Interaction between endocannabinoids and the adrenergic system before and after stress-exposure

Cold stress-induced analgesia (c-SIA) has been evaluated in male Wistar rats injected with cannabinoid receptors type 1 and a2-adrenergic receptor agonists and antagonists in different combinations before or after stress exposure. The aim of the study was to evaluate whether the endogenous cannabinoid and the adrenergic systems influenced c-SIA, and the patterns of their potential interaction. Exogenous administration of anandamide and Clonidine together, before or after stress exposure, increased c-SIA even with differences in the time of manifestation of the effect, its duration and the degree. The two systems differently contribute to c-SIA pathogenesis and mediation. Administered before stress exposure cannabinoids and the adrenergic system seem to oppose each other: the latter rather potentiates, while cannabinoids suppress c-SIA. Administered after stress exposure, instead, the two systems appear to exert a synergistic effect, and antagonization of each one of them abolishes the analgesic effect.


Introduction
Pathophysiology of nociception has been broadly investigated, given the negative sensory, emotional, cognitive, and social effects of pain experiencing. The endogenous cannabinoid and the adrenergic systems have been "traditionally" associated with pain perception (Nadal et al. 2013;Ellison 2017; for a review on endogenous cannabinoid system see Lu and Mackie 2016). Interactions be-tween the two systems have been described with implication for various pathologic conditions and disorders (Carvalho and Van Bockstaele 2012;Reyes et al. 2012;Gazarini et al. 2014;Nasehi et al. 2016;Meeran et al. 2021), as well in pain processing. Cannabinoid induced antinociception was inhibited by a 2 -adrenergic receptor antagonist yohimbine (Lichtman and Martin 1991) or destruction of catecholaminergic projections to the spinal cord (Gutierrez et al. 2003).
On the other hand, both the endocannabinoid and the adrenergic systems are engaged in the stress-response (Gorzalka et al. 2008;Yapıcı-Eser et al. 2018).
The aim of the present study was to evaluate whether the two systems affect stress-induced analgesia -a wellknown phenomenon, developing after stress exposure. Cannabinoid receptor type 1 (CB1R) and a 2 -adrenergic receptor (a 2 -AR) agonists and antagonists were administered in different combinations before and after stress exposure in order to estimate whether: • the two systems interact between them affecting c-SIA; • the potential interaction differs before and after stress. Administration before stress would impact on the pathogenesis of stress-induced analgesia, while once the stress has been induced a potential modulatory effect of the two systems would be observed instead.

Animals
In vivo experiments were conducted on adult male Wistar rats Rattus norvegicus weighing 200±20 g. The rats were kept at room temperature (22±1 °C), maintained under a 12h/12h light/dark regime, and supplied with standard chow and water ad libitum (Gordon et al. 2017). All experimental procedures were approved by the Ethics Research Commission of the Medical University-Sofia.

Acute model of cold stress
Acute cold stress was induced by placing the animals at low environmental temperature (4 °C) for 1 hour. During the time of cold exposure no food and water were allowed; the rats could move freely, allocated in individual cages without sawdust.

Nociceptive test
Paw-pressure test (PP; Randall-Selitto test): The changes in the mechanical nociceptive thresholds of the rats were measured by an analgesimeter (Ugo Basile). In brief, pressure was applied to the rat hind-paw and the pressure (g) required for eliciting a nociceptive response, such as a squeak or struggle, was taken as the mechanical nociceptive threshold (represented in arbitrary units, AU, according to scale). A cut-off value of 500 g was observed to prevent damage of the paw.

Statistical analysis
In vivo results were statistically assessed by one-way analysis of variance (ANOVA) followed by Newman-Keuls post-hoc comparison test. Values were mean ± S.E.M and these of p < 0.05 were considered to indicate statistical significance.
The same combinations (agonist+antagonist) were administered also before 1h CS: AEA+Yoh+1h CS and AM+Clo+1h CS (Fig. 2B). In the AEA+Yoh+1h CS-group a decrease in c-SIA was observed, while an increase during the whole estimated time resulted in the AM+Clo+1h CSgroup compared to controls, 1h CS and 1h CS+AEA+Clo.
Compared to animals with 1h CS, AEA+Clo administration both before and after stress led to a statistically relevant increase in PPTs, even though manifested at different time and to a different degree. Our experiments point at an interaction between the cannabinoid and the adrenergic systems in both mediation and modulation of c-SIA, but the two systems` interaction follows a different pattern before and after stress exposure.
Administration of AEA+Clo after stress led to a brief initial decline in PPTs followed by a substantial increase. It seems that both systems potentiate analgesia, being interdependent in c-SIA modulation, and inhibition of each one of them decreases PPTs. Studies have demonstrated that endocannabinoids tonically gate the stress response through the hypothalamic-pituitary-adrenal (HPA) axis, with their levels decreasing following acute stress, allowing activation of the HPA stress response (Gorzalka et al. 2008). HPA axis activation, on the other hand, leads to AEA levels increase (Di et al. 2005), and neurotransmitters, such as noradrenaline, adrenaline, and acetylcholine to be released (Bell 2018).
As to their participation in the pathogenesis of c-SIA (administration before 1h CS), it seems that the two systems have the opposite effects: endocannabinoids suppress, while the adrenergic system potentiates c-SIA.
Such results motivated us for an additional trial with administration of AEA, AM, Clo, and Yoh each one alone before 1h CS.
PPTs evaluation showed AEA alone administration decreased c-SIA after the 20 th min of the experiment -the curve slope resembled the one of the AEA+Clo+1h CS animals. Administration of Yoh along with AEA decreased c-SIA showing even a tendency toward hyperalgesia on the 30 th and 40 th min of the experiment (Fig. 3A).
On the contrary, AM and Clo potentiated c-SIA each one administered alone, and administration together led to a similar effect (Fig. 3B).
The results are consistent with experimental findings about cannabinoid induced inhibition of neurotransmitter release (Schlicker and Kathmann 2001;Szabo et al 2002;Doherty and Dingledine 2003), and particularly, a study demonstrating that pre-treatment with a cannabinoid receptor agonist diminishes stress-induced noradrenergic transmission (Reyes et al. 2012).
It seems that activation of cannabinoid receptors before and after stress exposure differently influences the stress-reaction.
Even though the usefulness of cannabinoids in the treatment of diseases is known back from ancient times (Baron 2015;Bridgeman and Abazia 2017), the underlying mech-anisms for such a therapeutic value were first described in the last decade (Kogan 2007;Atakan 2012;. The increased interest in the dynamics of cannabis and its derivatives in diseases and pathologic conditions treatment (Chiurchiù et al. 2018;Reddy et al. 2020) and even their implication in processes of aging  led to the concept of "medicinal cannabis" to become the focus of numerous studies and clinical trials. In this regard, understanding differences in cannabinoid receptors effects in physiologic and pathologic conditions, as well as their interactions with other receptors, could be of some help.

Conclusion
Exogenous administration of AEA and Clonidine together, before or after stress exposure, increased c-SIA even with differences in the time of manifestation of the effect, its duration and the degree.
The two systems participate (and interact) in both the pathogenesis (administered before stress) and in the modulation (administered after stress) of c-SIA.
The two systems differently contribute to c-SIA pathogenesis and mediation. As regards c-SIA-pathogenesis, endocannabinoids and the adrenergic system seem to oppose each other: the latter rather potentiates, while endocannabinoids suppress c-SIA. As to c-SIA-modulation, instead, the two systems appear to exert synergistic effect, and antagonization of each one of them abolishes the analgesic effect.

Conflict of interest
The authors declare no potential conflict of interest.