase in the number of licks. Unpunished licking count was also essentially unchanged by R--methylhistamine and immepip at 10 and 30 mg/kg (data not shown). Thus, two H3 agonists used in this study failed to show anxiolytic-like effects in benzodiazepine-sensitive models. We next performed pharmacological evaluation of H3 agonists in atypical anxiety models. Acute administration of R--methylhistamine dose dependently reduced the number of vocalizations induced by separation of guinea pig pups from their mother and mates (531.673.2 in 3 mg/kg, 252.3105.3 in 10 mg/kg, p <0.01, 152.9 104.4 in 30 mg/kg, p <0.01 vs. 693.187.0 in vehicle group;

Fig. 1 Effects of R--methylhistamine (RAMH), immepip, and diazepam on the time spent in the open arms in the rat elevated plus maze test. H3 agonists (10 and 30 mg/kg) and diazepam (5 mg/kg) were i.p. administered. Fifteen minutes (for H3 agonists) or 30 min (for diazepam) later, the exploring behavior was measured for 5 min. Data are represented as mean SEM (n =510). *p <0.05, significantly different from vehicle group

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Fig. 2 Effects of R--methylhistamine (RAMH), immepip, and diazepam on the licking responses in the rat Vogel conflict test. H3 agonists (10 and 30 mg/kg) and diazepam (5 mg/kg) were i.p. administered. Fifteen minutes (for H3 agonists) or 30 min (for diazepam) later, the number of licks was measured for 10 min. Data are represented as mean SEM (n =7). *p <0.05, significantly different from vehicle group

The effects of H3 receptor ligands, fluvoxamine, and desipramine on locomotor activities in mice were evaluated. Acute treatment with R--methylhistamine (counts/10 min, 226.922.9 in 15 mg/kg, 208.116.8 in 30 mg/kg, 236.4 25.5 in 60 mg/kg, vs. 250.026.8 in vehicle group; Fig. 7a) or immepip (counts/10 min, 199.021.3 in 10 mg/kg, 226.6 24.9 in 30 mg/kg, 223.129.1 in 100 mg/kg, vs. 202.424.9 in vehicle group; Fig. 7b) did not significantly alter the locomotion levels. Acute treatment with thioperamide was also ineffective in modifying the locomotor activities (counts/10 min, 218.528.8 in 10 mg/kg, 252.239.4 in 20 mg/kg, vs. 243.817.9 in vehicle group, n =6). Similarly, fluvoxamine and a low dose of desipramine did not change the locomotion levels (counts/10 min, 236.047.3 in fluvoxamine 20 mg/kg, 188.937.1 in fluvoxamine 40 mg/kg, 203.140.2 in desipramine 10 mg/kg, vs. 243.3 26.3 in vehicle group), whereas a significant reduction in

Fig. 3a). A similar tendency was observed by the administration of immepip, but statistically significant changes were detected only in the high dose group (345.884.5 in 30 mg/kg, p <0.01 vs. 673.847.3 in vehicle group; Fig. 3b). Fluvoxamine at 30 mg/kg and desipramine at 30 mg/kg also significantly decreased the number of vocalizations (278.7122.7 in fluvoxamine group, p <0.01, 264.239.8 in desipramine group, p <0.01 vs. 821.262.3 in vehicle group; Fig. 3c). In the residentintruder test in mice, both R--methylhistamine (30 mg/kg) and immepip (30 mg/kg) significantly attenuated the isolation-induced aggressive behavior by 44.1% (p <0.05) and 56.1% (p <0.01), respectively (Fig. 4a). Fluvoxamine (20 and 40 mg/kg) and desipramine (30 mg/kg) also significantly suppressed the duration of attack behavior (Fig. 4b and c; p <0.01 vs. vehicle, respectively). Figure 5a shows the effects of R--methylhistamine and immepip on the duration of freezing behavior in the rat conditioned fear stress test. Single treatment with R-methylhistamine at 30 mg/kg (92.317.7 s, p <0.01 vs. 194.516.2 s) and immepip at 10 mg/kg (111.822.0 s, p < 0.05 vs. 185.317.9 s) significantly reduced the freezing behavior observed in vehicle group. Thioperamide (10 mg/kg) alone had no significant effect on the freezing behavior (Fig. 5b), whereas the decreases in freezing time induced by R--methylhistamine (30 mg/kg) and immepip (10 mg/kg) were completely restored by combined treatment with thioperamide at 10 mg/kg (Fig. 5b; p <0.05 vs. each H3 agonist treatment group). Single treatment with fluvoxamine and desipramine was both ineffective at any of doses tested (30 and 60 mg/kg, p.o.; Fig. 6a). Repeated treatment with fluvoxamine (60 mg/kg, p.o.), however, significantly decreased the freezing behavior (161.917.1 s, p <0.05 vs. 234.110.2 s in vehicle group; Fig. 6b). Repeated treatment with desipramine (30 and 60 mg/kg, p.o.) was ineffective as in the case of single treatment (Fig. 6b).

