Interaction Between the Cannabinoid and Vanilloid Systems on Anxiety in Male Rats

Introduction: Previous studies have shown that the cannabinoid system is involved in anxiety. In addition, transient receptor potential vanilloid type-1 (TRPV1) channels are new targets for the development of anxiolytics. The present study investigated the possible interaction between the cannabinoid and vanilloid systems on anxiety-like behavior in rats. Methods: Four different groups of male Wistar rats received intraperitoneal (IP) injections of (1) vehicle (DMSO+saline), (2) cannabinoid receptor agonist WIN55212-2 (WIN) (1 mg/kg), (3) TRPV1 receptor antagonist capsazepine (CPZ) (5 mg/kg), or (4) combined WIN (1 mg/kg) and CPZ (5 mg/kg) treatment 30 minutes before testing in the elevated plus maze. Results: The results showed that compared to the control (vehicle), both WIN and CPZ increased the time spent and number of entries on the open arms. Co-administration of WIN and CPZ had a synergistic effect, i.e., the number of entries and time spent on the open arms was greater than that in the groups administered the two compounds alone. The total distance travelled by rats and total number of entries on to the arms did not significantly differ between groups. Conclusion: Acute neuropharmacological blockade of the TRPV1 receptor or stimulation of the CB1 receptor produced an anxiolytic effect. It seems that antagonism of the vanilloid system modulates cannabinoid gain that rises the anxiolytic effect. TRPV1 antagonism may amend generation of endocannabinoids, which in turn increases anxiolytic impact. These results suggest that two systems could act on or share a common signaling pathway affecting the expression of anxiety.

While data is available on the individual effects of the cannabinoid and vanilloid systems in the expression of anxiety, the simultaneous stimulation or inactivation of these two systems and their interactive mechanisms have not been studied for their effects on the expression of anxiety. Thus in this experiment, we intend to evaluate the hypothesis that the effects of the cannabinoid system on anxiety are mediated by the vanilloid system results, specifically through the activity of TRPV1 receptors. The interaction of these two neuromodulators in the modification of anxiety could have remedial significance in clinical situation. In the present experiment, we examined the consequences of co-administration of a cannabinoid agonist and a vanilloid antagonist on the expression of anxiety-like behaviors in rats.

Animals
Male Wistar rats (obtained from the Razi institute, Iran) weighing 200-250 g were used throughout these experiments. Animals were housed in groups of four per cage under a 12 h light/dark cycle (lights on at 07:00 h) at a constant temperature of 23±2°C and given ad libitum food and water. All experiments were carried out in a quiet room under controlled light conditions between 11:00 and 15:00. Animals were allowed to acclimate to vivarium conditions for 1 week prior to behavioral testing. The experimental protocols were approved by the Hamadan University of Medical Sciences and were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1985).

Elevated Plus-Maze (EPM)
The apparatus consisted of two opposing open arms (50×10 cm each) without side walls, adjacent to two enclosed arms (50×10×50 cm each), with a central platform common to all arms (10×10 cm). The apparatus was elevated 50 cm above the ground (Pellow et al., 1985;Pellow and File, 1986). Thirty minutes after drug administration, each animal was placed in the center of the maze, facing one of the open arms. The rats were allowed to explore the maze, and their behavior was recorded for 10 minutes by using a digital camera mounted above the maze. The time spent and percentage of entries on to the open and enclosed arms were recorded. Distance travelled was calculated and used as a measure of locomotion during the 10 minutes test period (Lister, 1987;Ganji et al., 2016). After each test, the apparatus was cleaned with 10% ethanol to eliminate the remaining odors. Each animal was used only once.

Statistical analysis
The statistical analysis of data was performed by one-way analysis of variance (ANOVA) followed by Tukey post-hoc analysis. All the results are presented in terms of mean±SEM. A p value less than 0.05 was considered to be significant.

Effects on the total distance covered by rats
The total distance covered by the WIN, CPZ, and WIN+CPZ treated rats during the 10 minutes test period was not significantly different from controls (Figure 1).

Effects on the percentage of entries on to the open arms
The

Effects on the time spent in open arms
Compared to the control group, rats in the WIN group showed a significant decrease in the time spent on the open arms (P<0.01) whereas those in the CPZ (P<0.05) and WIN+CPZ (P<0.01) groups showed a significant increase. Further, compared to the rats in the CPZ group, those in the WIN+CPZ group showed a significant increase in the time spent on the open arms (P<0.05) (Figure 3).

Effects on the closed arms entry
The number of entries on to the closed arms was not significantly different between WIN, CPZ, and WIN+CPZ groups compared to the control group (Figure 4).

