Oral haloperidol or olanzapine intake produces distinct and region-specific increase in cannabinoid receptor levels that is prevented by high fat diet

Accepted Manuscript Oral haloperidol or olanzapine intake produces distinct and region-specific increase in cannabinoid receptor levels that is prevented by high fat diet

Foteini Delis, Lauren Rosko, Aditya Schroff, Kenneth E. Leonard, Panayotis K. Thanos PII: DOI: Reference:

S0278-5846(17)30262-2 doi: 10.1016/j.pnpbp.2017.06.005 PNP 9127

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date: Revised date: Accepted date:

6 April 2017 2 June 2017 12 June 2017

Please cite this article as: Foteini Delis, Lauren Rosko, Aditya Schroff, Kenneth E. Leonard, Panayotis K. Thanos , Oral haloperidol or olanzapine intake produces distinct and region-specific increase in cannabinoid receptor levels that is prevented by high fat diet. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Pnp(2017), doi: 10.1016/j.pnpbp.2017.06.005

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ACCEPTED MANUSCRIPT Oral haloperidol or olanzapine intake produces distinct and region-specific increase in cannabinoid receptor levels that is prevented by high fat diet

Foteini Delis1 , Lauren Rosko2, Aditya Schroff 3 , Kenneth E. Leonard3, Panayotis K. Thanos 3*

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1 Department of Pharmacology, Medical School, University of Ioannina, 45110, Ioannina, Greece

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2 Georgetown University Medical Center, Georgetown University, Washington, DC, 20007, USA

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3 Behavioral Neuropharmacology and Neuroimaging Laboratory on Addictions, Research Institute on Addictions, University at Buffalo, Buffalo, NY, 14203, USA

* To whom correspondence is addressed: [email protected],

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1021 Main Street

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Phone: (716) 881-7520

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Buffalo, NY 14203-1016

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Running Head: Diet modulates CB1Rs in antipsychotic treatment

ACCEPTED MANUSCRIPT Abstract Clinical studies show higher levels of cannabinoid CB1 receptors (CB1R) in the brain of schizophrenic patients while preclinical studies report a significant functional interaction between dopamine D2 receptors and CB1Rs as well as an upregulation of CB1Rs after antipsychotic treatment. These findings prompted us to study the effects of chronic oral intake of

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a first and a second generation antipsychotic, haloperidol and olanzapine, on the levels and

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distribution of CB1Rs in the rat brain. Rats consumed either regular chow or high-fat food and drank water, haloperidol drinking solution (1.5 mg/kg), or olanzapine drinking solution (10

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mg/kg) for four weeks. Motor and cognitive functions were tested at the end of treatment week 3 and upon drug discontinuation. Two days after drug discontinuation rats were euthanized and

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brains were processed for in vitro receptor autoradiography. In chow-fed animals haloperidol and olanzapine increased CB1R levels in the basal ganglia and the hippocampus, in a similar,

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but not identical pattern. In addition, olanzapine had unique effects in CB1R upregulation in

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higher order cognitive areas, in the secondary somatosensory cortex, in the visual and auditory cortices and the geniculate nuclei, as well as in the hypothalamus. High fat food consumption

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prevented antipsychotic-induced increase in CB1R levels in all regions examined, with one exception, the globus pallidus, in which they were higher in haloperidol-treated rats. The results

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point towards the hypothesis that increased CB1R levels could be a confounding effect of

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antipsychotic medication in schizophrenia that is circumveneted by high fat feeding. Keywords: CB1, antipsychotic, high fat diet

ACCEPTED MANUSCRIPT 1.

Introduction Increased cortical and subcortical cannabinoid CB1 receptor (CB1R) binding has been

reported in human schizophrenia studies. In vivo brain imaging shows higher CB1R levels in frontal, temporal, parietal, medial cortices, the n. accumbens (NAC) (Ceccarini et al. 2013), and the pons (Wong et al. 2010) of schizophrenic patients, in agreement with post-mortem receptor

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binding studies also showing CB1R upregulation in dorsolateral prefrontal and cingulate cortices

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in schizophrenia patients (Dean et al. 2001; Zavitsanou et al. 2004; Newell et al. 2006; Dalton et al. 2011; Jenko et al. 2012).

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Studies have also shown that CB1R levels are modulated by the dopaminergic system and in particular, dopamine D2 receptors (D2R). Genetic ablation of D2Rs leads to a marked

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upregulation of cannabinoid CB1Rs in the cerebral cortex, the striatum, and the NAC (Thanos et

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al. 2011). Treatment with haloperidol, a first generation antipsychotic with high affinity for D2Rs, also leads to higher CB1R levels and CB1R activity in the basal ganglia (Andersson et al. 2005).

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On the other hand, studies have shown that that haloperidol decreases frontal cortical CB1R protein levels in women (Uriguen et al. 2009) and CB1R-mediated signaling in the frontal cortex

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of female rats (Wiley et al. 2008). CB1R levels increase in the hippocampus and the amygdala

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after risperidone treatment (Secher et al. 2010) and in the NAC after prolonged olanzapine withdrawal (Sundram et al. 2005). On the other hand, a selective decrease in CB1Rs in the

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arcuate nucleus and the dorsal vagal complex was shown after olanzapine treatment in female rats (Weston-Green et al. 2012). Still, other studies show no effects of haloperidol (Sundram et al. 2005) or olanzapine (Llorente-Berzal et al. 2012; Lazzari et al. 2017) treatment on CB1R levels. If antipsychotic treatment modulates brain CB1R levels, it would be necessary to study brain regions and circuits in which CB1Rs are most affected by antipsychotics, in order to clarify the potentially confounding effect of antipsychotic medication in humans.

