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Effects of Simultaneous Reinforcement of Endocannabinoid and Cholinergic Systems on Anxiety-Like Behavior in Mice

Objective Anxiety behavior is regulated by different neurotransmitter systems. There has been no direct relationship between endocannabinoid and cholinergic systems on anxiety in previous studies. This study investigated the effects of each of these systems separately and simultaneously using Donepezil (Cholinesterase inhibitor) and URB-597 (endocannabinoid degrading enzyme inhibitor) on anxiety-like behavior. Method Eighty-eight male mice were divided into eleven groups (n=8) including control (saline), diazepam (0.3 mg /kg), URB-597 (0.1, 0.3, or 1 mg /kg), donepezil (0.5, 1 or 2 mg/kg) and the combination of the two drugs at low, medium and high doses. All treatments were injected intraperitoneally 30 minutes before the elevated plus maze test.


Introduction
Anxiety is one of the most common disorders in psychiatry [1,2]. According to World Health Organization statistics, about 275 million people suffer from the disorder, with an estimated prevalence of approximately 42 million worldwide each year [3]. This disorder ranks ninth among all diseases and holds the highest prevalence among all mental disorders worldwide [4]. Anxiety disorders are characterized by a variety of neuroendocrine and neurotransmitter systems in the neuroanatomical different areas [5].
Contradictory reports have been published on the role of the cholinergic system in anxiety [6,7]. Therefore, administration of nicotinic or muscarinic antagonists decrease anxiety behavior induced by physostigmine [8] whereas enhanced cholinergic transmission, or activating M 1 receptors in the ventromedial prefrontal cortex induces anxiety [9] or gallamine (muscarinic receptor antagonist type 2) showed no effect in the elevated plus-maze [7]. Moreover, it is interesting that depressed and bipolar populations showed that scopolamine was also effective on anxiolytic effects (decrease anxiety symptoms); however, rats and mice administered with scopolamine showed increased anxiety in standard behavioural tests [10].
Endocannabinoid system has emerged as an important neuromodulatory system in the brain involved in a variety of physiological processes including pain, memory, and mood [11,12]. Although the physiological role of cannabinoid receptors (CB 1 and CB 2 ) has not yet been fully discovered, their wide distribution in different regions of the central nervous system indicates their signi cant role [13][14][15].
Anandamide is the most important and rst endogenous cannabinoids that have been identi ed [16].
Anandamide is rapidly eliminated after releasing in two steps, including intracellular resorption and intracellular degradation of arachidonic acid and ethanol amide by fatty acid amid hydrolase (FAAH) [17].
A selective inhibitor of FAAH is URB-597, which enhances and prolongs the effect of endogenous and exogenous anandamide [18].
The results of several studies have suggested that the cannabinoid system participates in the modulation of anxiety behaviors through CB 1 receptor [19][20][21] but its effects are inconsistent and dose-dependent.
According to distribution of the CB 1 receptor and acetylcholine in the prefrontal cortex, hippocampus, and amygdala of the mouse that overlap with anxiety responses are likely to interfere with this process as well [22]. Based on the previously described approach for the unclear role of the cannabinoid and cholinergic system in anxiety and their potential interference with this phenomenon, the aim of the present study was to determine the effect of coadministration of the cholinergic endogenous system (using donepezil, cholinesterase inhibitor) and the endocannabinoid (using URB 597, cannabinoid degrading enzyme inhibitor) on the anxiety model of the elevated plus maze in male mice.

Animals
Eighty-eight male mice (32-40g) were purchased from animal house of the Hamadan University of Medical Science. The animals were maintained in groups of four per cage under a 12-h light/dark cycle (lights on at 7:00 a.m.) in a temperature-controlled room. All mice were allowed to adapt to the new environment and handling for at least one week before experiments.

Experimental groups
Eighty-eight male SWISS mice were randomly assigned to eleven groups (n = 8 for each group): Control group (Group 1) Mice received intraperitoneal saline before being placed on the maze.

The Elevated plus maze (EPM) test
The elevated plus maze test is a valid test for the determination of the anxiety-like behavior of various drugs or compounds [25]. The elevated plus maze test consists of four arms (two open arms and two closed arms; 51 cm long 10 cm wide) centered perpendicular to each other forming a plus shape and extending 50 cm apart. Each mouse was placed in the center of the maze and allowed to explore all arms freely for 10 minutes [25,26]. After each test, the device was cleaned with 70% ethanol to eliminate residual odor.

