Short- and long-term effects of antipsychotic drug treatment on weight gain and H1 receptor expression

ARTICLE IN PRESS Psychoneuroendocrinology (2008) 33, 569–580

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Short- and long-term effects of antipsychotic drug treatment on weight gain and H1 receptor expression Mei Hana,b, Chao Denga,b, Thomas H.J. Burnec,d, Kelly A. Newella,b, Xu-Feng Huanga,b, a

Centre for Translational Neuroscience, School of Health Sciences, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia b Schizophrenia Research Institute, Sydney, NSW 2010, Australia c Queensland Centre for Mental Health Research, The Park Centre for Mental Health, Wacol, Queensland 4076, Australia d Neurobiology Program, Eskitis Institute for Cell and Molecular Therapy, Griffith University, Brisbane, Queensland 4111, Australia Received 9 August 2007; received in revised form 24 January 2008; accepted 29 January 2008

KEYWORDS Antipsychotics; Olanzapine; Histamine H1 receptor; mRNA expression; Hypothalamus; Weight gain

Summary The present study investigated body weight gain, food intake, open-field activity and brain histamine H1 receptor mRNA and protein expression in rats treated with three types of antipsychotics. Rats were divided into eight groups and treated with aripiprazole (2.25 mg/kg/day), olanzapine (1.5 mg/kg/day), haloperidol (0.3 mg/kg/day) or vehicle (as control) for 1 or 12 weeks. Administration of olanzapine for 1 week led to a threefold increase in body weight gain and a 35% increase in fat deposits compared to controls (po0.05). In the 12-week olanzapine treatment group, accumulative food intake was significantly higher in the first 7 weeks of treatment compared to controls (po0.018), while body weight gain was significantly greater in the first 8 weeks compared to controls (po0.045). Using in situ hybridization, we found that olanzapine treatment, but not aripiprazole or haloperidol treatment, significantly reduced H1 receptor mRNA expression in the arcuate hypothalamic nucleus (Arc: 18%, p ¼ 0.006, 1 week; 20%, p ¼ 0.008, 12 weeks) and ventromedial hypothalamic nucleus (VMH: 22%, p ¼ 0.006, 1 week; 19%, p ¼ 0.042, 12 weeks) compared to controls. The quantitative autoradiography data showed a reduction in VMH H1 receptor binding density after 1 (12%, p ¼ 0.040) and 12 (10%, p ¼ 0.094) weeks of olanzapine treatment. There were significant negative correlations between the levels of H1 receptor mRNA expression, and body weight gain and energy efficiency in the Arc and VMH after 1- and 12-week antipsychotic treatments in all groups. In addition, H1 receptor mRNA expression in the Arc showed a significant negative correlation with food intake and fat mass in all groups. Furthermore, there were negative correlations between H1 receptor binding density in the VMH and total fat mass and body

Corresponding author at: Centre for Translational Neuroscience, School of Health Sciences, University of Wollongong, Northfields Avenue,

Wollongong, NSW 2522, Australia. Tel.: +61 2 4221 4300; fax: +61 2 4221 4096. E-mail address: [email protected] (X.-F. Huang). 0306-4530/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2008.01.018

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M. Han et al. weight gain after 1 week of antipsychotic treatment. The present study suggests that downregulated VMH and Arc H1 receptor expression may be a key factor contributing to olanzapine-induced obesity. & 2008 Elsevier Ltd. All rights reserved.

1. Introduction A typical antipsychotics have been increasingly used as the first line of therapy for treating schizophrenia in place of conventional (or typical) antipsychotics. Compared with typical antipsychotics, such as haloperidol, atypical antipsychotics have been shown to be more efficient in controlling both positive and negative symptoms of schizophrenia and may also reduce the propensity to induce extrapyramidal symptoms (Conley and Kelly, 2005). However, some atypical antipsychotics, such as olanzapine, have been shown to lead to excessive body weight gain, diabetes and metabolic disorders (Nasrallah, 2003). This contributes to a serious lack of compliance in taking medication, which leads to symptom relapse plus a worsened long-term outcome, and results in a huge cost for society and families of patients (Bernstein, 1987; Fenton, 1997). In the clinical setting, patients treated with olanzapine can gain 4–5 kg within the first 10 weeks of treatment (Allison et al., 1999; Eder et al., 2001) and gain as much as 12 kg after 1 year of treatment (Nemeroff, 1997). This olanzapine-induced body weight gain has also been modelled in female rats (Huang et al., 2006; Arjona et al., 2004; Cooper et al., 2005). In comparison, aripiprazole, another atypical antipsychotic drug, does not induce significant weight gain in the clinical setting (Kasper et al., 2003). Antipsychotics are antagonists and agonists of multiple receptors, such as D2, H1, M15, 5-HT1A and 5-HT2A/2C. Some studies suggest that weight gain might be the result of antipsychotic action on a series of neurotransmitter systems, such as serotonin, histamine, dopamine and muscarinic (Allison and Casey, 2001; Deng et al., 2007; Huang et al., 2006). Recently a meta-analysis has shown a significant correlation between H1-histamine receptor occupancy and weight gain in clinical trials (Matsui-Sakata et al., 2005), while another study which correlated the receptor affinities of antipsychotic drugs with short-term weight gain data derived from a previous meta-analysis, also suggested that the H1-histamine receptor affinities of antipsychotic medications are positively correlated with weight gain (Kroeze et al., 2003). However, despite these exciting correlations from meta-analyses, no specific data showing changes at the molecular level have been reported and thus the molecular mechanisms of antipsychotic-induced weight gain via H1 histamine receptors are currently unknown. Specifically, it is not known which particular brain regions are involved in H1 receptor-mediated weight gain following antipsychotic drug treatment. H1 receptors are extensively expressed in the hypothalamus (Bouthenet et al., 1988). Evidence indicates the involvement of hypothalamic H1 histamine receptors in the regulation of body energy balance. For example, H1 receptor knockout mice are reported to have an increase in food intake and body weight gain (Masaki et al., 2004; Mollet