ACCEPTED MANUSCRIPT Second generation antipsychotic treatment is associated with overeating, weight gain, and related metabolic disorders (Eder et al. 2001; Gothelf et al. 2002; Minet-Ringuet et al. 2006; Allison et al. 2009; Muller et al. 2010; van der Zwaal et al. 2010; van der Zwaal et al. 2012; Daurignac et al. 2015). In addition, food intake and body weight regulation are directly related to the endocannabinoid system since genetic ablation and pharmacological antagonism of CB1Rs

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lead to leanness and suppress food intake (Verty et al. 2004; Salamone et al. 2007; de Kloet

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and Woods 2009; Pang et al. 2011; Lazzari et al. 2012; Manca et al. 2013; Mastinu et al. 2013). Most interestingly, CB1R antagonism normalizes olanzapine-induced changes in enzymes and

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metabolic parameters expressed in brain, liver, and blood (Lazzari et al. 2017). If antipsychotic treatment increases brain CB1R levels, it would be important to identify whether this increase is

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related to the amount and type of food intake.

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Based on the above, we studied the effects of haloperidol and olanzapine oral intake (1 month) on CB1R levels in the male rat brain. Effects of food intake (standard rodent chow vs.

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high fat (HF)) were also considered. A battery of motor and non-motor tasks was applied to the

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Animals

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Methods

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2.

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rats during- and post-treatment to assess the behavioral state of the animals.

Sixty adult male Sprague Dawley rats (Charles Rivers, Wilmington, MA, USA) were handled daily for two weeks prior to collection of baseline measurements. Animals were individually housed and kept on a reverse 12:12 L/D cycle with lights off at 07:00. Food and water were provided ad libitum. Animal weight, food, and fluid intake were measured daily. Animals were randomly assigned to 1 of 2 food groups (regular laboratory chow or regular chow + HF food) and 1 of 3 treatments (water, haloperidol solution (1.5mg/kg), or olanzapine solution (10mg/kg)).

ACCEPTED MANUSCRIPT All experiments were conducted and approved by the National Academy of Sciences Guide for the Care and Use of Laboratory Animals (NAS et al., 1996) and the University at Buffalo IACUC. 2.2.

Drugs

Haloperidol (H1512, Sigma Aldrich, US) and olanzapine (O253750, Toronto Research

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Chemicals, Canada) drinking solutions were prepared by dissolving the powder in 0.1N HCl, diluting with water, and fixing the pH to 7 with very dilute NaOH (Terry et al. 2007). The

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concentration of the solution was based on the animals’ average fluid intake of the previous day

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and on their body weight in order to formulate a drug dose of 1.5mg/kg/day for haloperidol and 10mg/kg/day for olanzapine. Drug solutions were prepared on a daily basis and no precipitate

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was observed in the bottles. Olanzapine has been previously shown to be stable in drinking water for at least 96 h (Terry et al. 2008). Drug doses were based on previous studies showing

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that at these doses the drugs, administered orally and chronically, have no more than 80%

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dopamine D2 receptor occupancy, thus being therapeutically relevant and without motor side-

Experiment outline

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effects (Kapur et al. 1999; Kapur et al. 2003; Terry et al. 2005; Barth et al. 2006).

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The experimental timeline is depicted in Figure 1, below. Drug treatment lasted 28 days, from

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Day0 to Day27. High fat (HF) food (Test Diets 58Y1 with 35% fat vs. regular chow Test Diets 5001 with 13% fat) was added to the Chow + HF groups on Day2. In this group, both foods were offered in the food container separated with a divider. Food position was switched every 34 days to prevent position bias; however all rats had 100% preference for the HF food from the first day it was presented until the end of the experiment. Motor and cognitive functions were assessed during treatment, in the AM (rats’ dark cycle, active phase, under red light) of Days 20 and 21, and post-treatment, during the AM of Day28 and29. The rats were euthanized in the AM of Day30.

ACCEPTED MANUSCRIPT Figure 1. Schematic outline of the experimental procedure. D: day; Tx: treatment; NOR: novel object recognition test; OFT: open field test; HF: high-fat food offered along with standard

Behavioral assessments

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2.3.

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diet, and preferred 100%.

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2.3.1. Catalepsy test

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The rats were placed on a grid that was inclined at 60o. We measured the rats’ latency to perform the first ambulatory movement with all four legs. Time is expressed in s.

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2.3.2. Treadmill

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To assess motor coordination, strength, and endurance we measured the maximum speed of the treadmill belt that the rats were able to follow. One day before the test the rats were placed

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on the treadmill at the lowest speed to habituate to the setup. On test day, the belt speed ranged from 22.5 cm/s to 50cm/s and increased by 2.5 cm/s every 1min. When a rat stopped running for more than 10s it was removed from the treadmill belt and the speed of the belt at that time was recorded. 2.3.3. Open Field Test

ACCEPTED MANUSCRIPT The open field test was performed in a cubic arena (edge: 40 cm) that records horizontal and vertical motion through a series of cross beams that are positioned 5cm apart, allowing for 2.5cm resolution (Tru Scan System, Coulbourn Instruments, PA, USA). The animal was placed in the center of the open field arena and each session lasted for 60min. We report ambulatory

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distance (cm) and vertical activity (rearing, number of vertical counts).

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2.3.4. Novel object recognition

The novel object recognition test, an example of a non-matching to target test (Ennaceur and

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Delacour 1988), was employed to assess short- and long-term memory at intertrial intervals T2=5min and T3=1hr, respectively. During trial T1, the rats were presented 2 identical objects

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[A,A] in a 40cm edge cubic arena, and left to explore them for 5min. Five min after the end of T1, they were re-placed in the arena (trial T2) and left to explore, for 5 min, objects [A,B] that

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had been placed in the same positions as [A, A]. One hour later, the rats were placed once

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more in the arena (trial T3) where 2 objects [A,C] had been placed in the same positions as in T1 and T2. The rats were videotaped with a camera that was hanging from the ceiling. Object

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exploration was defined as approaching the object at 2cm with the head facing the object, sniffing, whisking, or touching the object with the forepaws. Exploration of the novel (N) and

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familiar (F) objects were measured in s. Object Discrimination Index (DI) is expressed as the

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ratio (N-F)/(N+F) . Total object exploration time is also reported in s. Two different sets of objects, [A,B,C] and [A’,B’,C’], were used when the rats were tested during- and post-treatment, respectively. 2.4.

