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Olanzapine differentially affects 5-HT2A and 2C receptor mRNA expression in the rat brain
Behavioural Brain Research 171 (2006) 355–362
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Olanzapine differentially affects 5-HT2A and 2C receptor mRNA expression in the rat brain Xu-Feng Huang a,b , Mei Han a,b , Xin Huang a , Katerina Zavitsanou a,b,c , Chao Deng a,b,∗ a
Neurobiology Research Centre of Metabolic and Psychiatric Disorders, School of Health Science, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia b Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD), NSW, Australia c Australian Nuclear Science & Technology Organization (ANSTO), NSW, Australia Received 7 February 2006; received in revised form 28 March 2006; accepted 31 March 2006 Available online 22 May 2006
Abstract This study examined regional changes in rat brain mRNA levels encoding 5-HT2A and 5-HT2C receptors following chronic olanzapine treatment. The immediate effect (2 h after the last treatment) was a down-regulation of 5-HT2A receptor mRNA expression, predominantly in the hypothalamus, limbic system and striatum, while a rebound effect was observed 48 h later. 5-HT2C receptor mRNA expressions were decreased in the substantia nigra. Correlations between 5-HT2A receptor mRNA expression and total food intake, weight gain and energy efficiency were observed. © 2006 Elsevier B.V. All rights reserved. Keywords: Antipsychotics; Gene expression; Serotonin receptors; Body weight
Olanzapine is an atypical antipsychotic drug that has proven efficacy in ameliorating both positive and negative symptoms of schizophrenia [21]. Olanzapine, like other atypical antipsychotics, is also less likely to cause extrapyramidal side effects while a common side effect is significant body weight gain [1]. Olanzapine is a potent 5-HT2 receptor antagonist with high affinities to both 5-HT2A (Ki = 4.0 nM) and 5HT2C (Ki = 11 nM) receptor subtypes [4,14,28]. It also possesses antagonistic effects to histaminergic and dopaminergic systems [1,4,26]. In humans, PET studies show that 5-HT2 receptor occupancy is about 75% in a usual clinical dosage range after oral administration of olanzapine [15,22]. In rats, olanzapine occupancy at a dosage of 1 mg/kg is about 70 and 30% for 5-HT2A and D2 receptors, respectively, while it is around 76 and 41% at 2 mg/kg [18]. It was also noted that measurement of 5-HT2A receptor binding densities in response to olanzapine treatment was almost exclusively limited to the frontal cortex [30]. No data are available examining in situ regional changes of 5-HT2A and 5-HT2C receptors mRNA expression after olanzapine treatment in brain areas. Using a chronic olanzapine treated rat model,
∗
Corresponding author. Tel.: +61 2 42214934; fax: +61 2 42214096. E-mail address: [email protected] (C. Deng).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.03.040
this study aimed to test the following hypothesis: (1) the mRNA expression of 5-HT2A and 5-HT2C receptors respond differently to olanzapine treatment, across a wide range of brain structures; (2) there is a relationship between changes in gene expression in specific areas of the brain and energy intake/weight gain/energy efficiency. Twenty-four female Sprague–Dawley rats weighing ∼250 g were obtained from the Animal Resource Center (Perth, WA, Australia). They were housed individually in environmentally controlled conditions (temperature 22 ◦ C, light cycle from 06:00 to 18:00 h and dark cycle from 18:00 to 06:00 h), and allowed ad libitum access to water and standard laboratory chow diet (3.9 kcal/g; 10% fat, 74% carbohydrate and 16% protein by calories) throughout the study. Food and water intake was measured every 48 h throughout the entire course of this study. A weighed amount of food was given at the beginning of the dark cycle, and the remaining food and spillage collected and weighed at the end of each 48-h period. Animals were randomized, with 12 rats administered olanzapine (Eli Lilly, USA), and the remaining 12 rats were given vehicle. After 36 days of drug treatment, six rats from each group were sacrificed 2 h after the last drug administration to examine the effects of olanzapine treatment on 5-HT2A and 5-HT2C receptors mRNA expression. The other six rats from each group
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were sacrificed 48 h after the last drug administration to examine 5-HT2A and 5-HT2C receptors mRNA expression in response to 48 h of drug withdrawal. Olanzapine (in a small sweet pellet) was administered orally according to the method described previously [13]. All animals were administered with a pellet without drug for 1 week prior to the start of the studies. Based on previous studies, the dosage was 1.2 mg/kg, which is the ED50 dosage needed to reduce amphetamine-induced hyperactivity [2,20]. Rats were sacrificed with an overdose of sodium pentobarbitone anesthesia (120 mg/kg, i.p.) after a total of 36 days of drug treatment. All rats were sacrificed between 07:00 and 09:00 h, in order to minimize the variation of circadian mRNA expression. Brains were immediately removed after death and frozen in liquid nitrogen. Coronal brain sections (14 m) were cut at −17 ◦ C with a cryostat, and thaw-mounted onto baked microscope slides. Consecutive sections were taken and used for the detection of the mRNA expression. One set of sections was used for determining the probe specificity. Brain sections were immediately fixed in ice-cold 4% phosphate-buffered 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 in −70 ◦ C until use. Identification of neuroanatomical structures was according to a standard rat brain atlas [23]. The details of in situ hybridization methods have been described in our previous work [9]. In brief, the specific antisense hybridization probes for 5-HT2A receptor were 5 -atg gac acg ggc atg aca agg aaa ccc agc agc ata tca gct atg gca a-3 and 5 -tcc agc gag atg gcg cag agg tgc atg atg gag gcc gtg gag aag3 , and for 5-HT2C receptor were 5 -gcc tct ggg acg ctt ttc ttt
ctt ctt tcg acg tgg ttt ctg atc tgg-3 and 5 -aat cct ctc gct gac cac att aga ggg gtt gac tgg cag ctc taa gtt-3 . No sequences bearing significant homology to the designed probes were found in the GeneBank (NCBA). All oligonucleotide probes were terminally labeled using a 10-fold molar excess of [35 S] 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). Quantification of mRNA expression levels in various brain regions was performed by measuring the average density of each region in three adjacent brain sections. mRNA expression data for each brain area were analyzed by a Kruskal–Wallis test, followed by a Mann–Whitney U-test using the SPSS 11.5 program (Chicago, IL). 1. Energy Intake A two-way repeated ANOVA revealed significant main effects of treatment (F1,22 = 17.12, p < 0.001) and repeated measurement with days (F18,396 = 11.22, p < 0.001), and a significant interaction between the treatment and repeated measurement (F18,396 = 5.46, p < 0.001) on food intake. During the course of the drug treatment, olanzapine treatment increased food intake compared to the control group on a majority of the days (on days 2–12, 2.92 < t < 7.10, d.f. = 22, 0.008 > p > 0.001; days 20–24, 2.72 < t < 3.29, 0.12 > p > 0.003; day 32, t = 2.46, p = 0.022; Fig. 1A). Although there was no significant difference in energy intake on some of the days (days14–18, 26–30 and 34–36; t < 2.0, d.f. = 22, p > 0.05), the total amount of energy intake was significantly higher in the olanzapine treated rats than in the control group (827 ± 17 kcal/rat/36 days
Fig. 1. Body weight gain, food intake and water intake in every 48 h (A–C) and total (D–F) over 36 days of olanzapine treatment. *p < 0.05 vs. vehicle group.