et al., 2001). In addition data from both humans and animals indicate that H1-antihistamines increase appetite and body weight (Navarro-Badenes et al., 1992; Orthen-Gambill et al., 1988; Varsano et al., 1993; Wihl et al., 1985). Despite this evidence, the responses of H1 receptors to various antipsychotic drug treatments are still not known. Among the numerous nuclei of the hypothalamus, the ventromedial hypothalamic (VMH) and paraventricular nuclei play a significant role in the regulation of H1 receptor-mediated food intake (Fukagawa et al., 1989; Magrani et al., 2004), while other regions such as the lateral hypothalamic area, dorsomedial hypothalamic nucleus and preoptic anterior hypothalamus do not (Ookuma, 1989). In the present study we used short- and long-term rat models to examine if: (1) brain levels of H1 receptor expression differ between the rats treated with aripiprazole, olanzapine or haloperidol; (2) body weight gain, water intake, open-field activity, energy intake efficiency and fat storage vary between the rats treated with these three drugs; (3) any observed H1 receptor changes are brain region specific; and (4) whether relationships exist between changed levels of H1 receptor expression in specific areas of the brain and body weight gain/fatness, food intake and energy efficiency.

2. Methods 2.1. Animal and experimental procedures Female Sprague Dawley rats weighing 220–250 g were obtained from the Animal Resource Centre (Perth, WA, Australia). After arrival, rats were housed in individual cages under environmentally controlled conditions (temperature 22 1C, light cycle from 07:00 to 19:00 h and dark cycle from 19:00 to 07:00 h), with ad libitum access to water and standard laboratory chow. After a 1-week familiarization period, they were treated with aripiprazole (2.25 mg/kg/day, Otsuka, Japan), olanzapine (1.5 mg/kg/day, Eli Lilly, USA), haloperidol (0.3 mg/kg/day, Sigma, Australia), or vehicle for 1 or 12 weeks (Han et al., 2008). The daily dosage was divided into three equal amounts and all rats were treated three times a day (06:00, 14:00, 22:00 h) orally by specially prepared drug pellets as described previously (Huang et al., 2006; Huang-Brown and Guhad, 2002; Han et al., 2008). The minimum number of rats per group was 12. Food intake, water intake and body weight were recorded every week. All rats were sacrificed 48 h after the last drug treatment. The periovary, perirenal, and inguinal fat masses were weighed after the rats were sacrificed. Five rat brains from each group were used to determine the histamine H1 receptor mRNA and protein expression. All experimental procedures were approved by the Animal Ethics Committee, University of Wollongong, Australia, and complied with the

ARTICLE IN PRESS H1 receptor expression following short- and long-term antipsychotic drug treatment Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

2.2. Open-field test The open-field test was performed 1 week and 11 weeks after starting the treatment protocol in the 12-week treatment groups in order to determine if the antipsychotic drugs influence locomotor activity, which is related to energy expenditure. A single rat was placed in the centre of a grey rectangular arena (100  100 cm2 with 40 cm high walls) and behaviour was recorded for 5 min. Video recording of the behaviour was analysed with the Noldus software EthoVision Color-Pro. The following activities were examined: total distance travelled, distance travelled in the perimeter, latency to leave centre, frequency to cross between central/perimeter, frequency of entering and total time spent in the central zone and the peripheral zone.

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labelled probes in the respective hybridization solution. After hybridization, sections were washed in 1  SSC buffer at 55 1C three times for 20 min each followed by two washes in 1  SSC at room temperature for 1 h each. Finally, sections were dipped sequentially in Milli-Q water, 70% ethanol and 95% ethanol before air-drying and exposure to Hyper-b-max film (Amersham, UK). After 4 weeks of exposure, films were developed using standard procedures. The sections containing positive signals were dipped in emulsion solution (Amersham, UK) and then exposed for 6 weeks. This allowed a further examination of positive signals at the cellular level and confirmation of the results from the film. As in our previous work (Huang et al., 2004; Han et al., 2008), all films were analysed using a computer-assisted image analysis system, Multi-Analyst, connected to a GS-690 Imaging Densitometer (Bio-Rad, USA). Quantification of mRNA expression levels in various brain regions was performed by measuring the average density of each region. Values were then compared against a 14C-labelled autoradiographic standard (Amersham, UK).

2.3. Histology All rats were sacrificed using carbon dioxide asphyxiation between 07:00 and 09:00 h, in order to minimize the circadian-induced variation of mRNA expression. Brains were immediately removed after death and frozen in liquid nitrogen and then stored at 80 1C until sectioning. Coronal brain sections (14 mm) were cut at 17 1C with a cryostat, and thaw-mounted onto poly-lysine-coated slides. Sections for in situ hybridization were fixed in ice-cold phosphate-buffer containing 4% paraformaldehyde. Acetylation was carried out in 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0) for 10 min. Sections were then dehydrated in ethanol and stored at 20 1C until use. Identification of neuroanatomical structures was according to a standard rat brain atlas (Paxinos and Watson, 1997).