Receptor studies

2.4.1. Tissue preparation Rats were decapitated after isoflurane anesthesia, brains were rapidly extracted on ice, flashfrozen in methylbutane (-40oC), and stored at -70oC. Fifteen um-thick sections, prepared with a

ACCEPTED MANUSCRIPT cryostat (Leica CM30505), were thaw-mounted on clean charged glass slides. Every 3rd section was collected and the tissue was divided into 5 sets, which allows for a regular representation of the regions of interest every 225um. 2.4.2. In vitro receptor autoradiography

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Levels and distribution of D2Rs were determined with 2nM [3H]raclopride (specific activity 76 Ci/mmol) exactly as previously described (Tarazi et al. 1998). Levels and distribution of CB1Rs

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were determined with 5nM [3H]CP55940 (Specific activity 176 Ci/mmol) as previously described

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(Dalton et al. 2009), with an additional 5min wash in BSA-free buffer before the final dip in icecold distilled water.

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2.4.3. Visualization

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After drying, the slides were placed into a glass slide cassette along with tissue standards for

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image acquisition scanning using β-Imager 2000 (Biospace Measures, IN/US). Betavision+ software (Biospace Measures, IN/US) was used to outline regions of interest and to quantify

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radioactivity bound levels. Regions of interest were defined according to Paxinos and Watson,

Statistics

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2.5.

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6th edition, Rat brain atlas and according to the distribution of the ligands.

Receptor binding was analyzed with two-way ANOVA, with food and drug as between-subjects factors. Body weight, food, caloric, and water intakes, open field, treadmill, and catalepsy tests were analyzed with three-way repeated measures ANOVA, with food and drug as betweensubjects and time as within-subject factors. Novel object recognition data were analyzed with three way repeated measures ANOVA, with food and drug as between-subjects and trial as within subjects-factors, for each time point (during- and post-treatment) separately. Statistically

ACCEPTED MANUSCRIPT significant F- and p-values are reported in Table 1. When appropriate, multiple pairwise

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comparisons were done with the Tukey post-hoc test. Level of significance was set at p<0.05.

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Results

Statistically significant effects and interactions on behavioral and receptor measures are presented in Table 1. Table 1. Significant effects of food, drug, and time on behavioral and receptor binding measures

T FxD DxT

F(1,53)=11.7 F(2,53)=3.4, F(2,53)=4.6,

F(2,30)=34.9 F(2,30)=38.8 F(2,30)=31.9 F(2,30)=29.2

p<0.001 p<0.001 p<0.001 p<0.001

Striatum (continued) Dtail D Vtail D NACcore D NACshell D

F(2,30)=33.6 F(2,30)=37.54 F(2,30)=37.54 F(2,30)=37.54

p<0.001 p<0.001 p<0.001 p<0.001

CA1D CA1V CA2 CA3D CA3V DG DS VS

FxD FxD FxD FxD FxD FxD FxD FxD

F(2,30)=3.8 F(2,30)=7.4 F(2,30)=4.6 F(2,30)=5.5 F(2,30)=7.4 F(2,30)=2.1 F(2,30)=4.1 F(2,30)=8.9

p=0.03 p=0.002 p=0.01 p=0.009 p=0.002 p=0.04 p=0.01, P<0.001

V2 V1_1-3 V1_4 V1_5-6 Aud_1-3 Aud_4 Aud_5-6

FxD FxD FxD FxD FxD FxD FxD

F(2,30)=3.4 F(2,30)=3.4 F(2,30)=7.8 F(2,30)=6.3 F(2,30)=4.7 F(2,30)=6.6 F(2,30)=6.7

p=0.03 p=0.004 p=0.001 p=0.005 p=0.01 p=0.004 p=0.004

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Cerebral cortex PtA_1-3 PtA_4 PtA_5-6 S2_1-3 S2_4 S2_5-6 PR/IL Ocb Ins Cg Rspl

FxD FxD FxD FxD FxD FxD FxD FxD FxD FxD FxD

F(2,30)=5.5 F(2,30)=4.6 F(2,30)=5.5 F(2,30)=3.5 F(2,30)=3.6 F(2,30)=4.5 F(2,30)=4.4 F(2,30)=7.9 F(2,30)=7.9 F(2,30)=3.5 F(2,30)=4.6

p=0.009 p=0.01 p=0.009 p=0.04 p=0.04 p=0.02 p=0.02 p=0.001 p=0.001 p=0.04 p=0.02

Hypothalamus

FxD

F(2,26)=4.7

p=0.017

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Hippocampus

p=0.04 p=0.007 p=0.01 p=0.003 p=0.03 p=0.01 p=0.004 p=0.03

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Cannabinoid CB1 receptors Basal ganglia Striatum VL FxD F(2,30)=3.4 DL FxD F(2,30)=5.8 Med FxD F(2,30)=5.2 NAC FxD F(2,30)=6.9 GP FxD F(2,30)=3.8 SNprL FxD F(2,30)=5.1 SNprM FxD F(2,30)=6.6 EPN FxD F(2,30)=2.6

p<0.001 P<0.001 p=0.001 p=0.03 p<0.001

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Dopamine D2 receptors Striatum DL D VL D DM D VM D

F(53,1353)=2.1 F(32,1728)=11.2 F(2,53)=7.6 F(2,53)=3.6 F(4,104)=6.7

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Distance Vertical

p=0.001 p=0.04 p=0.01

Food FxDxT Water FxT Treadmill D Catalepsy DxT D.I. (during Tx) DxTr

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p<0.001

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F(33,1749)=16.4

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FxT

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Body weight

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Thalamus Geniculate_n.