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versus 731 ± 13 kcal/rat/36 days, t = 4.73, d.f. = 22, p = 0.000, Fig. 1D).
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Table 1 Body weight, fat deposits and soleus muscle mass of rats treated with olanzapine Olanzapine (n = 12)
2. Water Intake A two-way repeated ANOVA only revealed a significant main effect of repeated measurement on days (F18,396 = 9.046, p = 0.000; Fig. 1B). There was no main effect of treatment (F1,22 = 1.16, p = 0.30) and also no significant interaction between the treatment and repeated measurement (F18,396 = 1.71, p = 0.14). In fact, the total amount of water intake over 36 days of treatment was not significantly different between the olanzapine treated group (948 ± 65 ml/rat/36 days) and control group (878 ± 40 ml/rat/36 days; t = 0.92, d.f. = 22, p = 0.37; Fig. 1E). 3. Body weight gain and fat deposits A two-way repeated ANOVA revealed a significant main effect of treatment (F1,22 = 30.71, p = 0.000) and repeated measurement with days (F18,374 = 102.83, p = 0.000) on accumulated weight gain. There was no significant interaction between the treatment and repeated measurement (F18,374 = 2.49, p = 0.06). Although a consistent increase in body weight was observed in both groups (Fig. 1C), the body weight gain was 59% higher
Vehicle (n = 12)
Body weight (g) IBW FBW Weight gain
260 ± 3 305 ± 5 46 ± 3*
267 ± 3 295 ± 4 29 ± 3
Fat-pad masses (g) Periovary Perirenal Inguinal
4.54 ± 0.29 6.22 ± 0.37* 4.12 ± 0.24
4.02 ± 0.23 4.95 ± 0.40 3.91 ± 0.28
Total
14.89 ± 0.58*
12.88 ± 0.76
Muscle (g) Soleus
0.26 ± 0.01
0.27 ± 0.01
Others Total fat/IBW (%) Total fat/FBW (%)
5.73 ± 0.20* 4.87 ± 0.16
4.83 ± 0.27 4.34 ± 0.22
Data are mean ± S.E.M. * p < 0.05, IBW: initial body weight; FBW: final body weight.
in the chronic olanzapine-treated group than the control group (Table 1, Fig. 1F). The rats treated with olanzapine also had 40.5% higher feed efficiency (the ratio of accumulate body weight gain/the total food intake) than controls (olanzapine group 5.38 ± 0.22% versus the vehicle group 3.83 ± 0.42%,
Fig. 2. Photographs depict the mRNA expression of 5-HT2A (A –C ) and 5-HT2C (A –C ) receptors in the rat brain. Low-magnification film autoradiographs show the location of 5-HT2A and 5-HT2C receptors mRNA expression. The maps of A–C are adopted from a rat brain atlas [23] indicating where the levels of the mRNA expression were measured. Acb: accumbens nucleus, AHiPM: posteromedial part of the amygdalohippocampal area, AI: agranular insular cortex, APTD: dorsal part of the anterior pretectal nucleus, Arc: arcuate hypothalamic nucleus, Cg: cingulate cortex, CL: centrolateral thalamic nucleus, Cl: claustrum, CPu: Caudate putamen, IMD: intermediodorsal thalamic nucleus, LD: laterodorsal thalamic nucleus, LS: lateral septal nucleus, LSI: lateral septal nucleus, intermediate part, M1: primary motor cortex, MePD: posterodorsal part of medial amygdaloid nucleus, Pir: piriform cortex, PMCo: posteromedial cortical amygdaloid nucleus, RSG: retrosplenial granular cortex, S1: primary sensory cortex, SC: superior colliculus, SN: substantia nigra, Tu: olfactory tubercle nucleus, VMH: ventromedial hypothalamic nuleus, VTA: ventral tegmental area, ZI: zona incerta.
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t = 3.24, d.f. = 22, p = 0.004). A significant correlation between the total body weight gain and the total food intake was observed in the olanzapine treatment group (r = 0.79, p = 0.003, Pearson correlation) but not in the control group (r = 0.45, p = 0.15). The total fat accumulation (the sum of periovary, perirenal and inguinal fat masses) was significantly higher in the olanzapine group than the control group (+16%, p < 0.05). Among the different types of fat deposits, olanzapine appeared mainly to increase perirenal fat mass with the least affect on subcutaneous (inguinal) fat (Table 1). The ratio of total fat mass to initial body weight was significantly higher in the olanzapine group compared to the control group (+19%, p < 0.05; Table 1). Body fatness was also calculated as the ratio of total fat divided by final body weight, and was 12% higher in the olanzapine group than the control group (p = 0.059). Furthermore, total fat mass was significant correlated with final body weight (r = 0.74, p = 0.000, Pearson correlation) but not initial body weight (r = 0.20, p = 0.35).
4. Expression of 5-HT2A and 5-HT2C receptor mRNA Olanzapine appeared to have a very significant influence on the levels of 5-HT2A receptor mRNA expression. Interestingly, the levels of 5-HT2A receptor mRNA expression showed an opposing change between 2 and 48 h of the last olanzapine administration (Fig. 2; Table 2). Two hours after the last olanzapine treatment, the levels of 5HT2A receptor mRNA expression were significantly decreased in 13 of the 22 regions examined throughout the brain compared to the control group (Table 2). The regions showing a significant decrease were primarily the hypothalamus, limbic system and striatum (caudate putamen, CPu). In the hypothalamus, the rats treated with olanzapine had significantly decreased 5-HT2A receptor mRNA expression in the arcuate (Arc), dorsomedial hypothalamic nucleus (DMH) and ventromedial hypothalamic nucleus (VMH). In the limbic system, olanzapine significantly decreased 5-HT2A receptor mRNA in the nucleus accumbens (Acb), lateral septal nucleus (LS), amygdaloid nucleus (Amg)
Table 2 Effects of olanzapine treatment on the levels of 5-HT2A receptor mRNA expression in the brain regions shown Vehicle (mean ± S.E.M.)
Olanzapine 2 h (mean ± S.E.M.)
Bregmaa (mm)
48 h (mean ± S.E.M.)
