The outcomes of our study for the first time demonstrated that CB receptor ligands are able to potentiate the antidepressant activity of bupropion and moclobemide in the recognized behavioural tests in albino Swiss mice. The endocannabinoid system attracted attention of scientists as a promising target for the antidepressant therapy a long time ago. It turned out that both CB₁ and CB₂ receptors are localized peripherally and centrally, including these parts of the brain that are involved in emotion-related responses, such as anxiety, low mood, or stress (i.e. the hippocampus, amygdala, prefrontal cortex) [14, 15]. In fact, Smaga et al. [16] demonstrated that rats with experimentally induced childhood- or adult-like depression (i.e. the Wistar Kyoto or olfactory bulbectomized animals, respectively) presented diverse alterations in the endocannabinoid system, which included down-regulation of CB receptors, disturbances in the brain levels of anandamide and 2-arachidonoylglycerol, and changed expression of enzymes implicated in synthesis or metabolism of endocannabinoids (i.e. N-acyl phosphatidylethanolamine-specific phospholipase d, monoacylglycerol lipase, fatty acid amide hydrolase, diacylglycerol lipase α). Outcomes from clinical and post-mortem studies generally confirmed the pre-clinical ones, since abnormal levels of endocannabinoids were detected in serum of untreated female patients with major depression [17], whereas increased concentrations of endocannabinoids were found in the prefrontal cortex of both alcoholic [18] and depressed suicide victims [19]. Monteleone et al. [20] showed that polymorphism in the CB₁ receptor gene may be associated with a higher susceptibility to the development of depression in humans, whereas Onaivi and colleagues [21] found out that Japanese depressed and alcoholic patients presented polymorphism in cannabinoid CB₂ receptors. Additionally, Minocci et al. [22] demonstrated a significant association between polymorphism in the CB₂ receptor gene and occurrence of bipolar disorders.

Despite their opposite functional activity, agonists and inverse agonists/antagonists of CB receptors, including the ones used in our study, can exert the same responses in animal behavioural tests measuring an antidepressant-like or anxiolytic-like potential. Kruk-Słomka and colleagues [10] demonstrated that both CB₂ receptor agonist—JWH133 (0.5 and 1 mg/kg) and CB₂ receptor inverse agonist/antagonist—AM630 (0.5 mg/kg) when given at an acute dose induced anti-immobility effects in the FST in Swiss mice. Similar activity also produced the CB₁ receptor agonist—oleamide (10 and 20 mg/kg) [10] and inverse agonist/antagonist—AM251 administered at the single dose of (0.5 or 0.3 mg/kg) in NMRI mice [23]. Interestingly, the responses induced by oleamide and JWH133 in the FST were attenuated/reversed by per se ineffective doses of AM251 (0.25 mg/kg) and AM630 (2 mg/kg) [10].

In the present study, we showed that oleamide (5 mg/kg), AM251 (0.25 mg/kg), and AM630 (0.25 mg/kg) augmented the antidepressant activity of bupropion (10 mg/kg) in the FST and the TST, whereas JWH133 (0.25 mg/kg) did not have such a potential. Surprisingly, antidepressant effects of moclobemide (1.5 mg/kg) were considerably enhanced in both behavioural tests only by AM251 (0.25 mg/kg). Neither oleamide (0.5 mg/kg) nor JWH133 (0.25 mg/kg) or AM630 (0.25 mg/kg) increased activity of moclobemide sufficiently enough to be recognized as a significant interaction by two-way ANOVA. Since the same observations were made in two world widely used behavioural tests designed for measurement of the antidepressant-like activity of substances belonging to diverse chemical classes, our results should be considered as reliable. Moreover, we would like to emphasize that the outcomes were not confounded by hyper- or hypolocomotion of animals. The distance travelled by mice from all tested groups was comparable to the one covered by the vehicle-treated animals. Though altered motility of rodents can be expected after administration of cannabinoids [24, 25], our outcomes were in line with results published by Kruk-Słomka and colleagues [10].

It is quite interesting that the CB₁ receptor ligands acting oppositely, i.e. oleamide and AM251, influenced the activity of bupropion in the same manner. However, we should admit that such a bidirectional potentiation of the antidepressant action by cannabinoids had been observed before in our laboratory. The results of our previous studies demonstrated that oleamide and AM251 when administered at the sub-threshold concentrations in albino Swiss mice can potentiate the antidepressant activity of imipramine (15 mg/kg), escitalopram (2 mg/kg), and reboxetine (2.5 mg/kg) [6]. They also enhanced the antidepressant-like properties of biometals (i.e. magnesium, 10 mg/kg and zinc, 5 mg/kg) inhibiting the glutamatergic neurotransmission [12]. Additionally, Kruk-Słomka et al. [10] reported that all of the CB receptor ligands used in the present experiments augmented the anti-immobility effects of anticholinergic scopolamine (0.3 mg/kg) in the murine FST. Apart from the antidepressant-like effects, endo- and exocannabinoids are also able to produce the depressogenic (as well as anxiogenic and anxiolytic) ones in pre-clinical studies [26]. A similar trend can be observed in cannabis users—some of them confirm beneficial effects of cannabis in the depressive disorders [27], whereas the others experience a higher number of depressive symptoms [28]. Though scientists admit that the exact mechanism of this peculiar bidirectional activity of the CB₁ receptor ligands has not been discovered yet, they propose several explanations for this phenomena: (1) influence of testing conditions (i.e. animal strain, applied dose, environmental factors) that can induce either depressive- or antidepressant-like changes in the endocannabinoid system [29], (2) existence of the functional pools of CB receptors that are responsible for manifestation of the antidepressant or pro-depressive effects [26], (3) influence of other, non-CB₁ and non-CB₂, cannabinoid receptors [23], or (4) specific localization of the CB₁ receptors in the opposing, i.e. inhibitory (GABA-ergic) and excitatory (glutamatergic) terminals [30].