Statistical Analyses
The data were analyzed by SPSS version 16.0 using one-way ANOVA. Tukey's test was performed to determine the effects of various treatments. Results were presented as mean ± SEM for each group, and P < 0.05 was considered signi cance level.

Results
The number of entries to the open arms of the elevated plus maze Comparison of the number of entries to the open arms of the elevated plus maze using one-way ANOVA showed a signi cant difference between groups [F (10,77) = 13.88; P < 0.001]. Tukey's test revealed that the number of open arms entries in the groups receiving different doses of URB-0.1 mg/kg (P < 0.001), URB-0.3 (P < 0.001) or URB-1 (P < 0.01) were signi cantly higher than the control group. Also, there was no signi cant difference in the number of open arms entries between groups receiving different doses of URB 597 (Fig. 1.A).
Tukey's test showed that the number of open arm entries in the groups receiving donepezil at doses of 0.5mg/kg (P < 0.001), 1mg/kg (P < 0.01) or 2mg/kg (P < 0.05) was signi cantly higher than that in the control group. The number of open arm entries in the mice given 0.5 mg donepezil was higher than that in the mice given 2 mg donepezil (P < 0.05; Fig. 1

.A).
Comparison of the number of entries to the open arms of the elevated plus maze between the control group and the URB 597 + Donepezil (U + D) groups at low, moderate or high doses showed no signi cant difference between the groups. However, this parameter in the U + D (L) treatment signi cantly decreased compared with the lowest doses of URB 597 (P < 0.01) or donepezil (P < 0.05) alone. Moreover, in the U + D (M)-treated group, the number of open arm entries signi cantly reduced compared to the groups given URB-0.3 (P < 0.01) or Donepezil-1 alone (P < 0.05). In the U + D (H)-treated group, the number of open arm entries was lower than that in the groups given the high doses of URB 597 (P < 0.01) or Donepezil (P < 0.05) alone. It was also found that the number of open arm entries in the diazepam group was signi cantly higher than that in the control group (P < 0.001; Fig. 1.A).

The comparison of the number of entrances to closed arms
The comparison of the number of entries to closed arms using one-way ANOVA showed a signi cant difference between groups [F (10, 77) = 11.368; P < 0.001; Fig. 1.B]. The Tukey's test results showed that the number of closed arm entries was signi cantly lower in URB-0.1 (P < 0.01), URB-0.3 (P < 0.001) or URB-1 (P < 0.01) groups than in the control group. Also, donepezil-0.5 (P < 0.05), donepezil-1 (P < 0.001) or donepezil-2(P < 0.001) treatment showed signi cant decrease in this parameter compared to control group. The number of closed arm entries in the donepezil (2 mg/kg) group was lower than that in the donepezil-0.5 group (P < 0.05). group was signi cantly decreased compared to low dose (P < 0.01) or high dose (P < 0.01) URB treatment, as well as low dose (P < 0.01) or high dose (P < 0.05) Donepezil treatment. Also, signi cant decrease was also observed in the number of closed arms entries in U + D (M) compared to low dose (P < 0.05) or high dose (P < 0.05) URB treatment, as well as low dose (P < 0.001) Donepezil treatment, respectively. In addition, U + D (H) treatment lower than Donepezil drug (P < 0.05).
There were no signi cant differences between groups at low, medium or high dose of U + D. In addition, the number of closed arms entries in the diazepam receiving groups was signi cantly lower than the control group ( Fig. 1.B; P < 0.001).

Total arm entries in Elevated plus maze test
Several parameters considered for mouse locomotive activity evaluation in EPM. The number of entries in closed + open arms and the time spent in closed + open arms represent the animals ' locomotion status.

Time spent in open arms in the elevated plus maze
The results of one-way ANOVA revealed that between groups were signi cantly different in time spent in open arms [F (10, 77) = 6.253, P = 0.001; Fig. 3.A]. Tukey post hoc test revealed that time spent in open arms in URB-0.1 (P < 0.001) or URB-0.3 (P < 0.01) treatments was signi cantly more than control group. Also, the time spent in open arms in doses of donepezil-0.5 (P < 0.05), donepezil-1 (P < 0.05) or donepezil-2 mg/kg (P < 0.001) was signi cantly more than control group. This parameter was also decreased in the U + D (L) compared of URB-0.1(P < 0.01) or donepezil-2 (P < 0.01) groups. In addition, in the U + D (H) group lower than the URB-0.1 (P < 0.001) or Donepezil-2 (P < 0.001) groups, respectively. Diazepamtreated mice showed signi cant increase the time spent in open arms compared to control group (P < 0.001; Fig. 3