Hypothalamus Arc DMH LH VMH
5.8 5.8 9.5 15.7
± ± ± ±
1.8b 1.0b 3.8c 2.4b
32.5 39.4 28.3 43.5
± ± ± ±
7.4 10.0 6.9 4.0c
23.5 24.5 21.4 28.4
± ± ± ±
3.1 2.3 2.8 2.3
−3.14 −3.14 −3.14 −3.14
Limbic system Acb Amg Hip LS MePD PMCo
10.7 7.2 16.5 12.8 29.2 13.3
± ± ± ± ± ±
2.2c 1.1b 5.8 2.3b 4.1 5.4
44.4 35.2 40.6 53.1 69.3 26.0
± ± ± ± ± ±
10.0 6.8 9.7 8.7b 9.5b 10.1
22.1 22.9 25.4 27.0 38.7 22.5
± ± ± ± ± ±
2.1 2.8 2.7 1.0 2.8 1.7
0.70 −3.14 −3.14 0.70 −3.14 −5.30
22.6 ± 0.8
0.70
Striatum CPu
9.9 ± 2.1b
Cortex AI Cg Pir RSG
15.3 27.4 52.5 12.4
Brainstem SC SN VTA
20.9 ± 3.8 13.1 ± 4.8 21.3 ± 4.6
Other areas ChP TC Tu VDB
95.4 4.3 25.9 13.6
± ± ± ±
± ± ± ±
2.9c 6.8c 3.0 4.1c
9.3 1.3b 3.6 3.2b
40.1 ± 11.4 57.3 91.6 74.5 34.0
± ± ± ±
7.7b 11.6b 4.6c 6.3b
55.7 ± 5.0b 43.4 ± 9.2c 57.2 ± 5.3b 167.8 37.2 52.9 54.5
± ± ± ±
11.6b 8.9 8.4b 15.5
25.8 40.6 57.0 26.3
± ± ± ±
2.2 2.9 4.0 2.3
0.70 0.70 0.70 −3.14
29.0 ± 2.2 22.4 ± 1.4 31.7 ± 1.9
−5.30 −5.30 −5.30
± ± ± ±
0.70 −3.14 0.70 0.70
93.1 20.2 32.9 25.6
6.6 2.7 2.4 2.1
Note: Units of measurement are nCi/g tissue. Abbreviations—Amg: amygdaloid nucleus, ChP: choroids plexus, DMH: dorsomedial hypothalamic nucleus, Hip: hippocampus, LH: lateral hypothalamic area, TC: tuber cinereum area, VDB: vertical limb of the diagonal band. For other abbreviations see Fig. 2. a The location of the area was analyzed (Paxinos and Watson, 1997). b p < 0.01 vs. vehicle group. c p < 0.05 vs. vehicle group.
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and retrosplenial granular cortex (RSG). Furthermore, olanzapine treatment decreased the levels of 5-HT2A receptor mRNA expression in the agranular insular cortex (AI), tuber cinereum area (TC) and vertical limb of diagonal band (VDB). Forty-eight hours after the last olanzapine treatment, the levels of 5-HT2A receptor mRNA expression were significantly increased in most areas compared to the control group (Table 2). In the hypothalamus, the levels of 5-HT2A receptor mRNA were significantly increased in the VMH. In the limbic system, 5-HT2A receptor mRNA expression levels were significantly increased in the LS, cingulate cortex (Cg) and posterodorsal part of the medial amygdaloid nucleus (MePD). Furthermore, the levels of 5-HT2A receptor mRNA were significantly increased in the ventral tegmental area (VTA), AI, superior colliluculus nucleus (SC), substantial nigra (SN), olfactory tubercle nucleus (Tu) and choroids plexus (ChP). Expression of 5-HT2C receptor mRNA in the rat brain was more widespread than 5-HT2A receptor mRNA expression (Fig. 2; Table 3). However, the alterations induced by olanzapine were more limited. The most pronounced and significant
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changes in the level of 5-HT2C receptor mRNA expression caused by olanzapine treatment were in SN at 2 and 48 h after the last administration. Furthermore, olanzapine treatment significantly reduced 5-HT2C receptor mRNA expression in the posteromedial cortical amygdaloid nucleus (PMCo) while there was an increase in the Tu at 2 h after the last olanzapine treatment. There was also reduced 5-HT2C receptor mRNA expression in the posteromedial part of amygdalohippocampal area (AHiPM), and a slight but significant increase in 5-HT2C receptor mRNA expression in the ChP at 48 h after the last administration. Total food intake was negatively correlated with 5-HT2A receptor mRNA expression levels at 2 h after the last olanzapine administration in the CPu and VTA (Table 4). Total food intake was negatively correlated with levels of 5-HT2C receptor mRNA expression in the SN. Body weight gain was negatively correlated with levels of 5-HT2A receptor mRNA expression in the Arc, DMH, VMH and LS. The body weight gain was negatively correlated with 5-HT2C receptor mRNA expression levels in the SN. Energy efficiency was negatively correlated with levels of 5-HT2A receptor mRNA expression in the Arc,
Table 3 Effects of olanzapine treatment on the levels of 5-HT2C receptor mRNA expression in the brain regions shown Vehicle (mean ± S.E.M.)
Olanzapine 2 h (mean ± S.E.M.)
Bregmaa (mm)
48 h (mean ± S.E.M.)
Hypothalamus Arc LH VMH AHiPM
37.4 18.1 45.3 137.5
± ± ± ±
8.1 2.7 8.8 16.6
26.1 28.2 39.2 83.1
± ± ± ±
5.0 3.6 4.5 5.5b
29.9 26.6 48.3 120.2
± ± ± ±
2.1 3.3 3.2 5.0
−3.14 −3.14 −3.14 −5.30
Limbic system Acb CeL CeM LS MePD PMCo
52.8 125.8 43.6 73.9 184.0 29.8
± ± ± ± ± ±
5.0 9.8 2.2 6.7 12.0 2.6c
50.1 121.6 50.4 69.8 184.2 28.5
± ± ± ± ± ±
9.8 10.1 5.1 6.0 16.8 4.3c
46.8 114.2 44.0 62.8 169.5 44.0
± ± ± ± ± ±
2.6 6.4 3.4 3.9 11.8 3.0
0.70 −3.14 −3.14 0.70 −3.14 −5.30
Striatum CPu Cortex AI Cg Pir RSG Brainstem SC SN VTA Other areas ChP TC Tu VDB
37.0 ± 3.3 25.6 28.9 105.4 61.2
± ± ± ±
3.3 2.0 10.5 8.4
44.2 ± 4.5 14.5 ± 0.9c 49.4 ± 5.5 345.5 65.0 106.7 50.1
± ± ± ±
31.7 10.0 6.6b 5.1
36.2 ± 3.4 28.1 32.6 85.4 54.6
± ± ± ±
3.1 3.7 3.8 4.1
41.0 ± 5.3 13.6 ± 1.8b 39.9 ± 5.2 346.9 63.0 75.0 47.4
± ± ± ±
19.1b 7.5 6.2 5.1
31.5 ± 1.4 24.9 25.1 93.7 60.5
± ± ± ±
1.2 1.4 3.7 3.83
0.70 0.70 0.70 0.70 −3.14
54.1 ± 1.8 21.2 ± 1.6 49.8 ± 2.9
−5.30 −5.30 −5.30
± ± ± ±
0.70 −2.30 0.70 0.70
310.7 67.3 86.8 52.1
12.9 3.5 5.0 1.9
Note: Units of measurement are nCi/g tissue. Abbreviations—CeL: lateral division of central amygdaloid nucleus, CeM: medial division of the central amygdaloid nucleus. For other abbreviations see Fig. 2 and Table 2. a The location of the area was analyzed (Paxinos and Watson, 1997). b p < 0.05 vs. vehicle group. c p < 0.01 vs. vehicle group.