We assume that the observed oleamide–, AM251–, and AM630–bupropion interactions as well as the AM251–moclobemide interaction may be the result of enhanced monoaminergic neurotransmission in the brain. CB₁ receptors influence release of acetylcholine, dopamine, glutamate, γ-aminobutyric acid, noradrenaline, serotonin, and different hormones that are involved in the depression-related behaviour. CB₂ receptors reduce secretion of pro-inflammatory and enhance secretion of anti-inflammatory cytokines, whereas the immune responses are also implicated in the pathogenesis of depression [24, 31, 32]. Additionally, cannabinoids are known to have an impact on the activity of adreno- and serotoninergic receptors [32–34] as well as on the functioning of SERT and NAT, i.e. the serotonin and noradrenaline transporters, respectively [32, 35]. When given at high doses, they can inhibit the activity of monoamine oxidase [36]. We guess that the interplay between the cannabinoids applied in the present study and bupropion could have been particularly attributed to the intensified dopamine-related mechanisms. Bupropion is a dual inhibitor of dopamine and norepinephrine reuptake, and stimulation of CB receptors affects the secretion of dopamine [37]. However, it should be noted that the observed results are not a consequence of the simple potentiation of dopamine activity in the brain. As described in the medical literature [38], CB₁ receptor ligands can either increase or decrease dopamine synthesis and dopamine release. Thus, the final effect of enhanced dopamine neurotransmission is most probably a result of a complex interplay between the endocannabinoid system and other neurotransmitters (such as GABA, glutamate, and others). It is difficult to say whether the AM630–bupropion interaction was also at least partially attributed to the CB₁ receptor-dependent mechanisms or not. One the one side, several authors noted that AM630 can behave not only as a CB₂ receptor inverse agonist/antagonist, but it can also act as a CB₁ receptor inverse agonist [39]; thus, it may exert similar effects to AM251. On the other hand, JWH133, which does not bind exclusively to CB₂ receptors but also has a low affinity to CB₁ receptors [40], did not affect the activity of bupropion. Actually, since AM630 and JWH133 act oppositely at CB₂ receptors, their different activity towards antidepressant effects of bupropion could have been expected. Furthermore, a significant functional selectivity of the CB₂ receptor ligands found by several independent research teams [41–43] could have contributed to the observed interplays in the behavioural studies. It was revealed that certain CB₂ receptor ligands may activate diverse downstream intracellular pathways or may activate the overlapping ones but with different potency.

As for the AM251–moclobemide synergism of action, the potentiation of the serotoninergic signalling may be responsible. Monoamine oxidase inhibitors inhibit metabolism of serotonin and sympathomimetic amines, whereas AM251 does not directly interact with adrenergic receptors but it interplays with the serotoninergic ones. According to the literature data [44], the 5HT_1A receptors seem to be involved in the behavioural effects of AM251 treatment. Furthermore, Hill et al. [45] demonstrated that inhibition of monoamine oxidase (by tranycypromine) influenced cannabinoid receptor binding and changed the levels of endocannabinoids. We think that other mechanisms can also contribute to the drug–drug interactions observed in our study, since AM251 modulates the opioid signalling [23], whereas oleamide interplays with serotoninergic 5-HT_2C and 5-HT₇ receptors and affects the benzodiazepine receptor- and vanilloid receptor-dependent transmissions [46]. All of these pathways are directly and/or indirectly involved in the mood control.

Pharmacokinetic analyses carried out in the present study allowed us to assess concentrations of bupropion and moclobemide in mice brains after their combined administration with CB receptor ligands. This approach was aimed at determining drug–drug interactions involving changes in drug disposition. Augmentation of bupropion and/or moclobemide levels in the brain might have been an indication of the facilitated transport of these drugs through the blood–brain barrier [47] in the presence of alterations in the endocannabinoid signalling. However, though oleamide, AM251, and AM630 intensified the anti-immobility responses recorded after administration of bupropion, none of them augmented the brain levels of this antidepressant drug. Similarly, potentiation of moclobemide effects in the FST and in the TST by AM630 was not accompanied by an increase of moclobemide concentration in the mice brain. Based on the above-mentioned results we believe that the drug–drug interactions detected in the behavioural tests were due to processes at the cellular level, so they are pharmacodynamic in nature. Molecular studies are needed to explain the observed synergism of action.

The positive interaction between CB receptor ligands and bupropion and/or moclobemide found out in our study seems to be important from the clinical point of view, since concurrent administration of antidepressants and agents affecting the endocannabinoid system (particularly via CB₁ receptors) may improve the safety profile of the introduced treatment (due to dose reduction of either substances). Based on the literature data [9], we can also hypothesize that introduction of such a drug–drug combination may accelerate the alleviation of depressive symptoms (due to the fast onset of cannabinoids biological activity). However, further tests are needed to verify this supposition.