2.4. In situ hybridization The specific antisense hybridization probes for the histamine H1 receptor were 50 -GGG ACG TGT TTC CCT TTC CCC CTC TTG GCT GAA GAC AGT TGG AGA-30 (NM017018, encoding bases 850–894) and 50 -GGG GCG GCC CAA GGA CCA CTT AGT CAT GAT GAG ATA GAG GAT GTT-30 (NM017018, encoding bases 250–294). No sequences bearing significant homology to the designed probes were found in the Gene Bank (NCBA). All oligonucleotide probes were terminally labelled with 10-fold molar excess of [35S] dATP (specific activity: 1000 Ci/mmol, Amersham, Buckinghamshire, UK) and terminal transferase (Promega, Madison, WI), and purified over a MicroSpin G-50 column (Amersham, UK). The probe concentration was 107 pcm of [35S]-labelled probes in 750 ml hybridization solution. Hybridization was carried out by incubating sections in the hybridization buffer (50% deionized formamide, 4  SSC, 10% dextran sulphate, 1  Denhardt’s solution, 0.2% sheared salmon sperm DNA, 0.1% long-chain polyadenylic acid, 0.012% heparin, 20 mM sodium phosphate, pH 7.0, 106/75 ml of labelled probe and 5% DTT) at 37 1C for 16 h. Non-specific hybridization was determined by including 100-fold molar excess of non-

2.5. Receptor autoradiography H1 receptor binding was performed based on that described previously (Palacios et al., 1981; Ryu et al., 1995). The brain sections were incubated at room temperature for 1 h in 50 mM sodium potassium phosphate buffer (pH 7.4) containing 5nM [3H]pyrilamine (specific activity 27.0 Ci/mmol, Amersham Biosciences UK Limited). Nonspecific binding was determined by incubating consecutive sections in the presence of 2 mm triprolidine (Sigma). All sections were then washed four times for 2 min in ice-cold buffer. After a brief rinse in ice-cold distilled water, the slides were rapidly dried under a stream of cold air and exposed to Kodak BioMax MR film for 9 weeks. Films were then developed using standard procedures and all films were analysed using a computer-assisted image analysis system, Multi-Analyst, connected to a GS-690 Imaging Densitometer (Bio-Rad, USA).

2.6. Statistical analysis The data were analysed statistically using the SPSS 13.0 program (SPSS, Chicago, IL). Food intake, water intake and body weight gain for 1-week treatments were analysed by one-way ANOVA. Food intake, water intake, body weight gain and open-field behavior in the 12-week treatment groups were analysed by two-way repeated ANOVA (drug  treatment duration as repeated measure). Fat mass was analysed using a two-way ANOVA. H1 receptor mRNA expression and binding density were analysed by three-way ANOVA (drug  treatment duration  brain region as repeated measure). Multiple comparisons (post-hoc Tukey–Kramer-HSD test) were followed only when there was a significant ANOVA result. Pearson’s correlations were used to assess the relationships between H1 receptor expression with food intake, body weight gain, energy efficiency and fat mass. It was also used to assess the relationship between food intake with weight gain and fat mass.

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3. Results 100

3.2. Fat deposits Two-way ANOVA showed that there were significant effects of drug on the periovary (F[3, 93] ¼ 3.18, p ¼ 0.028) and total fat (F[3, 93] ¼ 2.89, p ¼ 0.040) masses. There were significant effects of treatment duration on the periovary fat (F[1, 93] ¼ 88.19, p ¼ 0.000) and total fat (F[1, 93] ¼ 64.83, p ¼ 0.000) masses. However, there was no significant interaction between the two factors in the periovary (F[3, 93] ¼ 0.44, p ¼ 0.998) or the total fat (F[3, 93] ¼ 0.15, p ¼ 0.997) masses. After 1 week of olanzapine treatment, a significantly increased fat accumulation was found in the periovary (+34%, po0.05), inguinal (+33%, po0.05) and total fat mass (the sum of periovary, perirenal and inguinal fat, +35%, po0.05) compared to the control group (Table 1). After 12 weeks of olanzapine treatment, a trend (po0.10) toward higher fat accumulation was found in the periovary (+16%), inguinal (+37%) and total fat (+22%) masses compared to the control group (Table 2). There were no effects of aripiprazole or olanzapine treatment on fat deposits.

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The olanzapine group gained three times more body weight compared to rats in the control group after 1 week of drug treatment (p ¼ 0.008), while the aripiprazole and haloperidol groups showed no effect. A two-way repeated measures ANOVA revealed significant effects of drug (F[3, 49] ¼ 3.17, p ¼ 0.032) and treatment duration (F[11, 539] ¼ 292.13, p ¼ 0.000) on accumulated body weight gain during the 12-week drug treatments. There was no significant interaction between the drug and treatment duration (F[33, 539] ¼ 1.21, p ¼ 0.203). Post-hoc tests showed that overall body weight gain during the 12-week period was significantly greater in the olanzapine group compared to controls (p ¼ 0.026; Figure 1). Further analyses showed that the significant increase in accumulated weight gain in the olanzapine group occurred in the first 8 weeks (0.001opo0.045; Figure 1). Therefore, the weekly weight gains in the first 8-week period were analysed and showed there were not only significant effects of drug (F[3, 49] ¼ 2.99, p ¼ 0.040) and treatment duration (F[7, 343] ¼ 223.73, p ¼ 0.000), but also a significant interaction between the drug and treatment duration (F[21, 343] ¼ 2.448, p ¼ 0.000). Weekly analyses showed that the olanzapine group had a significantly higher weekly weight gain than the controls only after the first week of treatment (p ¼ 0.001). The olanzapine group also had a significantly greater weekly weight gain than the aripiprazole group (p ¼ 0.007), but not the haloperidol group (p ¼ 0.770) after 1 week of treatment. The rats treated with olanzapine maintained their body weight at a high level through the treatment period (Figure 1), even though their weekly weight gains were not significantly different from the controls. There was no effect of aripiprazole or haloperidol treatment on body weight gain compared to controls.

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Figure 1 Accummulative body-weight gain (A), food intake (B), water intake (C) of rats treated with aripiprazole (ARP), olanzapine (OLZ) and haloperidol (HPD) for 12 weeks. *vs. control, po0.05.