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Table 4 The relationship between the total food intake, body weight gain and energy efficiency, and the levels of 5-HT2A receptor mRNA expression at 2 h after olanzapine administration Food intake
Body weight gain
Energy efficiency
Correlation
p value
Correlation
p value
Correlation
p value
−0.286 −0.627 −0.368 −0.433 −0.372 −0.574
0.321 0.022 0.230 0.122 0.172 0.032
−0.642 −0.562 −0.608 −0.599 −0.581 −0.324
0.013 0.046 0.027 0.024 0.023 0.259
−0.696 −0.424 −0.606 −0.459 −0.614 −0.054
0.001 0.149 0.028 0.099 0.015 0.855
5-HT2C receptor mRNA SN −0.672
0.006
−0.555
0.032
−0.355
0.194
5-HT2A mRNA Arc CPu DMH LS VMH VTA
CPu, DMH and VMH. No significant correlation was found between the energy efficiency, water intake and 5-HT2C receptor mRNA expression levels in any brain area examined (data not shown). This study found a down-regulation of 5-HT2A receptor mRNA expression at 2 h after last olanzapine administration, predominantly in the hypothalamus, limbic system and striatum. This confirms previous findings that, different from other Gprotein-coupled receptors, chronic blockade of 5-HT2A receptors by its antagonist leads not to an up- but an atypical downregulation [30]. The hypothalamus plays an important role in the regulation of both energy intake and expenditure. This study showed that the levels of 5-HT2A receptor mRNA expression were selectively decreased in the Arc, VMH and DMH at 2 h after last olanzapine administration. It is known that these three nuclei are critically involved in the regulation of energy balance [10]. This study found that the levels of 5-HT2A receptor mRNA expression were negatively associated with body weight gain and energy efficiency, but not with total energy intake and 24 h energy intake just before rats were killed in the Arc, VMH and DMH (Table 4). Increased energy efficiency was therefore a primary contributor to weight gain with prolonged olanzapine treatment. This may be due to reduced energy expenditure or a malfunction of the autonomic nervous system. A significant elevation of 5-HT2A receptor mRNA expression in the VMH, a classic satiety center, was seen 48 h after olanzapine treatment. The phenomenon may represent a rebound response in the receptor production at the gene transcription level, which suggested that chronic treatment of olanzapine did not cause permanent alteration of 5HT2A receptor mRNA expression. This might assist in the body weight reduction, as the phenomenon was observed in both humans and rodents after cessation of olanzapine treatment [2,25]. One of the main differences between atypical and typical antipsychotics is their ‘limbic-specific’ high ratio of 5-HT2 /D2 receptor binding [33]. It has been reported that the Acb, LS and Amg are common targets for antipsychotic actions [5,6,24]. Acb is a key structure in the mesolimbic dopamine system [32]. This study found a significant reduction in 5-HT2A receptor mRNA expression 2 h after the last olanzapine treatment.
While it is known that Acb forms part of circuits modulating feeding via its connections with a classic hunger center, the lateral hypothalamus [27], no significant correlation was found between the levels of 5-HT2A receptor mRNA expression and food intake, body weight gain or energy efficiency in this area. However, studies have shown that olanzapine induces the release of, and increases the level of dopamine concentration in the Acb, which is associated with reward or anti-depression effects [3,19]. LS receives direct projections from the amygdala and hypothalamus [17]. Although the involvement of the LS in energy balance regulation is not clear, it is known that it contains a number of neurotransmitters (such as agouti-related protein and neuropeptide Y-like immunoreactivity [7,16]) and receptors (such as the neuropeptide Y receptor subtype 2, agoutirelated protein and D4 mRNAs [8,11,16]) known to be critically involved in the regulation of energy balance and the development of obesity. The present study found that the levels of 5-HT2A receptor mRNA expression were associated with total food intake and body weight gain. The question of whether an interaction occurs between 5-HT2A receptors, melanocortin, neuropeptide Y and serotonin systems in the LS, and their relationship with the olanzapine treatment, will be an important area for further study. This study found that striatal 5-HT2A receptor mRNA expression was significantly down-regulated at 2 h after olanzapine treatment, and negatively correlated with food intake and body weight gain. The striatum is under regulatory control of the monoamines, dopamine and serotonin, and there is an interaction between the two systems, for example, olanzapine increases striatal dopamine release [19]. While the involvement of striatal dopamine D2 receptors have been reported in body weight regulation in humans and rodents [12,31], little information is available with respect to the involvement of striatal 5-HT2A receptor in body weight control. The present study confirms previous evidence that the 5HT2C receptor and its encoding mRNA are widespread in rodent brain. A consistent finding from this study was decreased 5HT2C receptor mRNA expression in the SN at 2 and 48 h after olanzapine treatment. The implications of this inhibition
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effect in the SN are presently unknown, but could be associated with the lower propensity to induced motor side effects, as there are extensive reciprocal connections between the SN and striatum. Furthermore, it has been reported that olanzapine, but not haloperidol, significantly inhibits the firing rate of the SN neurons [29]. While it has been reported that olanzapine decreases 5-HT2C receptor binding in hippocampus and striatum in rats [28], we did not find significant changes in these regions. In summary, the present study has shown for the first time the pattern of 5-HT2A and 5-HT2C receptors mRNA expression in the brain of rats treated with olanzapine. Olanzapine decreased the levels of 5-HT2A receptor mRNA expression in the striatum as well as hypothalamic and limbic nuclei relevant to the regulation of energy balance, reward, anxiety and motor function. A rebound effect at the gene transcription level of 5-HT2A receptors was observed 48 h after prolonged olanzapine treatment, which might contribute to weight reduction after cessation of olanzapine treatment. 5-HT2C receptor mRNA expression was mostly unaffected by olanzapine treatment. Finally, the olanzapine-induced weight gain may be due not only to high energy intake, but also to increased energy efficiency, particularly in the maintenance of body weight gain after chronic olanzapine treatment. Acknowledgments We wish to thank A/Prof. K. Russell (Department of Applied Statistics, University of Wollongong) for his suggestions regarding the statistical analysis. This study was supported by grants from the Research Committee of the University of Wollongong and the Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD), utilizing infrastructure funding from NSW Health. References [1] Allison DB, Casey DE. Antipsychotic-induced weight gain: a review of the literature. J Clin Psychiatry 2001;62(Suppl. 7):22–31. [2] Arjona AA, Zhang SX, Adamson B, Wurtman RJ. An animal model of antipsychotic-induced weight gain. Behav Brain Res 2004;152: 121–7. [3] Bymaster F, Perry KW, Nelson DL, Wong DT, Rasmussen K, Moore NA, Calligaro DO. Olanzapine: a basic science update. Br J Psychiatry Suppl 1999:36–40. [4] Bymaster FP, Calligaro DO, Falcone JF, Marsh RD, Moore NA, Tye NC, Seeman P, Wong DT. Radioreceptor binding profile of the atypical antipsychotic olanzapine. Neuropsychopharmacology 1996;14: 87–96. [5] Cohen BM, Cherkerzian S, Ma J, Ye N, Wager C, Lange N. Cells in midline thalamus, central amygdala, and nucleus accumbens responding specifically to antipsychotic drugs. Psychopharmacology 2003;167:403–10. [6] Deutch AY, Lee MC, Iadarola MJ. Regionally specific effects of atypical antipsychotic drugs on striatal Fos expression: the nucleus accumbens shell as a locus of antipsychotic action. Mol Cell Neurosci 1992;3:332–41. [7] Haskell-Luevano C, Chen P, Li C, Chang K, Smith MS, Cameron JL, Cone RD. Characterization of the neuroanatomical distribution of agouti-related protein immunoreactivity in the rhesus monkey and the rat. Endocrinology 1999;140:1408–15.
