Endocannabinoid System: behavioral modulation in murine models by Cannabinoids type 2 receptors

In the last decades, the eCB system has been highlighted by its neuro and immunomodulatory effects. Beyond CB1R effects in Central Nervous System (CNS), CB2R target drugs has been showed to be promising to mitigation of neuroinflammatory diseases in mouse models. However, it remains unknow the effects of CB2R target drugs on behavior. Therefore, we review the effects of CB2R on behavior in murine models by Pubmed website, selecting studies between 2001 to 2021. In this sense, many studies has demonstrated the effects of overexpression, lack, activation or antagonization of CB2R on Aggressive behavior, Memory-associated behaviors, Mood disorders and Reward behavior. Similarly, it is not clear yet how the eCB system modulates the behavior through CB2Rs present in neurons. Thus, in mouse models, although the pharmacological treatment with CB2R target drugs seems to be promising for neuroinflammatory diseases, on behavior there are few answers about the pathways of this modulation, as well as, it is fundamental the development and/or the update of behavioral tests that evaluate many parameters, then expose better interpretations in these tests.


Introduction Endocannabinoid System
The Endocannabinoid (eCB) system raised in the academic world in the end of the last century, since then its modulatory effects on other systems have been highlighted, mainly by its neuro and immunomodulatory effects.
In contrast, the presence of CB2R in neurons has motivated the research for neuromodulators effects of the systemic activation of CB2R to know the possibility of side effects, as well as to know the possibility of treatment of mood disorders, such as anxiety and depression (Chen, Gao, Gao, Su, & Wu, 2017). Therefore, the neuromodulators effects of eCB System by CB2R remain unknow, mainly in behavior. Thus, our goal was review and systematize the CB2R effects on behavioral Research, Society andDevelopment, v. 10, n. 13, e356101321491, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21491 3 modulation in murine models.

Methodology
The source of this review comprehends studies from PubMed website, which the keyworks was: "Cannabinoid receptor type 2"; "Endocannabinoid system"; and "Behavioral test". Thus, the first step was the select of studies between 2001 to 2021. Our goal was select experimental studies that have used behavioral tests in mouse models with overexpression, lack, activation or antagonization of CB2R, which includes CB2R target drugs (agonists and/or antagonists), CB2R-knockout mice, overexpression or lack of CB2R by other methodologies. The second step was select the main literature studied in behavioral clusters. The last step was including additional studies to explain our view point about our conclusions overall. According to Estrela, (2018) our study can be considerable a Narrative Revision (Estrela, 2018).

Results and Discussion
During our search, we systematize 12 methodologies, 33 effects/outcomes from 10 studies in 4 behavioral clusters: Aggressive behavior, Memory-associated behavior, Mood disorders and Reward behavior. Furthermore, some effects/outcomes appear more than once and can be linked to more than one of the 4 behavioral clusters (Table 1).

Aggressive behavior
Aggressiveness could be found in any mammal, likewise it stands as an important preserved behavior since mouse to humans. In this sense, Rodriguez-Arias, et al (2013) demonstrated that CB1R could be present in aggressive modulation in mice model (Rodriguez-Arias et al., 2013). Similarly, later the same group also showed that CB2R-knockout mice are more aggressive than wild type (WT), whereas acute administration of CB2R-agonist attenuate the aggressiveness in WT after isolation, while the CB2R-antagonist reversed it, in addition the CB2R-knockout mice did not have its aggressiveness changed by isolation, indicating a modulatory effect on aggressive behavior by CB2R (Rodriguez-Arias et al., 2015). Thus, supporting the eCB system as an aggressive behavior modulator by both CB1 and CB2 receptors.