3.3. Food intake In the 1-week treatment groups there were no significant effects of antipsychotic drug treatment on total food intake, although the olanzapine group tended to have a higher food intake than the control group (olanzapine vs. control: 133.475.0 g vs. 119.974.6 g, mean7SEM, p ¼ 0.066). In the 12-week treatment groups, a two-way repeated measures ANOVA revealed the significant effects of drug

ARTICLE IN PRESS H1 receptor expression following short- and long-term antipsychotic drug treatment

Table 1 Body weight and fat-pad masses of rats after aripiprazole, olanzapine and haloperidol treatments for 1 week. Control (n ¼ 12)

Aripiprazole (n ¼ 12)

Body weight (g) IBW 241.473.6 244.573.6 FBW 244.973.4 250.873.1 Fat-pad masses (g) Periovary 3.870.3 Perirenal 3.570.4 Inguinal 3.370.2 Total fat 10.670.8 Others Fat/IBW (%) Fat/FBW (%)

4.470.3 4.370.3

Olanzapine (n ¼ 12)

Haloperidol (n ¼ 12)

243.473.3 256.173.2

244.073.4 250.974.7

4.170.3 3.870.3 3.770.2 11.570.7

5.170.3* 4.870.6 4.470.3* 14.371.1*

4.070.2 3.870.3 4.170.2 11.970.5

4.770.3 4.670.3

5.970.4* 5.670.4*

4.970.2 4.770.2

Data are mean7SEM, IBW: initial body weight, FBW: final body weight. *po0.05 vs. control.

Table 2 Body weight and fat-pad masses of rats after aripiprazole, olanzapine and haloperidol treatments for 12 weeks. Control (n ¼ 14)

Aripiprazole (n ¼ 13)

Olanzapine (n ¼ 13)

Haloperidol (n ¼ 13)

243.073.2 318.278.8

245.273.4 307.075.3

6.870.4 6.870.6 5.970.6 19.471.5

7.870.8 7.671.1 6.770.8 22.172.6

7.070.5 6.770.6 6.270.5 19.971.5

7.470.4

8.070.6

9.071.0

8.170.6

5.970.2

6.370.4

6.870.6

6.470.4

Body weight (g) IBW 243.473.1 243.272.9 FBW 301.475.9 307.277.2 Fat-pad masses (g) Periovary 6.770.3 Perirenal 6.470.5 Inguinal 4.970.2 Total fat 18.071.0 Others Total fat/ IBW (%) Total fat/ FBW (%)

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aripiprazole and control groups (p ¼ 0.990). Further weekly analyses showed that the olanzapine group had significant higher weekly food intake than the control groups in weeks 1, 2 and 3 (olanzapine vs. control: Week 1, 148.474.0 g vs. 133.773.3 g; Week 2, 138.074.5 g vs. 120.871.9 g; Week 3, 186.6722.7 g vs. 130.475.4 g all po0.05).

3.4. Relationship between food intake, and body weight gain and fat mass after antipsychotic treatment Significant positive correlations were found between food intake and body weight gain in both 1- and 12-week treatment groups (r ¼ 0.424, p ¼ 0.003 and r ¼ 0.616, p ¼ 0.000). Food intake was also positively correlated with total fat mass after both 1- and 12-week treatments (r ¼ 0.425, p ¼ 0.003 and r ¼ 0.502, p ¼ 0.000) (Figure 2).

3.5. Water intake In the 1-week treatment groups there were no significant effects of antipsychotic drug treatment on total water intake. In the 12-week treatment groups however, a twoway repeated ANOVA showed significant effects of drug (F[3, 49] ¼ 7.56, p ¼ 0.000) and treatment duration (F[11, 539] ¼ 1606.29, p ¼ 0.000), and a significant interaction between the drug and treatment duration (F[33, 539] ¼ 8.29, p ¼ 0.000) on 12-week accumulative water intake. Significant reductions in accumulative water intake were found in the haloperidol group compared to the control group (weeks 1 and 3–12, 0.001opo0.027). The total water intake over 12 weeks was 38% less in the haloperidol group than the control group (Figure 1). There were no significant effects of olanzapine or aripiprazole treatment on water intake.

3.6. Open-field test

Data are mean7SEM. IBW: initial body weight, FBW: final body weight.

(F[3, 49] ¼ 8.63, p ¼ 0.000) and treatment duration on total food intake (F[11, 539] ¼ 5049.07, p ¼ 0.001). There was also a significant interaction between the drug and treatment duration (F[33, 539] ¼ 7.61, p ¼ 0.001) on food intake. In total, the olanzapine group had a significantly higher accumulative food intake than controls (p ¼ 0.042), while the haloperidol group tended to have a lower accumulative food intake (p ¼ 0.086). More specifically, during the course of the drug treatment a significant increase in accumulative food intake was found in the first 7 weeks in the olanzapine group compared to controls (0.001opo0.018). On the other hand, a significant decrease in accumulative food intake was found between weeks 9 and 12 in the haloperidol group compared to the control group (0.013opo0.042) (Figure 1). There was no significant difference in food intake between

Two-way repeated ANOVA revealed significant effects of drug (F[3, 48] ¼ 3.59, p ¼ 0.020) and treatment duration (F[1, 48] ¼ 87.18, p ¼ 0.000) on the total distance travelled in the 12-week treatment groups. There was a significant interaction between the drug and treatment duration (F[3, 48] ¼ 7.50, p ¼ 0.001) on distance travelled. Rats in the haloperidol group travelled less than controls at 1 week (96%, p ¼ 0.000), but not 11-weeks (14%, p ¼ 0.937) after starting drug treatments (Figure 3). However, there were no significant effects of drug and treatment duration on the other parameters measured in the open field. In addition, olanzapine and aripiprazole treatments had no effect on any parameters in the openfield test.