FxD

F(2,27)=9.2

P<0.001

F: food, D: drug, T: time, Tr: trials

Behavioral assessments

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3.1.

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3.1.1. Body weight

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Effects of food type and drug treatment on body weight over time are shown in Figure 2. HF-fed

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rats weighed more than chow-fed rats during the last 2 weeks of the experiment (p <0.05) and, eventually, gained more weight than chow-fed rats (100±4 vs. 80 ± 5 g, compared with baseline

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measures, p=0.005). No statistically significant effects of treatment on body weight were

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observed in either food group. 3.1.2. Food intake

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Food and caloric intakes over time are presented in Figure 2. Among chow-fed animals, olanzapine-treated ate more food than control rats on Days 3-4 and 7-20, (p<0.01) and more

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than haloperidol-treated on Days 9-20 (p<0.01). No consistent effects of antipsychotic treatment

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were observed among HF-fed rats. HF-fed rats ate the same as chow-fed rats until Day 15, but they significantly decreased their food intake thereafter (p<0.001). In contrast, their caloric

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intake was significantly higher than that of chow-fed of the same treatment until Day 15 (p<0.001), but it decreased and remained the same between the 2 food groups thereafter. 3.1.3. Water intake Water intake over time is presented in Figure 2. Water intake in HF-fed rats was less variable, compared with chow-fed rats, and lower, every 3rd day from Day 4 to Day 13 and almost every day from Day 15 to Day 28. Water intake was the same between the two food groups on the last day of the experiment. No treatment effects were observed in either food group.

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Figure 2. Effects of oral haloperidol and olanzapine treatments on body weight, food and water intakes in chow- and High Fat-fed rats. (i) Body weight (g: grams). (ii). Food intake (g food per kg body weight). (iii) Caloric intake (Kcal/kg b.w.: kilocalories per kg body weight). (iv) Water intake (g water/kg b.w.: g water per kg body weight). Vertical solid lines represent the beginning (Day0) and the end of drug treatments (Day27). Vertical dashed line represents high fat food introduction, maintained until the end of the experiment. C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA: oral olanzapine intake (10 mg/kg/day). ^: OLA compared with C and HAL, #: compared with corresponding Chow group, ↓: compared with Chow, main effect of food.

ACCEPTED MANUSCRIPT 3.1.3. Motor activity Effects of food type and drug treatment on motor activity are presented in Figure 3. During treatment, ambulatory distance was significantly lower in olanzapine-treated HF-fed rats, compared with control (p=0.01), while treadmill activity was lower in olanzapine-treated rats of both food groups, compared with control (p<0.001). In addition, during treatment vertical activity

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was significantly lower in haloperidol- and olanzapine-treated rats of both food groups,

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compared with control (p=0.01, p<0.01). After treatment was discontinued, latency to move on an inclined grid was higher in haloperidol and olanzapine-treated rats, regardless of food type,

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compared with control (p=0.001, p<0.001).

Figure 3. Effects of oral haloperidol and olanzapine on motor activity in chow- and high

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fat-fed rats during and post treatment. (i) Open field activity, (ii) Maximum treadmill speed. (iii) Catalepsy test: latency to move with all four limbs on an inclined grid. CHOW: standard

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laboratory diet; HF: high-fat food offered along with standard diet, and preferred 100%; Tx:

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treatment; C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA: oral olanzapine intake (10 mg/kg/day). T2: 5min intertrial interval, T3: 1hr intertrial interval. *:

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compared with respective C group.

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3.1.4. Novel object recognition

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Effects of food type and drug treatment on short- and long-term object recognition memory are

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depicted in Figure 4. Object discrimination index (DI) was lower in haloperidol- and olanzapinetreated rats in T3 (1h intertrial interval), regardless of the type of food consumed, compared with

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control (p<0.001), during treatment. No significant differences in total object exploration time

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during T1 were observed among the groups. Figure 4. Effects of oral haloperidol and olanzapine novel object recognition, in chowand high fat-fed rats during and post treatment. DI: Discrimination Index; CHOW: standard laboratory diet; HF: high-fat food offered along with standard diet, and preferred 100%; C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA: oral olanzapine intake (10 mg/kg/day). T1: 1st trial, with two identical objects; T2: 5min intertrial interval; T3: 1hr intertrial interval. *: compared with respective C group.

3.2.

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Receptor studies

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3.2.1. Dopamine D2 receptor binding

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D2R levels increased significantly after haloperidol and olanzapine treatment to the same degree (~30%) and in both food groups, in all divisions of the striatum and the NAC (p<0.001) (Figure 5).

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Figure 5. Effects of oral haloperidol and olanzapine treatments on dopamine D2 receptor

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levels in the basal forebrain. CHOW (left column): standard laboratory diet; High Fat (right

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column): high-fat food offered along with standard diet, and preferred 100%. C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA: oral olanzapine intake (10

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mg/kg/day). DL: dorsolateral striatum; VL: ventrolateral striatum; DM: dorsomedial striatum; VM:

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ventromedial striatum; D tail: dorsal tail of the striatum; V tail: ventral tail of the striatum; NACC: nucleus accumbens core; NACSH: nucleus accumbens shell; OT: olfactory tubercle. *:

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compared with respective C group.