Discussion
The results of the present study indicate that the CB1 agonist (WIN) and the TRPV1 antagonist (CPZ) show an anxiolytic profile in rats. Our findings demonstrated that Komaki (Pellow, Chopin, File, & Briley, 1985;Komaki et al., 2014b). In addition, co-administration of CPZ and WIN potentially increases the anxiolytic activity of these compounds in animals. Changes within the percentage of passage on to the open arena and time spent on these arena are regarded as reliable correlates of changes in the amount of anxiety in experimental models. Therefore, the EPM is advised as a validated and reliable device to reflect both anxiolyticand anxiogenic-like effects of various drugs (Pellow, et al., 1985;Pellow and File, 1986;Komaki, et al., 2014a;Komaki, et al., 2015b;Nemati, et al., 2015).
Recent studies suggest the involvement of vanilloid receptors in the expression of anxiety-like behavior and the anxiolytic properties of cannabinoid agonists in mice and rats (Kasckow, Mulchahey, & Thomas, 2004;Patel and Hillard, 2006;Roohbakhsh, Moghaddam, Massoudi, & Zarrindast, 2007;Rubino et al., 2007). Blockade of TRPV1 resulted in anxiolytic-like behavior in TRPV1 receptor-deficient mice, (Marsch et al., 2007) and rats treated with a TRPV1 antagonist into the medial prefrontal cortex showed a similar effect (Aguiar et al., 2009). Moreover, blockade of TRPV1 channels induces anxiolytic-like effects (Aguiar et al., 2009;Fogaça et al., 2012). TRPV1 promotes cellular depolarization and increases neuronal firing rate (Xing and Li, 2007). TRPV1 is a calcium permeable ligand-gated cation channel that is expressed in various peripheral non-neuronal tissues and throughout the central nervous system (Montell et al., 2002). Activation of TRPV1 channels can stimulate calcium influx, facilitate the release of neurotransmitters (Musella et al., 2009, Starowicz et al., 2007 and may facilitate anxiogenic behavior (Marsch et al., 2007). Different mechanisms are proposed to describe the interaction between the cannabinoid and vanilloid systems within the CNS. The attainable communication between TRPV1 and CB1 receptors on anxious status has been previously reported in hippocampus (Hakimizadeh and Oryan, 2012) or PAG (Campos and Guimaraes, 2009) regions. It has been suggested that lipid-based molecules such as anandamide-related compound may act on TRPV1 (di Marzo, et al., 1998;Beltramo and Piomelli, 1999), suggesting a possible interaction between the cannabinoid and vanilloid systems. Many studies have demonstrated that stimulation of CB1 and TRPV1 produces contrasting results in various research settings, including alteration in intracellular Ca 2+ levels (Szallasi and Di Marzo, 2000) and glutamate liberate within the substantia nigra pars compacta (Marinelli, et al., 2003). It has been shown that HU210, as a synthetic cannabinoid receptor agonist, block capsaicin-induced entry of Ca2+. The repressive effects of this synthetic agonist, in total, are in line with the results of cannabinoid blockage of capsaicin-evoked effects (Oshita, et al., 2005).
Stimulation of CB1 receptors might heighten activity by inhibiting TRPV1 receptors (Fogaca et al., 2012). Interaction between these two systems has also been reported. One mechanism for interactive effects with CB1 regarding TRPV1 is that arousal of TRPV1 increases intracellular Ca 2+ levels, which can trigger the Ca 2+ -related N-acyltransferase (NAT) (Reddy, et a., 1983). NAT regulates the rate-limiting stage in anandamide production and consequently, TRPV1 arousal may result in elevated anandamide production (Tóth et al., 2009). Anandamide could then trigger TRPV1 either directly or indirectly. Within the instance of direct arousal, anandamide may bind to and activate TRPV1. Direct arousal obviously happens once anandamide level is high. Low levels of anandamide may selectively trigger CB1 receptors (Tóth et al., 2009). Stimulation of CB1 receptors can result in desensitization, blockage, or potentiation of TRPV1 channels in neurons co-expressing CB1 receptors and TRPV1 channels (Hermann et al., 2003;Jeske et al., 2006;Evans, Scott, & Ross, 2007;Kim, et al., 2009;Patwardhan et al., 2006). Co-administration of CPZ and WIN possibly modifies calcium influx, release of neurotransmitters, and potentiates the anxiolytic-like response in rats.
In conclusion, our experiments showed that acute neuropharmacological stimulation of CB1 receptors or blockade of TRPV1 receptors produced anxiolytic activity. The synergic effect of CB1 receptor activation and blockade of the TRPV1 receptor potentiated their anxiolytic response. Endogenous cannabinoid signaling is possibly enhanced by co-administration of WIN and CPZ. The results of changing cannabinoid system and regulating vanilloid transmission suggest that these two networks might contribute in some common signal transduction pathways. It has been revealed that the antagonists of vanilloid system regulate cannabinoid products, which can have anxiolytic-like effects. Further studies of the interaction between the cannabinoid and vanilloid systems are warranted.