3.7. Expression of H1 receptor mRNA The H1 receptor was highly expressed in all brain regions examined, including regions of the hypothalamus, hippocampus and striatum and the distribution was similar to that reported previously (Lintunen et al., 1998). Three-way ANOVA showed there were significant effects of brain region

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Figure 2 Positive correlations were found between food intake with body-weight gain and total fat mass in both 1 and 12 weeks. J: control;  : aripiprazole; ,: Olanzapine; &: haloperidol. 3500

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Figure 3 Walking distance of rats in open field for 5 min. Rats were treated with aripiprazole (A), olanzapine (J), haloperidol (H) and vehicle (C). *vs. C, p ¼ 0.000.

(F[15, 420] ¼ 145.6, p ¼ 0.000) and significant interactions between brain region and drug (F[45, 420] ¼ 1.84, p ¼ 0.001), between brain region and treatment period (F[15, 420] ¼

13.8, p ¼ 0.012), and between all three factors (F[45, 420] ¼ 1.54, p ¼ 0.017) on H1 receptor mRNA expression. As presented in Tables 3 and 4, there are clear regional differences in H1 mRNA expression. Post-hoc tests showed that olanzapine treatment caused a significant reduction in H1 receptor mRNA expression only in the arcuate hypothalamic nucleus (Arc) and ventromedial hypothalamic nucleus (VMH). Arc: Compared to the control group, olanzapine treatment significantly decreased the levels of H1 receptor mRNA expression in both 1 week (olanzapine, 152.77 5.3 nCi/g tissue vs. control, 186.975.3, 18%, p ¼ 0.006) and 12-week groups (olanzapine, 156.873.2 vs. control, 194.778.7, 20%, p ¼ 0.008) (Figures 4 and 5). VMH: Compared to controls, the levels of H1 receptor mRNA expression were significantly decreased in the olanzapine group after 1-week (olanzapine, 149.673.3 vs. control, 192.875.4, 22%, p ¼ 0.006) and 12-weeks of treatment (olanzapine, 168.575.6 vs. control, 208.8710.6, 19%, p ¼ 0.042) (Figures 4 and 5).

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Figure 4 The levels of H1 receptor mRNA expression in the arcuate hypothalamic nucleus (Arc) and ventromedial hypothalamic nucleus (VMH) of rats treated with aripiprazole (ARP), olanzapine (OLZ), haloperidol (HPD) and vehicle (CONT). *vs. control, po0.05. White bar: 1-week treatment. Black bar: 12-week treatment.

Haloperidol and aripiprazole treatment had no significant effect on H1 receptor mRNA expression in any of the brain regions examined after 1 or 12 weeks of treatment (Tables 3 and 4).

3.8. Relationship between the levels of H1 receptor mRNA expression and food intake, energy efficiency, body weight gain and fat masses after 1 and 12 weeks of antipsychotic treatment Significant negative correlations were found between H1 receptor mRNA expression in the Arc and food intake, body weight gain, energy efficiency (the ratio of accumulated body weight gain/the total food intake), accumulative fat masses and total fat after both 1 and 12 weeks of drug treatment (0.629oro0.528, 0.004opo0.02). Significant negative correlations were also found between H1 receptor mRNA expression in the VMH with body weight gain and energy efficiency after both 1 and 12 weeks of drug treatment (0.555oro0.447, 0.014opo0.048).

3.9. H1 receptor binding density The finding of H1 receptor mRNA reductions in the Arc and VMH following olanzapine treatment prompted us to examine the H1 receptor binding density in these two brain regions. [3H]Pyrilamine was used to examine H1 receptor binding density in the Arc and VMH (Figure 6). Three-way ANOVA showed that there were significant effects of drug (F[3, 32] ¼ 5.8, p ¼ 0.029) and brain region (F[1, 32] ¼

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153.900, p ¼ 0.000) on H1 receptor specific binding densities and there was a borderline significant interaction between the two factors (F[3, 32] ¼ 2.694, p ¼ 0.063). However, there was no significant effect of treatment period (F[1, 32] ¼ 2.752, p ¼ 0.107), and no interaction between the treatment period and any other factors. Compared to the control group, H1 receptor-specific binding density was significantly decreased in the VMH of the olanzapine group after 1 week of treatment (olanzapine, 104.971.8 vs. control, 119.173.8, 12%, p ¼ 0.04) and remained reduced after 12 weeks of treatment (olanzapine, 99.671.7 vs. control, 111.274.1, 10%, p ¼ 0.094) (Table 5). There was a significant negative correlation between total fat mass and H1 receptor binding density in the VMH after 1 week of antipsychotic drug treatment (r ¼ 0.485, p ¼ 0.03) and a borderline significant correlation between total body weight gain and H1 receptor binding density after 1 week of drug treatment (r ¼ 0.42, p ¼ 0.066). There were no significant effects of aripiprazole or haloperidol treatment on [3H]pyrilamine binding density in the VMH. Furthermore, there were no significant effects of antipsychotic drug treatment on H1 receptor binding density in the Arc.

4. Discussion This study for the first time compared H1 receptor expression in the rat brain following short- and long-term administration of olanzapine, aripiprazole and haloperidol. A significant downregulation of H1 receptor mRNA expression was found in the VMH and Arc after olanzapine treatment for 1 and 12 weeks compared to controls. Similarly, downregulations in H1 receptor binding density were observed in the VMH following olanzapine treatment for 1 and 12 weeks compared to controls. Aripiprazole and haloperidol treatments had no effect on the levels of H1 receptor mRNA expression or binding density. H1 receptor mRNA expression in the VMH and Arc showed significant negative correlations with body weight gain and energy efficiency after 1 and 12 weeks of antipsychotic treatment, while H1 receptor mRNA expression in the Arc showed additional negative correlations with food intake and fat mass. Furthermore, total fat mass and body weight gain were negatively correlated with H1 receptor binding density in the VMH after 1 week of antipsychotic treatment. A major side-effect of olanzapine treatment in humans is weight gain (Allison et al., 1999). Generally, the most significant body weight gain is found in the first 7 weeks, then weight is gained gradually for up to 4 months and a high body weight plateau is maintained with chronic treatment (Nasrallah, 2003). Similarly, this study showed that rats treated with olanzapine had a significant increase in body weight in the first 8 weeks of treatment and this high body weight was then maintained until week 12 without any further significant increases in body weight between weeks 8 and 12. The detailed analyses in weekly weight gain showed that the extra weight in rats treated with olanzapine was gained mostly in the first week of treatment. In weeks 2 and 3, olanzapine-treated rats had slightly higher weekly weight gain than the controls, but this difference was not significant (all p40.05). Therefore, the weight gain