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Short communication
Olanzapine differentially affects 5-HT2A and 2C receptor mRNA expression in the rat brain Xu-Feng Huang a,b , Mei Han a,b , Xin Huang a , Katerina Zavitsanou a,b,c , Chao Deng a,b,∗ a
Neurobiology Research Centre of Metabolic and Psychiatric Disorders, School of Health Science, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia b Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD), NSW, Australia c Australian Nuclear Science & Technology Organization (ANSTO), NSW, Australia Received 7 February 2006; received in revised form 28 March 2006; accepted 31 March 2006 Available online 22 May 2006
Abstract This study examined regional changes in rat brain mRNA levels encoding 5-HT2A and 5-HT2C receptors following chronic olanzapine treatment. The immediate effect (2 h after the last treatment) was a down-regulation of 5-HT2A receptor mRNA expression, predominantly in the hypothalamus, limbic system and striatum, while a rebound effect was observed 48 h later. 5-HT2C receptor mRNA expressions were decreased in the substantia nigra. Correlations between 5-HT2A receptor mRNA expression and total food intake, weight gain and energy efficiency were observed. © 2006 Elsevier B.V. All rights reserved. Keywords: Antipsychotics; Gene expression; Serotonin receptors; Body weight
Olanzapine is an atypical antipsychotic drug that has proven efficacy in ameliorating both positive and negative symptoms of schizophrenia [21]. Olanzapine, like other atypical antipsychotics, is also less likely to cause extrapyramidal side effects while a common side effect is significant body weight gain [1]. Olanzapine is a potent 5-HT2 receptor antagonist with high affinities to both 5-HT2A (Ki = 4.0 nM) and 5HT2C (Ki = 11 nM) receptor subtypes [4,14,28]. It also possesses antagonistic effects to histaminergic and dopaminergic systems [1,4,26]. In humans, PET studies show that 5-HT2 receptor occupancy is about 75% in a usual clinical dosage range after oral administration of olanzapine [15,22]. In rats, olanzapine occupancy at a dosage of 1 mg/kg is about 70 and 30% for 5-HT2A and D2 receptors, respectively, while it is around 76 and 41% at 2 mg/kg [18]. It was also noted that measurement of 5-HT2A receptor binding densities in response to olanzapine treatment was almost exclusively limited to the frontal cortex [30]. No data are available examining in situ regional changes of 5-HT2A and 5-HT2C receptors mRNA expression after olanzapine treatment in brain areas. Using a chronic olanzapine treated rat model,
∗
Corresponding author. Tel.: +61 2 42214934; fax: +61 2 42214096. E-mail address: [email protected] (C. Deng).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.03.040
this study aimed to test the following hypothesis: (1) the mRNA expression of 5-HT2A and 5-HT2C receptors respond differently to olanzapine treatment, across a wide range of brain structures; (2) there is a relationship between changes in gene expression in specific areas of the brain and energy intake/weight gain/energy efficiency. Twenty-four female Sprague–Dawley rats weighing ∼250 g were obtained from the Animal Resource Center (Perth, WA, Australia). They were housed individually in environmentally controlled conditions (temperature 22 ◦ C, light cycle from 06:00 to 18:00 h and dark cycle from 18:00 to 06:00 h), and allowed ad libitum access to water and standard laboratory chow diet (3.9 kcal/g; 10% fat, 74% carbohydrate and 16% protein by calories) throughout the study. Food and water intake was measured every 48 h throughout the entire course of this study. A weighed amount of food was given at the beginning of the dark cycle, and the remaining food and spillage collected and weighed at the end of each 48-h period. Animals were randomized, with 12 rats administered olanzapine (Eli Lilly, USA), and the remaining 12 rats were given vehicle. After 36 days of drug treatment, six rats from each group were sacrificed 2 h after the last drug administration to examine the effects of olanzapine treatment on 5-HT2A and 5-HT2C receptors mRNA expression. The other six rats from each group
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were sacrificed 48 h after the last drug administration to examine 5-HT2A and 5-HT2C receptors mRNA expression in response to 48 h of drug withdrawal. Olanzapine (in a small sweet pellet) was administered orally according to the method described previously [13]. All animals were administered with a pellet without drug for 1 week prior to the start of the studies. Based on previous studies, the dosage was 1.2 mg/kg, which is the ED50 dosage needed to reduce amphetamine-induced hyperactivity [2,20]. Rats were sacrificed with an overdose of sodium pentobarbitone anesthesia (120 mg/kg, i.p.) after a total of 36 days of drug treatment. All rats were sacrificed between 07:00 and 09:00 h, in order to minimize the variation of circadian mRNA expression. Brains were immediately removed after death and frozen in liquid nitrogen. Coronal brain sections (14 m) were cut at −17 ◦ C with a cryostat, and thaw-mounted onto baked microscope slides. Consecutive sections were taken and used for the detection of the mRNA expression. One set of sections was used for determining the probe specificity. Brain sections were immediately fixed in ice-cold 4% phosphate-buffered 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 in −70 ◦ C until use. Identification of neuroanatomical structures was according to a standard rat brain atlas [23]. The details of in situ hybridization methods have been described in our previous work [9]. In brief, the specific antisense hybridization probes for 5-HT2A receptor were 5 -atg gac acg ggc atg aca agg aaa ccc agc agc ata tca gct atg gca a-3 and 5 -tcc agc gag atg gcg cag agg tgc atg atg gag gcc gtg gag aag3 , and for 5-HT2C receptor were 5 -gcc tct ggg acg ctt ttc ttt
ctt ctt tcg acg tgg ttt ctg atc tgg-3 and 5 -aat cct ctc gct gac cac att aga ggg gtt gac tgg cag ctc taa gtt-3 . No sequences bearing significant homology to the designed probes were found in the GeneBank (NCBA). All oligonucleotide probes were terminally labeled using a 10-fold molar excess of [35 S] 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). Quantification of mRNA expression levels in various brain regions was performed by measuring the average density of each region in three adjacent brain sections. mRNA expression data for each brain area were analyzed by a Kruskal–Wallis test, followed by a Mann–Whitney U-test using the SPSS 11.5 program (Chicago, IL). 1. Energy Intake A two-way repeated ANOVA revealed significant main effects of treatment (F1,22 = 17.12, p < 0.001) and repeated measurement with days (F18,396 = 11.22, p < 0.001), and a significant interaction between the treatment and repeated measurement (F18,396 = 5.46, p < 0.001) on food intake. During the course of the drug treatment, olanzapine treatment increased food intake compared to the control group on a majority of the days (on days 2–12, 2.92 < t < 7.10, d.f. = 22, 0.008 > p > 0.001; days 20–24, 2.72 < t < 3.29, 0.12 > p > 0.003; day 32, t = 2.46, p = 0.022; Fig. 1A). Although there was no significant difference in energy intake on some of the days (days14–18, 26–30 and 34–36; t < 2.0, d.f. = 22, p > 0.05), the total amount of energy intake was significantly higher in the olanzapine treated rats than in the control group (827 ± 17 kcal/rat/36 days
Fig. 1. Body weight gain, food intake and water intake in every 48 h (A–C) and total (D–F) over 36 days of olanzapine treatment. *p < 0.05 vs. vehicle group.