ACCEPTED MANUSCRIPT 3.2.2. Cannabinoid CB1 receptor binding In the basal ganglia (Figure 6A), haloperidol and, to a greater degree, olanzapine treatment increased CB1R binding in chow-fed rats, in all divisions of the striatum (p<0.02, p<0.001), in the NAC, (p<0.01), and the globus pallidus (GP) (p=0.02, p=0.003). Olanzapine treatment

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increased CB1R levels in the entopeduncular nucleus (EPN) (p=0.03) and the medial division of

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the substantia nigra (SNpr) (p<0.001), compared with control, and in the lateral SNPr, compared

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with control and haloperidol (p<0.001). In HF-fed rats, CB1R binding was affected in only one region, the GP of haloperidol-treated rats, where CB1R levels increased compared with control

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(p<0.001) and olanzapine (p=0.04).

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In hippocampus (Figure 6B), haloperidol and, to a greater degree, olanzapine treatments increased CB1R binding in chow-fed rats in the dorsal CA1 (CA1D) (p=0.01, p<0.001) and in

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the CA2 (p=0.01, p<0.001), compared with control. Olanzapine treatment increased CB1R

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levels in ventral CA1 (CA1V) (p=0.005), dorsal CA3 (CA3D) (p<0.001), ventral CA3 (CA3V) (p=0.01), dentate gyrus (DG) (p<0.001), and dorsal subiculum (DS) (p<0.001) compared with

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control and in ventral subiculum (VS) (p=0.03), compared with haloperidol and control. No

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treatment effects were observed in HF-fed rats. In all layers of the parietal association area (PtA), haloperidol and olanzapine increased CB1R

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binding in chow-fed rats, compared with control (p≤0.02), while in the secondary somatosensory cortex (S2) CB1R levels increased after olanzapine treatment (p≤0.04). No treatment effects were observed in HF-fed rats (Figure 6C).

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ACCEPTED MANUSCRIPT Figure 6. Changes in cannabinoid CB1 receptor levels induced by haloperidol and olanzapine treatments. A. Basal ganglia. B. Hippocampus. C. Higher order sensory cortical areas. CHOW (left column): standard laboratory diet; High Fat (right column): high-fat food offered along with standard diet, and preferred 100%. C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA: oral olanzapine intake (10 mg/kg/day). VL:

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ventrolateral striatum; DL: dorsolateral striatum; Med: medial striatum; NAC: n. accumbens; GP:

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globus pallidus; EPN: endopeduncular n.; SNL, SNM: substantia nigra pars reticulata, lateral and medial parts, respectively; CAD: cornu ammonis dorsal; CAV: cornu ammonis ventral; DG:

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dentate gyrus; DS, VS: subiculum dorsal and ventral, respectively; PtA: parietal association area; S2: secondary somatosensory cortex; 1-3: cortical layers I-III, 4: cortical layer IV, 5-6:

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treatment groups of the same food group.

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cortical layers V-VI. *: compared with respective C group; ^: compared with the other two

ACCEPTED MANUSCRIPT In frontal and midline cortical areas (Figure 7A), CB1R binding increased in chow-fed, olanzapine treated rats, compared with control and haloperidol: prelimbic/infralimbic (PL/IL), p<0.001, p=0.02,); orbital (Orb), p<0.001, p=0.03; Insular (Ins), p<0.001, p=0.003; Retrosplenial (Rspl), p<0.001, p=0.03) or compared with control only: Cingulate (Cg) p<0.001. No treatment

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effects were observed in HF-fed rats.

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CB1R binding to visual (V) and auditory (Aud) areas of the cerebral cortex (Figure 7B)

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significantly increased by olanzapine treatment in chow-fed rats, compared with control and haloperidol: (V2, p<0.001, p=0.002, V1 p≤0.001, Aud (p<0.001, p≤0.003). In the geniculate

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nuclei (Figure 7C), which are strongly connected to visual and auditory cortices, receptor binding increased after olanzapine treatment (p=0.01, p=0.03). No other statistically significant

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changes were observed in the thalamus. Binding to the hypothalamus increased after

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olanzapine treatment in chow-fed rats (p=0.01, p=0.03) (Figure 7C). No treatment effects were

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observed in HF-fed rats.

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ACCEPTED MANUSCRIPT Figure 7. Changes in cannabinoid CB1 receptor levels induced by olanzapine treatment. A. Frontal and medial cortical areas. B. Visual and auditory cortical areas. C. Hypothalamus and thalamus. CHOW (left column): standard laboratory diet; High Fat (right column): high-fat food offered along with standard diet, and preferred 100%. C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA: oral olanzapine intake (10 mg/kg/day). PL/IL:

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prelimbic-infralimbic; Orb: orbital; Ins: insular; Cg: cingulate; Rspl: retrosplenial; V2: secondary

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visual; V1: primary visual; Aud: auditory; Hypoth: hypothalamus; Ant: anterior thalamus; Lat/Ro: lateral rostral thalamus; Med: medial thalamus; Lat/Cau: lateral caudal thalamus; Gen:

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geniculate nuclei; PAG: periaqueductal gray; 1-3: cortical layers I-III, 4: cortical layer IV, 5-6: cortical layers V-VI. *: compared with respective C; ^: compared with the other two treatment

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groups of the same food group.

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No effects of food or drug treatment were observed in primary motor, secondary motor, and primary somatosensory areas of the cerebral cortex (Figure 8A,B). Neither was binding affected

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in regions of the limbic cortex or other limbic areas (Figure 8C).

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ACCEPTED MANUSCRIPT Figure 8. Motor, sensory, and limbic areas in which CB1 receptor levels were not affected by haloperidol or olanzapine treatments. A. Motor and sensorimotor cortical areas. B. Primary somatosensory cortical areas. C. Limbic areas. CHOW (left column): standard laboratory diet; High Fat (right column): high-fat food offered along with standard diet, and preferred 100%. C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA: oral

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olanzapine intake (10 mg/kg/day). M2: secondary motor; M1: primary motor; S/M: sensorimotor

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(forelimb and hindlimb regions); J:jaw; UL: upper lip; BF: barrel field (whisker sensory cortex); Tr: trunk; Ectorh: ectorhinal cortex; Perirh: perirhinal cortex; Entorh: entorhinal cortex; Piri:

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piriform cortex; Amy: amygdala; Olf bulb: olfactory bulb; Apir: amygdalopiriform transition area; 1-3: cortical layers I-III, 4: cortical layer IV, 5-6: cortical layers V-VI.