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Figure 5 Photographs depict histamine H1 receptor mRNA expression in rats treated with vehicle (A) or olanzapine (A0 ) for 1 week. Scale bar ¼ 2 mm. B and B0 are photographs with high magnification of the hypothalamic areas of A and A0 . C and C0 are high magnification emulsion autoradiographs showing the H1 receptor mRNA expression in the VMH. Scale bar ¼ 50 mm. D and D0 are high magnification emulsion autoradiographs showing the H1 receptor mRNA expression in the Arc. Scale bar ¼ 50 mm. Arc: arcuate hypothalamic nucleus, VMH: ventromedial hypothalamic nucleus.

in the first week is responsible for the changes observed during the week 1–8 period. Several short-term studies (p5 weeks) have also shown a rapid increase in body weight after olanzapine treatment in rats (Arjona et al., 2004; Hill and Young, 1980; Huang et al., 2006; Pouzet, 2003). However, it is not clear if weight gain is due to increased energy intake or decreased energy expenditure, which determines the energy balance regulation. In this study, rats treated with olanzapine showed increased weekly food intake in the first 3 weeks of treatment, while showing no differences in locomotor activities (i.e. distance travelled or travel velocity) after both 1 and 11 weeks of treatment, compared to controls. Since we did not measure the resting metabolic rate it is possible that reduced energy expenditure contributed to body weight gain in rats after olanzapine treatment. From this study however, we did show that increased energy intake contributed to the excessive energy storage as positive correlations were found between overall energy intake and body weight gain, and between overall energy intake and fat mass.

In the clinical setting olanzapine treatment causes an initial increase in body weight; body weight then stops increasing but remains at a high level while on the olanzapine treatment (Nasrallah, 2003). In the present study two time points that represent this situation are week 1, which showed the sharpest increase in body weight gain and week 12, where body weight was no longer increasing but was maintained at the high level (Figure 1A). These twotime points were therefore selected for the subsequent examination of H1 receptor expression. The present study found reduced H1 receptor mRNA expression in the Arc and VMH and reduced H1 receptor binding density in the VMH of olanzapine treated rats. Although we cannot exclude that this occurred via indirect downstream mechanisms, it is possible that this reduction is a consequence of olanzapine-induced H1 receptor antagonism since olanzapine has the highest binding affinity to H1 receptors among the three antipsychotic drugs (Kroeze et al., 2003). The exact mechanism of how H1 receptor antagonism downregulates H1 receptor expression is not

ARTICLE IN PRESS H1 receptor expression following short- and long-term antipsychotic drug treatment

Table 3 H1 receptor mRNA expression in different brain regions following 1-week treatment with aripiprazole, olanzapine and haloperidol.

AcbC AcbSh Arc CA1 CA2 CA3 Cpu DM DG HB LHA MEPV PVN RT VMH ZI

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Table 4 H1 receptor mRNA expression in different brain regions following 12-week treatment with aripiprazole, olanzapine and haloperidol.

Control (n ¼ 5)

Aripiprazole Olanzapine (n ¼ 5) (n ¼ 5)

Haloperidol (n ¼ 5)

Chronic

Control (n ¼ 5)

Aripiprazole (n ¼ 5)

Olanzapine (n ¼ 5)

Haloperidol (n ¼ 5)

143.177.1 137.274.9 186.975.3 207.274.0 190.177.6 249.4710.1 132.872.9 175.2711.6 251.076.0 228.7711.9 154.773.9 187.579.2 177.779.1 172.674.9 192.875.4 202.179.8

148.674.1 152.374.0 188.377.4 197.273.9 173.079.1 240.274.1 148.675.1 165.472.5 243.376.3 225.576.4 159.774.0 205.879.9 180.279.5 174.575.4 196.076.7 189.179.0

146.675.0 147.375.4 165.074.9 187.4710.4 167.876.4 238.378.1 139.274.4 161.577.6 237.479.6 215.978.3 155.077.2 208.4714.6 180.376.8 174.278.0 192.6714.0 187.7711.0

AcbC AcbSh Arc CA1 CA2 CA3 CPu DM DG HB MePV LHA PVN RT VMH

141.276.8 138.175.4 194.778.7 180.475.0 155.175.8 227.4710.3 128.975.7 160.0711.2 224.379.0 189.478.8 187.878.8 148.573.4 185.677.2 160.774.1 208.8710.6

138.079.9 137.177.9 198.279.8 191.878.1 160.878.5 224.379.3 127.178.5 145.7712.1 231.378.4 217.879.8 194.2711.1 145.674.3 169.178.9 161.878.9 199.177.8

147.975.6 144.175.6 192.774.3 190.074.0 168.878.7 227.877.1 138.575.3 155.577.6 231.276.7 197.672.3 202.377.7 148.477.7 177.4711.6 161.174.0 201.8713.0

ZI

185.577.6

178.7713.1

131.575.0 134.074.3 156.873.2* 200.479.8 178.2712.7 231.0710.6 121.775.7 147.0711.9 241.2717.9 214.6723.0 194.6715.8 146.079.1 180.0711.7 155.5711.3 168.575.6** 199.1717.3