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versus 731 ± 13 kcal/rat/36 days, t = 4.73, d.f. = 22, p = 0.000, Fig. 1D).
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Table 1 Body weight, fat deposits and soleus muscle mass of rats treated with olanzapine Olanzapine (n = 12)
2. Water Intake A two-way repeated ANOVA only revealed a significant main effect of repeated measurement on days (F18,396 = 9.046, p = 0.000; Fig. 1B). There was no main effect of treatment (F1,22 = 1.16, p = 0.30) and also no significant interaction between the treatment and repeated measurement (F18,396 = 1.71, p = 0.14). In fact, the total amount of water intake over 36 days of treatment was not significantly different between the olanzapine treated group (948 ± 65 ml/rat/36 days) and control group (878 ± 40 ml/rat/36 days; t = 0.92, d.f. = 22, p = 0.37; Fig. 1E). 3. Body weight gain and fat deposits A two-way repeated ANOVA revealed a significant main effect of treatment (F1,22 = 30.71, p = 0.000) and repeated measurement with days (F18,374 = 102.83, p = 0.000) on accumulated weight gain. There was no significant interaction between the treatment and repeated measurement (F18,374 = 2.49, p = 0.06). Although a consistent increase in body weight was observed in both groups (Fig. 1C), the body weight gain was 59% higher
Vehicle (n = 12)
Body weight (g) IBW FBW Weight gain
260 ± 3 305 ± 5 46 ± 3*
267 ± 3 295 ± 4 29 ± 3
Fat-pad masses (g) Periovary Perirenal Inguinal
4.54 ± 0.29 6.22 ± 0.37* 4.12 ± 0.24
4.02 ± 0.23 4.95 ± 0.40 3.91 ± 0.28
Total
14.89 ± 0.58*
12.88 ± 0.76
Muscle (g) Soleus
0.26 ± 0.01
0.27 ± 0.01
Others Total fat/IBW (%) Total fat/FBW (%)
5.73 ± 0.20* 4.87 ± 0.16
4.83 ± 0.27 4.34 ± 0.22
Data are mean ± S.E.M. * p < 0.05, IBW: initial body weight; FBW: final body weight.
in the chronic olanzapine-treated group than the control group (Table 1, Fig. 1F). The rats treated with olanzapine also had 40.5% higher feed efficiency (the ratio of accumulate body weight gain/the total food intake) than controls (olanzapine group 5.38 ± 0.22% versus the vehicle group 3.83 ± 0.42%,
Fig. 2. Photographs depict the mRNA expression of 5-HT2A (A –C ) and 5-HT2C (A –C ) receptors in the rat brain. Low-magnification film autoradiographs show the location of 5-HT2A and 5-HT2C receptors mRNA expression. The maps of A–C are adopted from a rat brain atlas [23] indicating where the levels of the mRNA expression were measured. Acb: accumbens nucleus, AHiPM: posteromedial part of the amygdalohippocampal area, AI: agranular insular cortex, APTD: dorsal part of the anterior pretectal nucleus, Arc: arcuate hypothalamic nucleus, Cg: cingulate cortex, CL: centrolateral thalamic nucleus, Cl: claustrum, CPu: Caudate putamen, IMD: intermediodorsal thalamic nucleus, LD: laterodorsal thalamic nucleus, LS: lateral septal nucleus, LSI: lateral septal nucleus, intermediate part, M1: primary motor cortex, MePD: posterodorsal part of medial amygdaloid nucleus, Pir: piriform cortex, PMCo: posteromedial cortical amygdaloid nucleus, RSG: retrosplenial granular cortex, S1: primary sensory cortex, SC: superior colliculus, SN: substantia nigra, Tu: olfactory tubercle nucleus, VMH: ventromedial hypothalamic nuleus, VTA: ventral tegmental area, ZI: zona incerta.
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t = 3.24, d.f. = 22, p = 0.004). A significant correlation between the total body weight gain and the total food intake was observed in the olanzapine treatment group (r = 0.79, p = 0.003, Pearson correlation) but not in the control group (r = 0.45, p = 0.15). The total fat accumulation (the sum of periovary, perirenal and inguinal fat masses) was significantly higher in the olanzapine group than the control group (+16%, p < 0.05). Among the different types of fat deposits, olanzapine appeared mainly to increase perirenal fat mass with the least affect on subcutaneous (inguinal) fat (Table 1). The ratio of total fat mass to initial body weight was significantly higher in the olanzapine group compared to the control group (+19%, p < 0.05; Table 1). Body fatness was also calculated as the ratio of total fat divided by final body weight, and was 12% higher in the olanzapine group than the control group (p = 0.059). Furthermore, total fat mass was significant correlated with final body weight (r = 0.74, p = 0.000, Pearson correlation) but not initial body weight (r = 0.20, p = 0.35).
4. Expression of 5-HT2A and 5-HT2C receptor mRNA Olanzapine appeared to have a very significant influence on the levels of 5-HT2A receptor mRNA expression. Interestingly, the levels of 5-HT2A receptor mRNA expression showed an opposing change between 2 and 48 h of the last olanzapine administration (Fig. 2; Table 2). Two hours after the last olanzapine treatment, the levels of 5HT2A receptor mRNA expression were significantly decreased in 13 of the 22 regions examined throughout the brain compared to the control group (Table 2). The regions showing a significant decrease were primarily the hypothalamus, limbic system and striatum (caudate putamen, CPu). In the hypothalamus, the rats treated with olanzapine had significantly decreased 5-HT2A receptor mRNA expression in the arcuate (Arc), dorsomedial hypothalamic nucleus (DMH) and ventromedial hypothalamic nucleus (VMH). In the limbic system, olanzapine significantly decreased 5-HT2A receptor mRNA in the nucleus accumbens (Acb), lateral septal nucleus (LS), amygdaloid nucleus (Amg)
Table 2 Effects of olanzapine treatment on the levels of 5-HT2A receptor mRNA expression in the brain regions shown Vehicle (mean ± S.E.M.)
Olanzapine 2 h (mean ± S.E.M.)
Bregmaa (mm)
48 h (mean ± S.E.M.)