Memory-associated behavior
Memory could determinates our decisions and consequently our behavior. Therefore, murine models have been used to quantify it by behavioral tests. In this sense, Garcia-Gutierrez, et al (2013) reported an impairment in the short and long term contextual memory in CB2R-knockout mice and in CB2R-antagonist acute treatment, while the CB2R-agonist acute treatment improved it in Step-down inhibitory avoidance test (SDIA), however it was not clear if these differences are in contextual memory itself or in the exploratory behavior: CB2R-knockout mice, for example, expressed difference against WT in the train part of SDIA in this study, suggesting then an exploratory behavior modulation which may have confounded the results (Garcia-Gutierrez et al., 2013).
Similarly, Lim and Kim (2016a) had controversial results to linked the lack of CB2R to contextual memory either, CB2R-knockout mice showed a contextual memory impairment by decreased of freezing in contextual test without difference in cued test of Fear conditioning behavior test (FCBT), as well as the CB2R antagonist acute administration did not promote any difference in FCBT or Y-maze test (YMT), furthermore CB2R-knockout mice showed increased in spontaneous alteration in YMT (Li & Kim, 2016a). Later, the same group brought out an elegant study, they reported that the overexpression of CB2R in pyramidal cells or interneurons from CA1 area of hippocampus did not promote any change in YMT, FCBT or Novel object test (NOT), whereas the lack of CB2R in pyramidal cells from the same area by CRISPR (Clustered regularly interspaced short palindromic repeats) promotes increased in spontaneous alteration in YMT, as well as, by the same methodologies, the overexpression of CB2R in microglial cells from CA1 area of hippocampus promotes increased of freezing in contextual text of FCBT, whereas the lack of CB2R promotes a decreased of freezing in the same test, however this lack also promotes an improvement in NOT (Li & Kim, 2017). Indicating then, an improving of spatial work memory by the lack of CB2R, however it is not clear yet a CB2R modulation on fear memory or spatial memory, as well as it also not clear if that modulation is mediated by neurons or microglial cells.
In addition, in mice models of neural diseases, the CB2R-agonist chronic treatment has not showed any difference in spatial memory in Morris water maze test (MWM) in Alzheimer's disease model (J. Wu et al., 2013), and has showed a memory-associated recognition impairment in NOT in a stroke model (Ronca et al., 2015). Thus, despite the CB2R-agonist treatment attenuate damages in these and others neural diseases in mice models, it has not showed good outcomes in memoryassociated behavior. In the other hand, the eCB system stands present in memory-associated behavior, especially in fearassociated behavior and this modulation is more associated with the CB2Rs present in microglial cells, thus it must to be considerate in adoption of treatments with CB2R target drugs.

Mood disorders
Beyond the neuroinflammatory diseases, the eCB system has been also investigated over the mood disorders, which the murine models are large used in these models. Thereby, García-Gutiérrez, et al (2012) has showed that synthetic CB2Rantagonist acute-treatment has an anxiogenic effect, whereas the synthetic CB2R-agonist acute-treatment has no effect in In the other hand, Li & Kim (2016a) has showed that CB2R-knokout mice and CB2R-agonist acute-treatment does not have effect in the exploratory behavior into aversive areas of typical tests of anxiety-like behavior (Li & Kim, 2016a).
However, the use of knockout mice to research for eCB system roles is questionable because eCB system is present in Central nervous system (CNS) development (Harkany et al., 2007). In addition, it is unclear whether the typical tests of anxiety-like behavior in mouse models, such as OFT, EPM, LDB and Zero-maze test (ZMT) evaluate precisely this behavior (Ennaceur & Chazot, 2016). Moreover, recently it has showed that the overexpression of CB2Rs in glutamatergic neurons from CA1 area of hippocampus also induced increased of activity in the central area of OFT, without alteration in ZMT (Li & Kim, 2017). It suggests that the increased of exploratory activity in aversive area of OFT could be does not associated with anxiety-like behavior. Thus, it must to be more investigated.
Furthermore, over the depression-like behavior, Bahi, et al (2014) also demonstrated that BCP acute-treatment decreased the latency to eat in Novelty suppressed feeding test (NSFT), and decreased the immobility time in Forced swim test (FST) and in Tail suspension test (TST) in mice model, whereas the action of acute-treatment with CB2R-antagonist reversed these results (Bahi et al., 2014). It supports the use of CB2R as a pharmacological target to mood disorders, however there are few data to corroborate it yet. Nonetheless, the BCP treatment has other targets beyond CB2R (Gertsch et al., 2008;Hashiesh et al., 2020), which means it is necessary an investigation for its outcomes in mice models of depression-like behavior.

