The present study showed that THCV can block the discriminative stimulus effects of THC and partially substitute for THC in a dose-dependent manner. Thus, THCV may act as a partial agonist on the same receptors that THC produces its effects. Also, THCV increases the response rate at the dose it partially substitutes THC, which implies it can have stimulant properties. Finally, the observed effects were free from a possible interaction resulting in a change in blood levels of THC and its active metabolite 11-OH THC.

In vitro studies characterized THC as a partial agonist at both CB1 and CB2 receptors with moderate affinity and with no apparent receptor subtype selectivity. Thus, the activation of these target receptors will depend on the presence and the coupling efficiency of other ligands, reserve receptor density, and signal transduction efficiency. A wide variety of behavioral effects of THC is observed in vivo when other factors, such as biased agonism and endocannabinoid system regulatory activities, are considered. Similarly, THCV presents a complex pharmacological profile as indicated in numerous in vitro and in vivo assays [17,21,22,23]. In a previous review by McPartland et al. (2015) [6], THCV was suggested as a neutral antagonist of CB1 receptors and a partial agonist on CB2 receptors while it may show an indirect agonism at CB1 receptors by inhibiting endocannabinoid tone. A recent study confirmed that it has CB1 antagonist, but not agonist, effects in vitro [23]. However, there are studies implying THCV produces some in vivo activity through inverse agonism at CB1 receptors, and shows some opposing effects compared to other CB1 antagonists depending on the model, dose, and the availability of the other ligands [6]. Using the drug discrimination method provided a unique opportunity to compare the “interoceptive” effects of THCV and THC that represents a final behavioral outcome of the complex interplay between different interactions [24].

There are only a few studies that have tested THCV in animals trained to discriminate THC. In contrast to our results, a recent study by Moore et al. (2023) [17] indicated that THCV did not substantially alter THC’s discriminative stimulus effects across a dose range of 3–30 mg/kg, but it slightly decreased THC-associated responding at the higher doses in some cases, which the authors attributed to possible sedative effects rather than a direct modulation of THC’s interoceptive effects. We observed the sedative effects of THCV at 6 mg/kg dose when it was combined with THC 1.5 mg/kg. When it was given alone at the substitution experiments the response rate with the same dose (6 mg/kg) of THCV was not different from the control but there was a decreasing trend in lever pressing rate, and we did not try a higher than 6 mg/kg dose of THCV in these experiments.

In comparing our findings with those of Moore et al. (2023) [17], several key procedural differences may account for the contrasting results regarding THCV’s interaction with THC in a drug discrimination model. First, we performed our experiments in male Sprague-Dawley rats while Moore at al. used both male and female Long Evans rats. Our use of Sprague-Dawley rats, as opposed to the Long Evans rats employed in Moore et al., may have contributed to the observed differences, as strain-specific responses to cannabinoids have been documented in the literature. For instance, Deiana et al. (2007) [25] demonstrated that Long Evans and Lister Hooded rats, but not Sprague-Dawley rats, acquired and maintained self-administration of the cannabinoid CB1 receptor agonist WIN 55212-2, indicating significant strain differences in cannabinoid reinforcement behaviors.

Second, an important difference between the two studies was the timing of the drugs. Moore et al. administered THC 45 min before the session, followed by THCV (or another minor cannabinoid) given 15 min prior to the session. In contrast, in our study THC was administered 30 min before the session, while THCV was administered 20 min prior. This modified timing might create greater overlap in the peak activity of both compounds, allowing us to observe a dose-dependent partial substitution for THC by THCV at certain doses and an antagonistic effect at higher doses, where THCV reversed THC-associated responding.

In a study by Zagzoog et al. (2020) [9], the affinities of different cannabinoids were evaluated for the CB1 and CB2 receptors in order to further understand their pharmacodynamics. The compound CP55940, which has equal affinity to CB1 and CB2 binding, was used to test the characteristics of the cannabinoids. It was found that THCV partially displaced CP55940 from the CB1 receptor which indicated binding at an independent site on the receptor, or non-competitive binding. The same findings were concluded for the CB2 receptor as well. This aligns with our observation that THCV exhibits partial substitution effects at lower doses while antagonizing THC’s effects at higher doses. These findings emphasize the notion that THCV’s actions are modulated not only by direct receptor interactions but also by the presence of other ligands such as THC in the cannabinoid system components. Moreover, our findings support that THCV’s pharmacological effects are highly dose-dependent and influenced by complex receptor interactions, as seen in both in vitro studies and in vivo behavioral assays.