Hypothalamus Arc DMH LH VMH
5.8 5.8 9.5 15.7
± ± ± ±
1.8b 1.0b 3.8c 2.4b
32.5 39.4 28.3 43.5
± ± ± ±
7.4 10.0 6.9 4.0c
23.5 24.5 21.4 28.4
± ± ± ±
3.1 2.3 2.8 2.3
−3.14 −3.14 −3.14 −3.14
Limbic system Acb Amg Hip LS MePD PMCo
10.7 7.2 16.5 12.8 29.2 13.3
± ± ± ± ± ±
2.2c 1.1b 5.8 2.3b 4.1 5.4
44.4 35.2 40.6 53.1 69.3 26.0
± ± ± ± ± ±
10.0 6.8 9.7 8.7b 9.5b 10.1
22.1 22.9 25.4 27.0 38.7 22.5
± ± ± ± ± ±
2.1 2.8 2.7 1.0 2.8 1.7
0.70 −3.14 −3.14 0.70 −3.14 −5.30
22.6 ± 0.8
0.70
Striatum CPu
9.9 ± 2.1b
Cortex AI Cg Pir RSG
15.3 27.4 52.5 12.4
Brainstem SC SN VTA
20.9 ± 3.8 13.1 ± 4.8 21.3 ± 4.6
Other areas ChP TC Tu VDB
95.4 4.3 25.9 13.6
± ± ± ±
± ± ± ±
2.9c 6.8c 3.0 4.1c
9.3 1.3b 3.6 3.2b
40.1 ± 11.4 57.3 91.6 74.5 34.0
± ± ± ±
7.7b 11.6b 4.6c 6.3b
55.7 ± 5.0b 43.4 ± 9.2c 57.2 ± 5.3b 167.8 37.2 52.9 54.5
± ± ± ±
11.6b 8.9 8.4b 15.5
25.8 40.6 57.0 26.3
± ± ± ±
2.2 2.9 4.0 2.3
0.70 0.70 0.70 −3.14
29.0 ± 2.2 22.4 ± 1.4 31.7 ± 1.9
−5.30 −5.30 −5.30
± ± ± ±
0.70 −3.14 0.70 0.70
93.1 20.2 32.9 25.6
6.6 2.7 2.4 2.1
Note: Units of measurement are nCi/g tissue. Abbreviations—Amg: amygdaloid nucleus, ChP: choroids plexus, DMH: dorsomedial hypothalamic nucleus, Hip: hippocampus, LH: lateral hypothalamic area, TC: tuber cinereum area, VDB: vertical limb of the diagonal band. For other abbreviations see Fig. 2. a The location of the area was analyzed (Paxinos and Watson, 1997). b p < 0.01 vs. vehicle group. c p < 0.05 vs. vehicle group.
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and retrosplenial granular cortex (RSG). Furthermore, olanzapine treatment decreased the levels of 5-HT2A receptor mRNA expression in the agranular insular cortex (AI), tuber cinereum area (TC) and vertical limb of diagonal band (VDB). Forty-eight hours after the last olanzapine treatment, the levels of 5-HT2A receptor mRNA expression were significantly increased in most areas compared to the control group (Table 2). In the hypothalamus, the levels of 5-HT2A receptor mRNA were significantly increased in the VMH. In the limbic system, 5-HT2A receptor mRNA expression levels were significantly increased in the LS, cingulate cortex (Cg) and posterodorsal part of the medial amygdaloid nucleus (MePD). Furthermore, the levels of 5-HT2A receptor mRNA were significantly increased in the ventral tegmental area (VTA), AI, superior colliluculus nucleus (SC), substantial nigra (SN), olfactory tubercle nucleus (Tu) and choroids plexus (ChP). Expression of 5-HT2C receptor mRNA in the rat brain was more widespread than 5-HT2A receptor mRNA expression (Fig. 2; Table 3). However, the alterations induced by olanzapine were more limited. The most pronounced and significant
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changes in the level of 5-HT2C receptor mRNA expression caused by olanzapine treatment were in SN at 2 and 48 h after the last administration. Furthermore, olanzapine treatment significantly reduced 5-HT2C receptor mRNA expression in the posteromedial cortical amygdaloid nucleus (PMCo) while there was an increase in the Tu at 2 h after the last olanzapine treatment. There was also reduced 5-HT2C receptor mRNA expression in the posteromedial part of amygdalohippocampal area (AHiPM), and a slight but significant increase in 5-HT2C receptor mRNA expression in the ChP at 48 h after the last administration. Total food intake was negatively correlated with 5-HT2A receptor mRNA expression levels at 2 h after the last olanzapine administration in the CPu and VTA (Table 4). Total food intake was negatively correlated with levels of 5-HT2C receptor mRNA expression in the SN. Body weight gain was negatively correlated with levels of 5-HT2A receptor mRNA expression in the Arc, DMH, VMH and LS. The body weight gain was negatively correlated with 5-HT2C receptor mRNA expression levels in the SN. Energy efficiency was negatively correlated with levels of 5-HT2A receptor mRNA expression in the Arc,
Table 3 Effects of olanzapine treatment on the levels of 5-HT2C receptor mRNA expression in the brain regions shown Vehicle (mean ± S.E.M.)
Olanzapine 2 h (mean ± S.E.M.)
Bregmaa (mm)
48 h (mean ± S.E.M.)
Hypothalamus Arc LH VMH AHiPM
37.4 18.1 45.3 137.5
± ± ± ±
8.1 2.7 8.8 16.6
26.1 28.2 39.2 83.1
± ± ± ±
5.0 3.6 4.5 5.5b
29.9 26.6 48.3 120.2
± ± ± ±
2.1 3.3 3.2 5.0
−3.14 −3.14 −3.14 −5.30
Limbic system Acb CeL CeM LS MePD PMCo
52.8 125.8 43.6 73.9 184.0 29.8
± ± ± ± ± ±
5.0 9.8 2.2 6.7 12.0 2.6c
50.1 121.6 50.4 69.8 184.2 28.5
± ± ± ± ± ±
9.8 10.1 5.1 6.0 16.8 4.3c
46.8 114.2 44.0 62.8 169.5 44.0
± ± ± ± ± ±
2.6 6.4 3.4 3.9 11.8 3.0
0.70 −3.14 −3.14 0.70 −3.14 −5.30
Striatum CPu Cortex AI Cg Pir RSG Brainstem SC SN VTA Other areas ChP TC Tu VDB
37.0 ± 3.3 25.6 28.9 105.4 61.2
± ± ± ±
3.3 2.0 10.5 8.4
44.2 ± 4.5 14.5 ± 0.9c 49.4 ± 5.5 345.5 65.0 106.7 50.1
± ± ± ±
31.7 10.0 6.6b 5.1
36.2 ± 3.4 28.1 32.6 85.4 54.6
± ± ± ±
3.1 3.7 3.8 4.1
41.0 ± 5.3 13.6 ± 1.8b 39.9 ± 5.2 346.9 63.0 75.0 47.4
± ± ± ±
19.1b 7.5 6.2 5.1
31.5 ± 1.4 24.9 25.1 93.7 60.5
± ± ± ±
1.2 1.4 3.7 3.83
0.70 0.70 0.70 0.70 −3.14
54.1 ± 1.8 21.2 ± 1.6 49.8 ± 2.9
−5.30 −5.30 −5.30
± ± ± ±
0.70 −2.30 0.70 0.70
310.7 67.3 86.8 52.1
12.9 3.5 5.0 1.9
Note: Units of measurement are nCi/g tissue. Abbreviations—CeL: lateral division of central amygdaloid nucleus, CeM: medial division of the central amygdaloid nucleus. For other abbreviations see Fig. 2 and Table 2. a The location of the area was analyzed (Paxinos and Watson, 1997). b p < 0.05 vs. vehicle group. c p < 0.01 vs. vehicle group.