Fig. 3 Effects of R--methylhistamine (RAMH; a), immepip (b), and reference drugs (c) on isolation-induced vocalization in guinea pig pups. R--methylhistamine (3, 10, and 30 mg/kg) and immepip (3, 10, and 30 mg/kg) were i.p. administered 15 min before measurement of vocalization for 5 min. Fluvoxamine (30 mg/kg) and desipramine (30 mg/kg) were i.p. injected 30 min before the test. Data are represented as mean SEM (n =812). **p <0.01, significantly different from vehicle group

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effective range (Figs. 7a,), suggesting that these H3 agonists do not induce hypolocomotion which may, in general, result from suppression of histaminergic neurotransmission (Haas and Panula 2003). These locomotion data enable us to exclude the possibility that false-positive efficacy is detected in the mouse residentintruder model due to non-specific inhibition of aggressive behavior (Fig. 4a). At the same time, these may exclude the idea of an artifact that any changes in general behaviors or activities contribute to apparent anti-freezing effects of H3 agonists in the conditioned fear stress test (Fig. 5a). In consideration of the anxiolytic-like effects of H3 agonists commonly observed in atypical models across different species, it seems unlikely that an altered locomotor activity in animals affects their efficacies despite the limitation of locomotion data obtained from the single species (mice) in the present study. There have been several studies indicating the functional relationship between anxiety and histaminergic neurotrans-

Fig. 4 Effects of H3 agonists (a), fluvoxamine (b), and desipramine (c) on isolation-induced aggressive behavior in mice. R--methylhistamine (RAMH, 10 and 30 mg/kg) and immepip (10 and 30 mg/kg) were i.p. administered 15 min before measurement of duration of attacks for 10 min. Fluvoxamine (20 and 40 mg/kg) and desipramine (10 and 30 mg/kg) were i.p. injected 30 min before the test. Data are represented as mean SEM (n =815). *p <0.05, **p <0.01, significantly different from vehicle group

locomotion levels (96.136.9, p <0.05) was observed at high dose of desipramine (30 mg/kg; Fig. 7c).

Discussion Here, we performed a wide variety of in vivo pharmacological and behavioral evaluation and clearly indicated the efficacy profiles of H3 agonists in animal anxiety models. This study provided evidence, for the first time, that H3 agonists have certain anxiolytic-like effects exclusively in atypical models, which are qualitatively similar to those of SSRI, but separate from those of benzodiazepine anxiolytics that are active in classical models. R--methylhistamine and immepip did not affect spontaneous locomotor activity even at doses much higher than those in the pharmacologically

Fig. 5 Suppression by R--methylhistamine (RAMH) and immepip of freezing behavior (a) and its reversal by combined treatment with thioperamide (THIO; b) in rat conditioned fear stress test. R-methylhistamine (10 and 30 mg/kg) and immepip (3 and 10 mg/kg) were i.p. administered, and after 15 min, freezing behavior was measured for 5 min. In co-administration test, H3 agonists and thioperamide (10 mg/kg) were i.p. administered 15 min before the test. Data are represented as mean SEM (n =711). *p <0.05, **p <0.01, significantly different from vehicle group. #p <0.05, significantly different from vehicle + H3 agonist group