Our study also identified stimulant-like properties of THCV at doses where partial substitution for THC was observed, as evidenced by increased response rates. This finding challenges the conventional view of THCV as a cannabinoid devoid of psychoactive effects [10]. However, we did not perform additional tests specifically designed to assess psychostimulant activity, such as open field tests, locomotor activity assays, or detailed behavioral observations, to further explore this effect, and our claim is based solely on the observed increase in lever pressing rates during the test sessions. Another explanation for the increased response rate can be an anxiogenic or obsessive-like behavior exacerbation. This is consistent with a recent finding that THCV increased the number of marbles buried in a marble burying test [26]. Increased number of intrusions in a verbal recall task after a five-day dosing of THC and THCV combination in healthy cannabis users could indicate a possible pattern of obsessive or perseverative behavior [7]. Moreover, a recent placebo-controlled study investigating the acute effects of oral THCV treatment reported that euphoric mood was the most reported adverse effect [8]. Further studies employing similar dosing regimens or combinations with cannabis/THC in different human participants (i.e., healthy participants, chronic cannabis users) are essential to better understand the behavioral and physiological implications of these findings. Moreover, a better understanding of the effects of major and minor cannabinoids on cognitive processes could also clarify the increased response rates [27]. Lastly, combining these behavioral studies with measures of oscillatory activity could further inform our study of the neural basis of some of the perplexing behavioral interactions between THC and THCV [28].

In the present study, the observed behavioral effects are not due to an altered metabolism of THC or through its active metabolite 11-OH THC. The absence of such pharmacokinetic interactions is noteworthy given that cannabinoids often exhibit complex metabolic interactions, including competition for cytochrome P450 enzymes or modulation of drug-metabolizing enzymes [29]. This finding is consistent with previous reports that THCV does not significantly alter THC metabolism at the doses tested [7]. However, our results reflecting the peripheral blood concentrations do not rule out potential local interactions within the brain or other tissues. Regional differences in drug distribution or metabolism, particularly in areas dense in CB1 receptors, and receptor-level competition could still play a significant role in the observed behavioral outcomes [30].

While our findings advance our understanding of THCV’s discriminative effects, several limitations should be noted. First, our use of only male Sprague-Dawley rats greatly limits generalizability of our findings due to the previously documented strain- [25] and sex-dependent [19,31,32] effects of cannabinoids. Moreover, previous studies have demonstrated that male rats develop less tolerance to the effects of THC in the tetrad test. Moreover, anxiogenic effects were observed in female rats after THC treatment, whereas THC decreases anxiety in male rats [30]. The blood and brain concentrations of THC as well as its active metabolite 11-OH-THC were also found to be higher in female rats after edible THC consumption [19]. There is limited data on sex- and strain-dependent effects of THCV, but future studies focusing on these factors are warranted to determine whether similar patterns emerge for this compound. Second, we used food restrictions to maintain operant responding, and a sucrose pellet was used as the reward, both common practices in drug discrimination studies. Both THC and THCV have known effects on feeding and satiety. While THCV has been reported to suppress appetite [10], THC exhibits an orexigenic effect and increases food intake initially; however, tolerance to this effect may develop in rats after chronic treatment [33]. These interactions with feeding-related pathways could modulate motivational or interoceptive states and potentially influence discrimination performance independently of cannabinoid-like subjective effects. Finally, our study did not further evaluate whether the partial substitution of THCV could be reversed by CB1 or CB2 antagonists such as rimonabant or SR144528. These studies would be required to confirm that the observed effects are mediated by a cannabinoid receptor-mediated mechanism. However, since both THC and THCV display complex pharmacology, including partial agonist and dose-dependent effects at CB1 receptors, as well as activity at CB2 and non-cannabinoid targets, the antagonist combination studies would become particularly challenging to interpret. Moreover, the combinatorial outcome may depend not only on dose, but also on the relative efficacy, affinity, and dynamic balance between agonist and antagonist actions at multiple sites. While antagonist studies are an important future direction, the present findings provide a critical first step in establishing that THCV can partially substitute for THC in a drug discrimination paradigm.