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Table 4 The relationship between the total food intake, body weight gain and energy efficiency, and the levels of 5-HT2A receptor mRNA expression at 2 h after olanzapine administration Food intake
Body weight gain
Energy efficiency
Correlation
p value
Correlation
p value
Correlation
p value
−0.286 −0.627 −0.368 −0.433 −0.372 −0.574
0.321 0.022 0.230 0.122 0.172 0.032
−0.642 −0.562 −0.608 −0.599 −0.581 −0.324
0.013 0.046 0.027 0.024 0.023 0.259
−0.696 −0.424 −0.606 −0.459 −0.614 −0.054
0.001 0.149 0.028 0.099 0.015 0.855
5-HT2C receptor mRNA SN −0.672
0.006
−0.555
0.032
−0.355
0.194
5-HT2A mRNA Arc CPu DMH LS VMH VTA
CPu, DMH and VMH. No significant correlation was found between the energy efficiency, water intake and 5-HT2C receptor mRNA expression levels in any brain area examined (data not shown). This study found a down-regulation of 5-HT2A receptor mRNA expression at 2 h after last olanzapine administration, predominantly in the hypothalamus, limbic system and striatum. This confirms previous findings that, different from other Gprotein-coupled receptors, chronic blockade of 5-HT2A receptors by its antagonist leads not to an up- but an atypical downregulation [30]. The hypothalamus plays an important role in the regulation of both energy intake and expenditure. This study showed that the levels of 5-HT2A receptor mRNA expression were selectively decreased in the Arc, VMH and DMH at 2 h after last olanzapine administration. It is known that these three nuclei are critically involved in the regulation of energy balance [10]. This study found that the levels of 5-HT2A receptor mRNA expression were negatively associated with body weight gain and energy efficiency, but not with total energy intake and 24 h energy intake just before rats were killed in the Arc, VMH and DMH (Table 4). Increased energy efficiency was therefore a primary contributor to weight gain with prolonged olanzapine treatment. This may be due to reduced energy expenditure or a malfunction of the autonomic nervous system. A significant elevation of 5-HT2A receptor mRNA expression in the VMH, a classic satiety center, was seen 48 h after olanzapine treatment. The phenomenon may represent a rebound response in the receptor production at the gene transcription level, which suggested that chronic treatment of olanzapine did not cause permanent alteration of 5HT2A receptor mRNA expression. This might assist in the body weight reduction, as the phenomenon was observed in both humans and rodents after cessation of olanzapine treatment [2,25]. One of the main differences between atypical and typical antipsychotics is their ‘limbic-specific’ high ratio of 5-HT2 /D2 receptor binding [33]. It has been reported that the Acb, LS and Amg are common targets for antipsychotic actions [5,6,24]. Acb is a key structure in the mesolimbic dopamine system [32]. This study found a significant reduction in 5-HT2A receptor mRNA expression 2 h after the last olanzapine treatment.
While it is known that Acb forms part of circuits modulating feeding via its connections with a classic hunger center, the lateral hypothalamus [27], no significant correlation was found between the levels of 5-HT2A receptor mRNA expression and food intake, body weight gain or energy efficiency in this area. However, studies have shown that olanzapine induces the release of, and increases the level of dopamine concentration in the Acb, which is associated with reward or anti-depression effects [3,19]. LS receives direct projections from the amygdala and hypothalamus [17]. Although the involvement of the LS in energy balance regulation is not clear, it is known that it contains a number of neurotransmitters (such as agouti-related protein and neuropeptide Y-like immunoreactivity [7,16]) and receptors (such as the neuropeptide Y receptor subtype 2, agoutirelated protein and D4 mRNAs [8,11,16]) known to be critically involved in the regulation of energy balance and the development of obesity. The present study found that the levels of 5-HT2A receptor mRNA expression were associated with total food intake and body weight gain. The question of whether an interaction occurs between 5-HT2A receptors, melanocortin, neuropeptide Y and serotonin systems in the LS, and their relationship with the olanzapine treatment, will be an important area for further study. This study found that striatal 5-HT2A receptor mRNA expression was significantly down-regulated at 2 h after olanzapine treatment, and negatively correlated with food intake and body weight gain. The striatum is under regulatory control of the monoamines, dopamine and serotonin, and there is an interaction between the two systems, for example, olanzapine increases striatal dopamine release [19]. While the involvement of striatal dopamine D2 receptors have been reported in body weight regulation in humans and rodents [12,31], little information is available with respect to the involvement of striatal 5-HT2A receptor in body weight control. The present study confirms previous evidence that the 5HT2C receptor and its encoding mRNA are widespread in rodent brain. A consistent finding from this study was decreased 5HT2C receptor mRNA expression in the SN at 2 and 48 h after olanzapine treatment. The implications of this inhibition
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effect in the SN are presently unknown, but could be associated with the lower propensity to induced motor side effects, as there are extensive reciprocal connections between the SN and striatum. Furthermore, it has been reported that olanzapine, but not haloperidol, significantly inhibits the firing rate of the SN neurons [29]. While it has been reported that olanzapine decreases 5-HT2C receptor binding in hippocampus and striatum in rats [28], we did not find significant changes in these regions. In summary, the present study has shown for the first time the pattern of 5-HT2A and 5-HT2C receptors mRNA expression in the brain of rats treated with olanzapine. Olanzapine decreased the levels of 5-HT2A receptor mRNA expression in the striatum as well as hypothalamic and limbic nuclei relevant to the regulation of energy balance, reward, anxiety and motor function. A rebound effect at the gene transcription level of 5-HT2A receptors was observed 48 h after prolonged olanzapine treatment, which might contribute to weight reduction after cessation of olanzapine treatment. 5-HT2C receptor mRNA expression was mostly unaffected by olanzapine treatment. Finally, the olanzapine-induced weight gain may be due not only to high energy intake, but also to increased energy efficiency, particularly in the maintenance of body weight gain after chronic olanzapine treatment. Acknowledgments We wish to thank A/Prof. K. Russell (Department of Applied Statistics, University of Wollongong) for his suggestions regarding the statistical analysis. This study was supported by grants from the Research Committee of the University of Wollongong and the Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD), utilizing infrastructure funding from NSW Health. References [1] Allison DB, Casey DE. Antipsychotic-induced weight gain: a review of the literature. J Clin Psychiatry 2001;62(Suppl. 7):22–31. [2] Arjona AA, Zhang SX, Adamson B, Wurtman RJ. An animal model of antipsychotic-induced weight gain. Behav Brain Res 2004;152: 121–7. [3] Bymaster F, Perry KW, Nelson DL, Wong DT, Rasmussen K, Moore NA, Calligaro DO. Olanzapine: a basic science update. Br J Psychiatry Suppl 1999:36–40. [4] Bymaster FP, Calligaro DO, Falcone JF, Marsh RD, Moore NA, Tye NC, Seeman P, Wong DT. Radioreceptor binding profile of the atypical antipsychotic olanzapine. Neuropsychopharmacology 1996;14: 87–96. [5] Cohen BM, Cherkerzian S, Ma J, Ye N, Wager C, Lange N. Cells in midline thalamus, central amygdala, and nucleus accumbens responding specifically to antipsychotic drugs. Psychopharmacology 2003;167:403–10. [6] Deutch AY, Lee MC, Iadarola MJ. Regionally specific effects of atypical antipsychotic drugs on striatal Fos expression: the nucleus accumbens shell as a locus of antipsychotic action. Mol Cell Neurosci 1992;3:332–41. [7] Haskell-Luevano C, Chen P, Li C, Chang K, Smith MS, Cameron JL, Cone RD. Characterization of the neuroanatomical distribution of agouti-related protein immunoreactivity in the rhesus monkey and the rat. Endocrinology 1999;140:1408–15.
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