134.879.5 133.9710.0 152.775.3* 176.876.5** 168.274.0 223.678.9 127.477.3 148.074.4 219.879.0 198.7713.3 137.173.7 163.572.4 175.4711.0 163.074.8 149.673.3** 164.674.7

Mean7SEM. Units of measurement are nCi/g tissue. AcbC, accumbens nucleus, core; AcbSh, accumbens nucleus, shell; Arc, arcuate hypothalamic nucleus; CA1-3, fields of CA1-3 of hippocampus; Cpu, caudate putamen; DM, dorsomedial hypothalamic nucleus; DG, dentate gyrus; HB, habenular nucleus; LHA, lateral hypothalamic area; MePV, medial amygdaloid nucleus, posteroventral part; PVN, paraventricular hypothalamic nucleus; RT, reticular thalamic nucleus; VMH, ventromedial hypothalamic nucleus; Zi, zona incerta *po0.01 vs. control. **po0.05 vs. control.

known. Normally, administration of an antagonist will upregulate its down-stream receptor. Recently however, an exception was seen in a number of G-protein-coupled receptors such as 5-HT2A and 5-HT2C (Huang et al., 2006; Van Oekelen et al., 2003). Furthermore, aripiprazole and haloperidol, which have low affinities for the H1 receptor had no effect on H1 receptor expression. The VMH plays a significant role in the regulation of food intake and energy expenditure (King, 2006). A lesion to the VMH results in decreased sympathetic activity, hyperphagia and obesity in rodents accompanied by hyperinsulinemia and increased metabolic efficiency. Our group also showed that the VMH plays an important role in the regulation of energy balance in chronic high-fat diet-induced obesity (Huang et al., 2003; Huang and Wang, 1998). The present study showed a significant down-regulation of H1 receptor expression in the VMH in the olanzapine-treated group compared to the control group. An important negative correlation between H1 receptor mRNA expression in the VMH with body weight gain and energy efficiency was also found, in addition to negative correlations between H1 receptor binding density in the VMH with body weight gain and total fat mass. These results suggest that a decreased H1 receptor expression in the VMH might contribute to body weight gain and increased energy efficiency in rats treated with olanzapine. In addition to the VMH, this study reports a downregulation of neuronal histamine H1 receptor mRNA expres-

187.8712.3

Mean7SEM. Units of measurement are nCi/g tissue. AcbC, accumbens nucleus, core; AcbSh, accumbens nucleus, shell; Arc, arcuate hypothalamic nucleus; CA1-3, fields of CA1-3 of hippocampus; Cpu, caudate putamen; DM, dorsomedial hypothalamic nucleus; DG, dentate gyrus; HB, habenular nucleus; LHA, lateral hypothalamic area; MePV, medial amygdaloid nucleus, posteroventral part; PVN, paraventricular hypothalamic nucleus; RT, reticular thalamic nucleus; VMH, ventromedial hypothalamic nucleus; Zi, zona incerta *po0.01 vs. control. **po0.05 vs. control.

sion in the Arc in the olanzapine group following both 1- and 12-week treatments, compared to controls. Moreover, there was a significant negative correlation between H1 receptor mRNA expression in the Arc and body weight gain, food intake, energy efficiency and accumulative fat mass. In support of this finding, in humans the H1 receptor agonist, betahistine is reported to reduce food intake and prevent body weight gain when it was co-administered with olanzapine (Poyurovsky et al., 2005; Rossi et al., 1999). Clearly, Arc H1 receptors are, at least partially, responsible for weight gain associated with olanzapine treatment. Despite the fact that we found no change in H1 receptor binding density in this brain region, this finding does not rule out that these receptors may have decreased function. Arc is an area containing numerous key neurotransmitters and receptors regulating body weight, such as neuropeptide Y, agouti-related peptide, POMC and CART (Huang et al., 2003; Schwartz et al., 2000). The relationship between H1 receptor and these neuromodulators needs further study in order to obtain a full understanding of the H1 receptor’s involvement in weight regulation. The majority of the olanzapine-induced increase in body weight in the present study occurred in the first 8 weeks of treatment and this high weight was then maintained until 12 weeks without further increases in body weight. We observed a downregulation in H1 receptor expression at week 1, when there was a clear increase in body weight gain in the olanzapine group, but we also observed a

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Figure 6 Line drawing adapted from (Paxinos and Watson, 1997) indicates the location of the quantified VMH and Arc (A). Photographs depict total (B) and non-specific (B) [3H]pyrilamine binding. Scale bar ¼ 2 mm. Table 5 H1 receptor specific densities in Arc and VMH following treatment with aripiprazole, olanzapine and haloperidol. Control (n ¼ 5)

Aripiprazole Olanzapine (n ¼ 5) (n ¼ 5)

Arc 1 wk 97.174.7 91.872.8 12 wks 98.774.7 94.774.3

93.773.6 93.472.8

Haloperidol (n ¼ 5)

95.374.4 8473.0

VMH 1 wk 119.173.8 110.973.8 104.971.8* 107.673.7 12 wks 111.274.1 106.473.2 99.671.7 101.973.5 Mean7SEM. Units of measurement are fmole/mg. Arc, arcuate hypothalamicnucleus; VMH, ventromedial hypothalamic nucleus. *po0.05 vs. control.