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Correlation analysis

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Pearson correlation analysis between total food intake and regional CB1R levels showed a significant positive correlation in the NAC (r=0.59, p=0.01) and the EPN (r=0.61, p=0.008) in

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chow-fed, but not in HF-fed rats (Figure 9).

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Figure 9. CB1R specific binding in the n. accumbens and the endopeduncular n. was significantly correlated with total food intake in chow-fed rats. CHOW (left column):

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standard laboratory diet; High Fat (right column): high-fat food offered along with standard diet, and preferred 100%. C: control, water intake; HAL: oral haloperidol intake (1.5 mg/kg/day); OLA:

Discussion

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oral olanzapine intake (10 mg/kg/day).

Here we show that rat motor activity, alert exploratory behavior, and object discrimination are impaired during chronic oral intake of haloperidol or olanzapine both under a normal lab diet and under an HF diet. At a 2-day washout period, these behavioral deficits are no longer present. At this time, basal ganglia dopamine D2 receptors are upregulated by both antipsychotic treatments in both food groups. A widespread cannabinoid CB1R upregulation in a

ACCEPTED MANUSCRIPT drug- and region-specific pattern is observed. This antipsychotic-induced CB1R upregulation is prevented by high fat feeding in the vast majority of the brain regions examined. It is known that stimulation of the endocannabinoid system is associated with psychosis (Andreasson et al. 1987) and increases the risk of psychotic symptoms in young persons with

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(Henquet et al. 2005) or without (Hurst et al. 2011) predisposition for psychosis, in agreement

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with studies in rodents (Rubino and Parolaro 2014). Therefore, elevated CB1R density in the

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brain of schizophrenic patients (Dean et al. 2001; Zavitsanou et al. 2004; Newell et al. 2006; Dalton et al. 2011; Jenko et al. 2012) may be part of the biological substrate of the disease. On

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the other hand, the current study shows that CB1R density is also elevated by antipsychotic treatment. This finding has two implications: first, CB1R upregulation observed in schizophrenic

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patients may be a result of their medication and second, effective antipsychotic treatment may

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be hampered by antipsychotic-induced CB1R upregulation. These considerations, along with

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possible counteracting mechanisms mobilized by high-fat feeding, are discussed below. Behavioral assessments

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Overall, most behavioral observations in the current study were in agreement with

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previous or expected findings. In parallel with previous studies, olanzapine treatment increased food intake in chow-(van der Zwaal et al. 2010) but not in HF-fed rats (van der Zwaal et al.

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2012), without affecting body weight in either food group (Minet-Ringuet et al. 2006; Muller et al. 2010; Llorente-Berzal et al. 2012). Endogenous or exogenous cannabinoids increase appetite and stimulate feeding, an effect that is mediated by CB1Rs since it is blocked by CB1R antagonist treatment (Berry and Mechoulam 2002; Williams and Kirkham 2002). Thus, higher food intake levels in chow-fed, olanzapine-treated rats are consistent with their higher brain CB1R levels. In agreement with this reasoning, HF-fed olanzapine-treated rats neither presented with higher CB1R levels nor did they have higher food intake compared with the

ACCEPTED MANUSCRIPT corresponding control group. We should note here that olanzapine treatment did not lead to weight gain, in agreement with previous studies in male rats (Minet-Ringuet et al. 2006; Muller et al. 2010; Llorente-Berzal et al. 2012). We should also note that body weight and metabolism are strongly affected in olanzapine-treated females (Coccurello et al. 2006; Weston-Green et al.

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2012) and, therefore, it is unclear how this treatment would influence behavioral and CB1R

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measures in female rats.

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Normal ambulatory activity in chow-fed treatment groups suggests that, in agreement with previous studies (Bernardi et al. 1981), tolerance to antipsychotic treatment is induced by

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the applied oral intake protocols and that, in accordance with previous studies (Kapur et al. 1999; Terry et al. 2007; Terry et al. 2008), at the applied doses, D2R occupancy is comparable

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between the drugs and therapeutically relevant, without inducing gross motor side effects.

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Impaired ambulation in HF-fed rats and impaired treadmill motor activity in both food

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groups that received olazapine are indicative of the unfavorable effect of this antipsychotic on energy balance, fat deposition, and metabolic markers. Finally, the fewer vertical counts

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observed in all groups during treatment are indicative of the detrimental effects of both first- and second generation antipsychotic drugs on alert exploratory behavior. These motor deficits were

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not observed when the rats were re-tested post-treatment, which shows that this treatment

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protocol has no residual effects on basal horizontal and vertical motor activity at a two-day washout period. However, both antipsychotic treatment groups, regardless of their food regimen, exhibited a significant increase in their latency to move when tested 24 hrs posttreatment, which is likely due to the observed D2R upregulation, in agreement with previous findings (Gulati et al. 1988; Kinon and Kane 1989; Geurts et al. 1999; Vasconcelos et al. 2003). Cognitive function, as determined by object discrimination memory assessment, was affected by both antipsychotic treatments and in both food groups during drug treatment, at the

ACCEPTED MANUSCRIPT longer itertrial interval (1hr). This was not an unspecific effect of impaired object exploratory behavior since total object exploration time during T1 was the same for all groups. Our findings are in broad agreement with previous studies in chow-fed rats showing that oral haloperidol (Terry et al. 2007) and chronic or acute intraperitoneal olanzapine treatments (Orsetti et al. 2007; Mutlu et al. 2011) impair object discrimination. The current study extends these effects to

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HF-fed rats. Both haloperidol and olanzapine treatments impaired object discrimination,

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compared with control, without any residual effects post-treatment. Notably, HF feeding alone had no effect on object discrimination, in agreement with previous studies (Heyward et al. 2012;

CB1 receptor upregulation after olanzapine and haloperidol treatments

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4.2.