Reward behavior
The eCB system has been showed in mesolimbic pathway, thus in the modulation of reward behavior, which is a fundamental target in the mitigation of side effects from psychoactive drugs. In this sense, the chronic activation of CB2Rs in

Systemic CB2R-agonist chronic-treatment
It Reversed the effect of agonist the hippocampus increased synaptic transmission (Kim & Li, 2015). Completely, CB2R-kockout mice has decreased of excitatory synapse in the hippocampus (Li & Kim, 2016b). As well as, the strength of hippocampus nucleus-accumbens (NAc) synapse drives conditioned place preference, similarly as the use of anti-depressive drugs is linked to the increased of this pathway, and differently as the chronic stress is linked to the decreased of this pathway (LeGates et al., 2018). The NAc has an important role in mesolimbic pathway about the regulation of dopamine levels, motor activity and NAc is also linked to behavior changes induced by drugs abuse intake, however NAc roles remains unknown, but the hippocampus-NAc input has showed important modulatory effect in reward behavior (Perez & Lodge, 2018;Scudder, Baimel, Macdonald, & Carter, 2018).
Therefore, we suggest that the increased in central area of OFT by CB2R-acute treatment (Bahi et al., 2014) or overexpression of CB2Rs in glutamatergic neurons from CA1 area of hippocampus (Li & Kim, 2017) would be linked to a eCB system neuro-modulatory effect in reward behavior by an increase of exploration, instead of anxiety-like behavior.
In addition, the eCB system has also linked to mesolimbic pathway by presence of CB2Rs in dopaminergic neurons of ventral tegmental area (Zhang et al., 2014). Completely, the systemic, the intranasal or the intra-accumbens acute treatment with CB2R-agonist inhibits the cocaine self-administration and inhibits the increased of hyper activity induced by cocaine, whereas this drug effect is absent in CB2R-knockout mice (Xi et al., 2011). Similarly, Lopes, et al (2019) also demonstrated that acute administration of CB2R-agonist inhibits hyper activity induced by cocaine, whereas the CB2R-antagonist reversed it (Lopes, Bastos, Costa, Aguiar, & Moreira, 2020). It supports an interesting application in mitigation of side effects of abuse drugs by pharmacological treatment of CB2R target drugs.

Conclusion
In summary, after few years of eCB system discovered, there are a range of studies and reviews about its modulatory effect on CNS, and it is clear a large potential of the use of eCB system as a pharmacological target for many diseases, especially in immune and neural diseases, however, it remains unknow these effects on behavior. In this review we systematize 12 methodologies, 33 effects/outcomes from 10 studies in 4 behavioral clusters (Table 1). Thus, we conclude the eCB system modulates behaviors by CB2R in mouse models, which includes Aggressive behavior, Memory-associated behavior, Mood disorders and Reward behavior, as well as, the CB2R target drugs has a large potential to be a pharmacological treatment in Mood disorders and mitigation of side effects promoted by abuse drugs. However, in spite of a considerable number of studies that linked the presence, the lack, the activation or the antagonization of CB2R to behavioral changes, there are a few answers about which other behaviors could be modulated by it, as well as, there is not sure about all consequences of acute or chronic treatment with agonists or antagonists of CB2R. This lack of answers is promoted both by the complexity of eCB system and the controversial interpretations in behavioral tests. In this sense, it is necessary future researches to multi target treatments into eCB system, including receptor side effects and physiology and pharmacology behavioral effects modulated by CB2R. As well as, the development and/or the update of behavioral tests that can evaluate the correlation of behavioral parameters and eCB system should be provide.