downregulation in H1 receptor expression at week 12 when body weight had stopped increasing and had plateaued at a high level. A previous study has found that withdrawal of olanzapine treatment causes rapid weight loss in rats (Goudie et al., 2002). Therefore we suggest the continuous H1 downregulation at 12 weeks is related to maintaining the body weight at the high level. It would be interesting in future studies to examine the effects of a drug withdrawal period to body weight and H1 receptor expression to determine if H1 expression returns to normal along with body weight and if they remain correlated. Clinically, both aripiprazole and haloperidol do not induce significant weight gain (Allison et al., 1999; Marder et al., 2003; Wirshing, 1999). It is also known that both have significantly lower H1 receptor binding affinities than olanzapine, 15 and 900 times, respectively (Kroeze et al., 2003). We did not observe a significant gain of body weight and fat mass with either of these drug treatments. Furthermore, no differences in the VMH and Arc H1 mRNA expression or H1 receptor binding density were found in either aripiprazole or haloperidol treated groups for 1 and 12 weeks when compared to controls. This supports a previous study suggesting that H1 receptor affinity of antipsychotic drugs can be used to predict short-term weight gain (Kroeze et al., 2003). In agreement with human studies, we showed that haloperidol treatment in rats did not induce body-weight gain. However, this does not mean that haloperidol has not affected the regulation of body metabolism. This is because we observed a trend of decreased food intake from weeks 1 to 8 and a significant decrease in food intake from weeks 9

to 12 compared to the control group. On the other hand, the rats treated with haloperidol had a significantly lower spontaneous locomotor activity in the first week open-field test and a trend toward lower activity in the week 11 test. However, they showed no change in anxiety-related parameters of the open-field test. Therefore, a consequence of low-energy intake as well as low activity may have led to no change in body weight. It should be noted that this study has examined multiple brain regions (16 brain regions for H1 receptor mRNA and 2 brain regions for H1 receptor binding). Statistically multiple comparisons without correction of significance level have a potential to increase Type I error. To control for the Type I error, multiple comparisons were performed only after a significant ANOVA effect. Furthermore, olanzapineinduced alterations of H1 receptor mRNA expression and receptor binding were found specifically in the two key hypothalamic nuclei (Arc and VMH) for food intake and body weight regulation and these changes were significantly correlated with a number of metabolic parameters, which did imply the involvement of these hypothalamic H1 receptors in olanzapine-induced weight gain. However, due to this limitation of non-correction for multiple comparisons, a further study is important to verify these findings. The drug dosages used in the literature for olanzapine, aripiprazole and haloperidol vary significantly. Similar doses to that used in this study have been used in the literature previously and have been shown to be behaviourally effective. For example, aripiprazole has recently been used at a dose of 2.5 mg/kg (Bruins Slot et al., 2005), while haloperidol has frequently been utilized at a dose of 0.3 mg/kg (Pouzet et al., 2003; Wiley, 2008). In addition, doses of olanzapine ranging from 1.2 (Arjona et al., 2004; Huang et al., 2006) to 2.0 mg/kg (Cooper et al., 2005) have consistently been used in the literature and have been shown to produce the obesity phenotype in rats (Arjona et al., 2004; Cooper et al., 2005; Huang et al., 2006). Furthermore, these selected doses all share a D2 occupancy of approximately 70–80% in rats (Kapur et al., 2003; Natesan et al., 2006a, b). While serum levels of the antipsychotic drugs were not measured in the present study, we have shown previously that the doses of the drugs used in this study do affect central receptor systems relative to their pharmacological profile (Han et al., 2006; Huang et al., 2006), indicating the effectiveness of these doses. However, it should be noted that while the doses used in the present study have been shown to be behaviourally and pharmacologically active in rats, they do not necessarily equate to doses used in the clinical setting.

ARTICLE IN PRESS H1 receptor expression following short- and long-term antipsychotic drug treatment Future studies to validate the importance of the H1 receptor in olanzapine-induced obesity are recommended. While it is known that H1 receptor knockout mice are obese, it would be important in future studies to use the same dosing regimen as in the present study in H1 knockout animals to determine if olanzapine exacerbates the level of obesity. A recent study by Kim et al. (2007) examined the food-intake regulator AMPK in the hypothalamus of antipsychotic-treated H1 knockout mice. This study concluded that olanzapine acts via H1 receptors to activate AMPK in the hypothalamus, as in H1 knockout mice this activation is absent, further indicating the role of H1 receptors in olanzapine-regulated energy balance. In summary, aripiprazole, olanzapine and haloperidol differentially regulate H1 receptor expression in rat hypothalamus. Olanzapine can down-regulate the H1 receptor mRNA and protein expression in the hypothalamic VMH, a nucleus critically involved in the regulation of food intake, while aripiprazole and haloperidol do not. A similar phenomenon was seen in the Arc with a reduction in H1 receptor mRNA expression, but not protein binding after olanzapine treatments. It appears that olanzapine-induced weight gain is largely due to an increase of energy intake and no change in spontaneous locomotor activity, while other factors contributing to the energy balance equation need further studies, such as resting metabolic rate and active/willingness activities. Whilst meta-analysis studies have previously shown a correlation between H1 receptor affinity and weight gain (Kroeze et al., 2003; Matsui-Sakata et al., 2005), we have specifically shown for the first time that the atypical antipsychotic drug olanzapine, which has high H1 antagonist properties and induces significant weight gain, downregulates the H1 receptor exclusively in the hypothalamic areas of VMH and Arc. Therefore, it is suggested that new novel antipsychotic drugs be screened to eliminate this target on H1 receptors in the hypothalamus.

Role of the funding source Funding for this study was provided by the University of Wollongong Research Committee and the Schizophrenia Research Institute. Both funding bodies had no role in study design, the collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the paper for publication.

Conflict of interest There are no actual or potential conflicts of interests from any of the authors of the manuscript.

Acknowledgments This study was supported by the University of Wollongong Research Committee and by the Schizophrenia Research Institute, Australia, utilizing infrastructure funding from NSW Health. The Schizophrenia Research Institute is formerly known as the Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD). The EthoVision system

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was purchased through a Ramaciotti Foundations Biomedical Research Award to C. Deng.

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