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Kosari et al. 2012; Tucker et al. 2012), but see (Camer et al. 2015) for opposite findings.

Oral antipsychotic treatment produced a region-specific CB1R upregulation, mainly in

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chow-fed rats. The results predict that olanzapine and, to a smaller degree, haloperidol affect

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basal ganglia function at input (striatum, NAC), intermediate (GP) and output (EPN, SNpr) stuctures via CB1R, thereby essentially interfering with various aspects of brain function

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including habit formation, responses to stimuli, and processing of rewards. Of particular interest are two areas where CB1R levels were positively correlated with food intake, the NAC, a key

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structure in reward-related brain processing, and the EPN, a basal ganglia output strucutre

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receiving converging inputs from regions of the striatum related to goal-directed behaviors and habit formation.

In addition, the results predict that olanzapine and, to a smaller degree, haloperidol influence hippocampal function via CB1Rs, thus affecting declarative memory and spatial orientation via the hippocampal endocannabinoid system. Olanzapine-induced upregulation of CB1Rs in higher order frontal (PL/IL, Orb, Ins, Cg, Rspl) and parietal areas (PtA, S2), as well as in visual (V2, V1, geniculate nuclei) and auditory areas, suggests that this antipsychotic

ACCEPTED MANUSCRIPT modulates executive function and sensory processing, spatial orientation, directed attention, and spatial learning (Reep et al. 1994; Shibata et al. 2004), via CB1R, thus bridging previous studies on antipsychotic-induced (Terry et al. 2008; Muller et al. 2010) and cannabinoid-induced cognitive effects (Varvel et al. 2001; Abush and Akirav 2010; Galanopoulos et al. 2014; Ruhl et

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al. 2014). A narrower CB1R-mediated effect of haloperidol on these functions is expected since

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CB1Rs are affected by haloperidol only in higher order parietal areas, PtA and S2.

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CB1R upregulation after haloperidol or olanzapine treatments in chow-fed rats is in agreement with previous findings of increased basal ganglia CB1R levels after haloperidol

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treatment (Andersson et al. 2005) and is consistent with recent findings that CB1R antagonists neutralize the metabolic effects of olanzapine (Lazzari et al. 2017) and with previous findings of

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D2R and CB1R interactions. Genetic ablation of either receptor from the beginning of

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development leads to upregulation of the other receptor (Houchi et al. 2005; Thanos et al. 2011). Simultaneous activation of D2Rs and CB1Rs results in receptor heteromer formation,

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mediated by the CB1R carboxyterminal and the D2R third intracellular loop (Khan and Lee

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2014) , in increased second messenger production, and in a switch in the G-protein specificity of the CB1R (Glass and Felder 1997; Jarrahian et al. 2004; Kearn et al. 2005). Finally, partial

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decrease in either receptor striatal levels leads to a similar decrease in the levels of the other receptor (Blume et al. 2013). Based on the above, we may propose that the CB1R upregulation

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observed in the current study is linked to the D2R upregulation induced by antipsychotic treatment in chow-fed rats. Interestingly, olanzapine treatment produced higher and more widespread increase in CB1R levels, compared with haloperidol, in spite of their similar effects on D2R levels. In addition to D2Rs, olanzapine binds to many different receptors, including serotonin 5HT receptors (5HTRs), adrenergic, histamine, and muscarinic receptors (Bymaster et al. 1996). Electrophysiological (Best and Regehr 2008; Burattini et al. 2014), neurochemical (Parrish and

ACCEPTED MANUSCRIPT Nichols 2006; Zarate et al. 2008), pharmacological (Devlin and Christopoulos 2002), and behavioral (Dogrul et al. 2012; McLaughlin et al. 2012) studies point to the existence of 5HTR/CB1R interactions in a variety of brain regions and functions. Chronic olanzapine treatment is known to increase 5HT1aR and decrease 5HT2aR in prefrontal cortex and to have variable effects on 5HT2cR levels in the hippocampus (Tarazi et al. 2002; Padin et al. 2006;

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Lian et al. 2013). Changes in 5HTRs may account for the unique effects of olanzapine

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administration on CB1R upregulation in the prefrontal cortex and the hippocampus in the current study.

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In humans, studies converge to an upregulation of brain CR1R in sc hizophrenia (Wong et al. 2010; Jenko et al. 2012) that is reportedly not correlated with medication exposure

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(Zavitsanou et al. 2004; Newell et al. 2006; Ceccarini et al. 2013), possibly more strongly

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associated with paranoid-type schizophrenia (Dalton et al. 2011). On the other hand, unchanged (Deng et al. 2007; Koethe et al. 2007) or lower (Eggan et al. 2008; Eggan et al.

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2010) brain CB1R levels in schizophrenia have also been reported, which could be related to

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methodological issues or to region-specific effects. In several animal models of schizophrenia (isolation rearing, disrupted-in-schizophrenia-

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1, DISC1; methylazoxymethanol, MAM; sponteneously hyperactive rat, SHR), lower CB1R

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levels or lower CB1R agonist-stimulated G-protein activation have been shown in basal ganglia, frontal cortical, and limbic regions of the brain (Adriani et al. 2003; Malone et al. 2008; Zamberletti et al. 2012; Kaminitz et al. 2014; Ballinger et al. 2015) and psychotic-like symptoms are reversed after cannabinoid agonist treatment (Almeida et al. 2014; Gomes et al. 2014; Levin et al. 2014). This suggests that the therapeutic effects of antipsychotics in these models (Bakshi et al. 1998; Cilia et al. 2001; Powell and Geyer 2002; Le Pen et al. 2011) may be mediated by the induced increase in CB1R levels.