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methylhistamine (10 mg/kg) did not change the time spent on the open arm in the same test (Perez-Garcia et al. 1999). These findings strongly suggest the substantial contribution of postsynaptic (H1 receptor inhibition) but not presynaptic (H3 receptor stimulation) modulation of histamine-dependent anxiogenic responses in the benzodiazepine-sensitive models. One potential clue for interpretation is the existence of complex postsynaptic histamine receptor systems consisting of anxiogenic-like H1 and anxiolytic-like H2 receptors, as demonstrated by the classical animal model (mouse light/dark test; Malmberg-Aiello et al. 2002). Changes in the release of endogenous histamine by H3 agonists may therefore affect postsynaptic signal transduction and subsequent expression of anxiety responses via these receptors in a highly complex manner. Future research should explore this issue furthermore. In clinical practice, antidepressants such as SSRIs and TCAs are now becoming the first-line therapy for treating a

Fig. 6 Effects of acute (a) and repeated (b) treatment with fluvoxamine (30 and 60 mg/kg) and desipramine (30 and 60 mg/kg) on freezing behavior in rat conditioned fear stress test. In acute experiments, fluvoxamine and desipramine were p.o. administered 60 min before measurement of freezing behavior for 5 min. In chronic experiments, fluvoxamine and desipramine were p.o. administered once a day for 5 days. Data are represented as mean SEM (n =710). *p <0.05, significantly different from vehicle group

mission in the classical animal models. An H1 receptor antagonist, chlorpheniramine, improved anxiety in the rat elevated plus maze test and the open field behavioral test (Hasenohrl et al. 1999). Yanai et al. (1998) reported that anxiety was decreased in the elevated plus maze test of the mice lacking H1 receptors. Furthermore, the anxiolytic drugs such as diazepam and serotonin 5-HT1A agonist buspirone significantly inhibited the brain histamine turnovers in rodents (Oishi et al. 1992; Oishi et al. 1986). Diazepam also inhibited an anxiogenic-like effect induced by thioperamide in the mouse light/dark test (Imaizumi and Onodera 1995). Taken in concert, these findings support the notion that the histaminergic system enhances an anxiogenic-like response mainly via activation of H1 receptors and inhibition of H3 receptors. In the present study, however, stimulation of H3 receptors by R-methylhistamine and immepip, at either of the tested doses (10 and 30 mg/kg), did not exhibit anxiolytic-like effects in both the elevated plus maze test (Fig. 1) and the Vogel type conflict test (Fig. 2), as opposed to diazepam. The result in the former was consistent with the previous report that R--

Fig. 7 Effects of R--methylhistamine (RAMH; a), immepip (b), and reference drugs (c) on spontaneous locomotion in mice. R-methylhistamine (RAMH; 15, 30, and 60 mg/kg) and immepip (10, 30, and 100 mg/kg) were i.p. administered 15 min before measurement of the locomotor activities for 10 min. Fluvoxamine (20 and 40 mg/kg) and desipramine (10 and 30 mg/kg) were i.p. injected 30 min before the test. Data are represented as mean SEM (n =8). *p <0.05, significantly different from vehicle group

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variety of anxiety disorders. In marked contrast to this, these antidepressants fundamentally exhibit no therapeutic efficacy in classical anxiety models like elevated plus maze test and Vogel type conflict test, where pharmacological validation of benzodiazepine anxiolytics has often been conducted (Griebel et al. 1997; Petersen and Lassen 1981). Current findings may challenge these issues and vocalizations in mammals triggered by maternal separation have been regarded as an appropriate model that may detect the anxiolytic-like efficacy of antidepressants. In the rat maternal separation studies, the serotonergic antidepressants are effective in reducing the number of vocalizations (Olivier et al. 1998), but the drugs enhancing noradrenergic neurotransmission are shown to rather worsen the anxiety responses (Winslow and Insel 1990). Accordingly, these appear to be not so suitable as to evaluate anxiolytic-like actions of antidepressants in general. However, when it is the case with guinea pig pups, the similar efficacy has been demonstrated in common with various types of antidepressants, including TCAs, SSRIs, SNRIs, and monoamine oxidase inhibitors (Borsini et al. 2002). We indeed confirmed the reduction of the number of vocalizations in isolated guinea pig pups by fluvoxamine and desipramine (Fig. 3c), which are consistent with the previous reports (Molewijk et al. 1996). Both R--methylhistamine and immepip dose dependently suppressed vocalizations (Fig. 3a), suggesting that H3 agonists as well as antidepressants function as an anxiolytic. Although there has been no direct evidence that histamine induces vocalizations in guinea pig pups via its postsynaptic receptors, an H1 antagonist mepyramine suppressed foot shock-dependent vocalizations in rats (Sanchez 2003). Combined with our data, there may be a causal relationship between histaminergic neurotransmission and vocalization responses triggered by anxiety or stress. These points should also be addressed in the future pharmacological studies. To more precisely determine the neurobehavioral phenotype of H3 agonist-induced anxiolytic-like effects and, in particular, their similarities to the serotonergic and noradrenergic drugs, we next performed the mouse resident intruder test and the rat conditioned fear stress test. According to an earlier study, citalopram (SSRI) and clomipramine (TCA with relatively high affinity for 5-HT transporter) are effective equally in both the resident intruder test and the forced swimming test at the tested doses, whereas an effective dose of venlafaxine (SNRI) and reboxetine (NRI) in residentintruder test is higher than that in forced swimming test (Millan et al. 2001). Thus, one can assume that the residentintruder test is relatively sensitive to the serotonergic antidepressants. Consistent with the previous studies (Sanchez and Hyttel 1994), fluvoxamine reduced isolation-induced aggression (Fig. 4b) accompanied by no significant changes in locomotor activities