.A).
The time spent on the closed arms of elevated plus maze Post-hoc Tukey showed this parameter in U + D (L) mice decreased compared with donepezil-0.5 (P < 0.01) or donepezil-2 (P < 0.001) groups. In addition, in U + D (M) group lower than Donepezil-2 (P < 0.01) group. The U + D (H) group was signi cantly lower than all of Donepezil dosage (P < 0.001). Also, the time spent in closed arms in diazepam receiving groups was signi cantly lower than the control group (P < 0.001; Fig. 3.B).
The ratio of open arm entries/time to the closed arm entries/time There was a signi cant difference in the percentage of open / closed arms entry between groups [F (10, 77) = 8.612, P < 0.001]. The U + D (H) treated mice spent signi cantly lower this ratio than donepezil-2 treated animals (P < 0.01; Fig. 4

.A).
Results regarding the ratio of the time spent on open arm to that spent on closed arm time are shown in Fig. 4.B. One-way ANOVA showed that there was a signi cant difference in this ratio between groups [F (10, 77) = 4.927, P < 0.001]. Tukey's test showed that this parameter in the URB-597 + donepezil (L) was lower than that in URB-0.1 (P < 0.01) or in URB-597 + donepezil (H) was lower than that in URB-0.1 (P < 0.05). This parameter was signi cantly higher in the diazepam group than in the control group (P < 0.001; Fig. 4.B).

Discussion
In this study, we investigated the effects of enhancement of endocannabinoid and cholinergic neurotransmitter system separately and simultaneously using donepezil (cholinesterase inhibitor) and URB-597 (endocannabinoid degrading enzyme inhibitor) on anxiety-like behavior. Key ndings of this study included: (1) Injection of three doses of URB-597(cannabinoid-degrading enzyme inhibitor) reduced anxiety. (2)  These results are consistent with the other results regarding a role in the modulation of anxiolytic effects of cannabinoids by type 1 receptor (CB 1 ) [19]. Another study showed contradictory result for example cannabinoid receptor agonists (CP55940, WIN55212-2) produced anxiolytic effects in mice only at low doses [20] whereas at higher doses had anxiogenic effect [21]. Other contradictions are the natural compound in the cannabis plant (Delta 9-tetrahydrocannabinol) that produced an anxiolytic effect in mice in the light-dark test [27] while in the same test had anxiolytic effect [28].
It has also been reported that WIN 55,212-2 drug in mice reduces anxiety by intervention in GABAergic transmission, whereas it increases anxiety through intervention in glutamatergic synaptic transmission [29]. The anxiolytic effects created by URB-597 are also similar to the anxiolytic effect of diazepam. According to the pervious report, administration of URB-597 may interfere with GABAergic transmission [30]. Overall, donepezil as a cholinergic system, has anxiolytic effect. These results are consistent with previous studies in which administration of scopolamine (a muscarinic receptor antagonist) in mice produced anxiogenic effect [31,32]. In contrast, administration of type 1 muscarinic (Xanomeline) [33] or nicotine receptors agonists has anxiolytic effect [34]. Further studies are needed to clarify the contribution of each of these receptors more precisely.
The results of coadministration URB-597 with donepezil at low, medium and high doses, did not affect the number of open arms entries, while reducing the number or spending time of closed arms entry. The anxiolytic effect observed in the simultaneous administration of the above drugs is lower than the anxiolytic effect of each drug alone. There is no study about interaction these systems in anxiety-like behavior. In memory studies showed the interaction between cannabinoid (WIN-2; a synthetic cannabinoid) and cholinergic systems (acetylcholinesterase inhibitor) on memory, showed that WIN-2 signi cantly decreased rates of hippocampal principal cells performance and that rivastigmine reversed these short memory de cits and normalized hippocampal discharge rates [35].

Conclusion
The present report con rms that in reduction anxiety-like behavior via acetylcholine and anandamide secretion with activation of cholinergic and endocannabinoid receptors. Operations of one neurotransmitter system alone reduce anxiety while interaction of the two systems have less effect on reduction in anxiety. Future studies include additional molecular and histological experiments designed to elucidate the mechanisms underlying interaction between these systems that is more effective on anxiety-like behavior.
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