ACCEPTED MANUSCRIPT On the other hand, in the phencyclidine (PCP) model of schizophrenia, CB1R levels or/and endocannabioid levels increase (Hajos et al. 2008; Vigano et al. 2009; Seillier et al. 2010) and psychotic-like symptoms are worsened with cannabinoid agonist (Vigano et al. 2009) and reversed with cannabinoid antagonist treatment (Hajos et al. 2008; Seillier et al. 2010; Guidali et al. 2011; Seillier and Giuffrida 2016). Based on the findings of the current study, we

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could predict that haloperidol and olazapine do not reverse the behavioral deficits induced by

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PCP, since thay change CB1R levels towards the same direction as PCP. Indeed, previous studies show that haloperidol and olanzapine do not reverse cognitive deficts incuced by PCP

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(Bruins Slot et al. 2005; Dunn and Killcross 2006; Beraki et al. 2008; Goetghebeur and Dias 2009; Brown et al. 2014; Yamazaki et al. 2014). Therefore, the regulation of CB1R levels in

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response to antipsychotic treatment should be of major importance as it might mediate their therapeutic action or, on the other hand, contribute to treatment-resistant conditions (Molins et

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al. 2016; Gillespie et al. 2017), with the outcome depending on the the combination of the

HF feeding prevents antipsychotic-induced CB1R upregulation in most brain

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applied treatment and the subtype of the disease.

regions

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The current results show that HF diet prevents CB1R upregulation induced by

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antipsychotic treatment, which suggests that dietary factors may interfere with the regulation of CB1R levels in the brain. Brain endocannabinoid levels undergo specific changes in response to variations in dietary fat composition. High fat (Higuchi et al. 2011), arachidonate-rich (Berger et al. 2001), and oleic acid-rich (Artmann et al. 2008) feeding have been reported to increase endocannabinoid or their precursor levels in the brain, which is expected to downregulate CB1Rs given that inhibition of endocannabinoid catabolizing enzymes, leads to lower CB1R activity (Schlosburg et al. 2014). In agreement, CB1R stimulation by high agonist concentrations also decreases CB1R mRNA (Corchero et al. 1999) and protein levels (Sim-Selley and Martin

ACCEPTED MANUSCRIPT 2002), leading to receptor desensitization (Lazenka et al. 2013). Thus, we postulate that changes in brain endocannabinoid content, as a result of a HF diet, may prevent or reverse the antipsychotic-induced upregulation of CB1Rs in the brain, possibly by interfering in CB1R interactions with other receptors, including D2Rs and 5-HTxRs. Quantification of regional brain

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studies in HF-fed rats would be required to confirm this hypothesis.

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endocannabinoid levels, assessment of receptor downstream signaling, and receptor interaction

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Our findings also suggest that dietary factors may interfere in CB1R regulation in a region-specific way since CB1Rs were not downregulated by HF diet in the GP of haloperidol-

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treated rats. The GP is part of the indirect basal ganglia pathway that exerts an inhibitory effect on the thalamocortical projection. Inhibition of the indirect pathway, as predicted by the

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upregulated CB1Rs on the striatopallidal terminals, is expected to facilitate the thalamocortical

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projection. At this point it is unclear how this brain response is associated with the animals’ behavior since post-treatment motor and cognitive measures of the HF haloperidol group did not

Summary and Conclusions

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differ from the other treatment groups in the current study.

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The behavioral and dopaminergic profile of the applied oral antipsychotic intake protocol was in agreement with well-documented observations of previous studies. In addition,

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detrimental effects on motor and cognitive functions were largley absent as soon as treatment was discontinued, an indication that the applied doses were therapeutically relevant. In chowfed rats, CB1R levels were upregulated in the basal ganglia and the hippocampus by both haloperidol and olanzapine treatments, while olanzapine-specific increases in CB1Rs were observed in higher order areas of the cerebral cortex and in the visual and auditory system as well. CB1R upregulation was completely prevented by HF feeding in all brain regions but one.

ACCEPTED MANUSCRIPT Based on these results we propose that increased CB1R levels could be a confounding effect of antipsychotic medication in schizophrenia that is circumveneted by high fat feeding. Obviously, imposing a high-fat diet, particularly to patients receiving antipsychotic medication, is not acceptable. However, the results suggest that there exists an interaction between antipsychotic treatment and metabolism that modulates cannabinoid receptor expression

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throughout the brain, meriting further study. In addition, these findings point towards future

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experiments that would assess antipsychotic–induced CB1R regulation in animals models of schizophrenia so as to rule out a differential effect of antipsychotic treatment in health and

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disease. Ethics concerns and conflicts of interest

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All experiments were performed according to the National Institutes of Health guide for the care and use of Laboratory animals, including maximizing information published and minimizing unnecessary studies. The authors have no conflict of interest to declare.

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All authors have contributed to design, data collection, data analysis, and interpretation of the finidngs.

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The manuscript, approved by all authors, is not under consideration for publication elsewhere. Should it be accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.

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Acknowledgement: This research was funded by the Research Foundation of New York, Q0942017.

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Zavitsanou K., Garrick T., Huang X. F., 2004. Selective antagonist [3H]SR141716A binding to cannabinoid CB1 receptors is increased in the anterior cingulate cortex in schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 28: 355-360.

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Brain CB1R levels are assessed after chronic oral haloperidol or olanzapine intake Effects of normal diet or high-fat diet are also considered Both antipsychotic treatments upregulate CB1Rs in a drug- and region-specific way CB1R upregulation is prevented by high-fat diet An interaction between antipsychotic treatment and metabolism modulates CB1Rs

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