(Fig. 7c). Likewise, R--methylhistamine and immepip suppressed aggressive behavior induced by social isolation (Fig. 4a). In concert with the previous finding that H1 receptor-deficient mice showed reduced aggressive behavior (Yanai et al. 1998), the efficacy of R--methylhistamine and immepip is probably derived from decrease in both histamine release and its subsequent action to H1 receptors, at least in this model. Although the noradrenergic antidepressant desipramine (30 mg/kg) significantly suppressed the duration of attack behavior (Fig. 4c), it also induced hypolocomotion at the apparently effective dose (Fig. 7c), highlighting relative importance of serotonergic neurotransmission for the specific expression of anxiolytic-like action in the residentintruder test. Differential responses between the two reference drugs (fluvoxamine and desipramine) in the conditioned fear stress test appear to partially coincide with a previous report that SSRIs but not classical noradrenergic antidepressants such as maprotiline exhibited the anxiolytic-like efficacy in this test (Hashimoto et al. 1996). Comparable results supporting our data are also obtained where the effects of fluvoxamine on conditioned freezing were enhanced by its chronic treatment (Li et al. 2001). Under these experimental conditions, single treatment with R--methylhistamine and immepip significantly reduced freezing time (Fig. 5a), and these were completely antagonized by thioperamide (Fig. 5b). Contextual fear conditioning implies learning the relationship between aversive events and the environmental stimuli that predict such events. Also, H3 receptor ligands influence memory depending on the brain region, the nature of the cognitive task involved, and the pharmacological profiles of tested compounds. Indeed, the cognitive performance of rats in object recognition and a passive avoidance response, both of which are possibly related to the function of the frontal cortex, was strongly impaired by R-methylhistamine (Blandina et al. 1996), while thioperamide improved the response latency in the passive avoidance test (Meguro et al. 1995). Conversely, a beneficial effect of R-methylhistamine has been reported in rodent spatial learning and memory in a water maze test that primarily reflect the function of the hippocampus in rodents (Smith et al. 1994). Thus, such differences in the behavioral tasks and their relevant brain regions may explain this discrepancy. As for the contextual fear conditioning, previous studies showed that local administration of H3 agonists and H3 antagonists (or inverse agonists) immediately after the fear conditioning into the basolateral amygdala enhanced (Cangioli et al. 2002) and impaired (Passani et al. 2001) memory consolidation, respectively, as demonstrated by prolonged and shortened freezing time at retention test. These results are suggestive of a potential modulatory influence on cognition rather than anxiety of H3 receptor ligands that were administered in memory

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185

acquisition phase. Compared with our studies, the most different is the timing of H3 agonist administration. When considering our dosing condition (just before the test), the critical point is whether the H3 agonists administered impair the recall of memory. In addition to the forementioned report (Smith et al. 1994), another (Rubio et al. 2002) and our pilot study (unpublished data) also showed no amnesic effects of R--methylhistamine regarding the recovery of spatial memory in the rat water maze. These findings now led us to conclude that the observed responses by H3 agonists administered in the retention phase possibly seem to reflect genuine anxiolytic-like properties of the compounds in the conditioned fear stress test. The exact mechanisms whereby H3 agonists exert their action in animal anxiety models have to await full elucidation. H3 agonists behave as a presynaptic heteroreceptor to suppress the release of 5-HT (Schlicker et al. 1988), noradrenaline (Schlicker et al. 1994), dopamine (Schlicker et al. 1993), -aminobutyric acid (Garcia et al. 1997), and acetylcholine (Clapham and Kilpatrick 1992). Meanwhile, diazepam, one of the reference drugs used in this study, is reported to directly or indirectly inhibit neurotransmission of 5-HT, noradrenaline, dopamine, and acetylcholine (Danneberg and Weber 1983). Our observations that the H3 agonists failed to show anxiolytic-like effects in the elevated plus maze and the Vogel type conflict tests (Figs. 1, 2), therefore, suggest the importance of aminobutyric acid receptor activation per se in diazepamsensitive classical models. On the other hand, significant anxiolytic-like effects in three atypical models appeared to be shared between the H3 agonists and fluvoxamine (Figs. 3, 4, 5, and 6). Whereas the basic mechanism of action of SSRIs depends on the increase of 5-HT in the synaptic cleft (Bosker et al. 1995), H3 agonists rather inhibit the release of 5-HT in the brain (Schlicker et al. 1988). Thus, it is far less likely that the changes in monoamine signals, in particular the reduction of serotonergic neurotransmission, underlie the anxiolytic-like effects of H3 agonists. Other considerations to help explain our observations are the following findings regarding an intimate association between histamine and corticotrophin-releasing factor (CRF) in the regulation of anxiety and stress-related responses. Originally described as a pivotal mediator of acute neuroendocrine responses to stress, CRF is currently envisioned as a peptide neurotransmitter involved in the pathogenesis of anxiety and depressive disorders (Hauger et al. 2006). Anxiety behavior can be triggered by central injection of CRF (Swerdlow et al. 1986), whereas CRF1 receptor antagonists exhibit anxiolytic-like activities in the isolation-induced vocalization test (Hodgson et al. 2007), the residentintruder test (Farrokhi et al. 2004), and the conditioned fear stress test (Hikichi et al. 2000). In fact, selective CRF1 receptor antagonists have been developed as

a novel therapeutic approach for the treatment of anxiety and depression (Steckler and Dautzenberg 2006). On the other hand, earlier studies strongly suggested the pathophysiological importance of histamine by demonstrating that a variety of stress exposures, including restraint, cold temperature, and foot shock increase brain histamine turnover (Taylor and Snyder 1971; Yoshitomi et al. 1986). An intracerebroventricular infusion of histamine increased the level of CRF mRNA in the hypothalamic paraventricular nucleus (Kjaer et al. 1998) and the subsequent release of ACTH and corticosterone into plasma (Kjaer et al. 1994), suggesting positive regulation of CRF system by histamine. Moreover, stimulation of H3 receptor by R-methylhistamine, which is known to reduce the release of endogenous histamine (Ferretti et al. 1998), inhibited the ACTH and prolactin responses to restraint stress (Knigge et al. 1999). These observations enable us to propose that the putative mechanism of anxiolytic effects of H3 agonists depends on their functional modulation of CRF neurons via inhibition of histamine release. The present study clearly demonstrated that R-methylhistamine and immepip are effective in all of the atypical anxiety models tested, and these appear to be qualitatively analogous to the profiles of SSRI but not benzodiazepine. Although the underlying mechanisms remain to be elucidated, the observed competitive interaction between the two H3 agonists and thioperamide may guarantee that the primary action of R--methylhistamine and immepip resides in the activation of H3 receptors. Additional mechanistic studies using H3 receptor knockout mice will be supportive for a further substantiation of this notion. The present outcome may also provide an opportunity to further investigate the pathophysiological roles of H3 receptor in anxiety and stress-related responses and simultaneously raise the possibility that the H3 agonists offer an effective therapeutic option for the treatment of some forms of anxiety disorders in which SSRIs are commonly prescribed.

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