As far as we know, this is the first study to examine the interplay of cannabis use and the endocannabinoid receptor genes on functional neuroimaging-derived phenotypes in patients with a first-episode of psychosis. We did not detect an effect of cannabis use on brain activity when performing the n-back task. Instead, our data indicate that genetic modulation has an impact on the effect of cannabis use on brain response.

Regarding the analysis of the main effect of cannabis use on the n-back task-related brain activity, we did not observe differences between CU and CNU. These findings diverge from previous fMRI studies on schizophrenia, which showed higher activity rates in CU as compared to CNU in multiple brain areas associated with different cognitive dimensions (executive functions, verbal processing and attention, emotional processing and visuospatial abilities) [40,41,42]. However, our results should be interpreted in light of several aspects.

First, these previous findings were based on different tasks (e.g., emotional memory task, mental rotation task, cognitive task-present and task-absent conditions). Second, the design based on first-episode psychosis cases limits the main cannabis use effect on brain function before the onset of the psychosis, while schizophrenia-based samples (such as in previous fMRI studies) do not exclude the impact of cannabis use after the illness onset. Third, these previous studies include patients with an illness duration longer than ten years, which hampers the direct comparison of the result because duration-related illness factors can also affect brain activity. Fourth, general population-based fMRI studies performed with CU in the non-intoxicated state predominantly show the opposite effects to those observed in chronic patients with schizophrenia. Thus, CU show attenuated brain activity or activation of compensatory regions compared to CNU (for review, see [57]). In contrast, the scarce fMRI literature about patients diagnosed with schizophrenia suggests that CUtend to show better preserved functioning in areas associated with the task than non-users [40,41,42].

Concerning our cannabis × gene approach, our data seem to support the idea that the individual genetic background of the ECBS may influence the effect of cannabis on the brain’s functional response to specific cognitive demands, which could be linked to the participation of this system in the vulnerability for psychosis [44,45]. Notably, we found evidence of a genotype × cannabis use interaction for both endocannabinoid receptor genes (CNR1 and CNR2) in the 2-back vs. 1-back contrast, suggesting a modulation effect of genetics on neuronal dynamics related to working memory after cannabis exposure.

For CNR1, we observed an interplay between rs1049353 and cannabis use in areas that are known to have reward-related functions [58,59]. In line with our results, the ECBS has been implicated in reward-processing and reward-seeking behaviour (reviewed in [60]), which is also supported by the fact that CB1R is densely expressed in areas associated with reward processing (e.g., hippocampus, amygdala, prefrontal cortex, anterior cingulate, striatum or VTA). In this sense, substance abuse is associated with increased activity of dopaminergic neurons, which, in turn, release endocannabinoids through the activation of CB1Rs in GABA or glutamate-containing neurons [18]. Then, alterations in the ECBS by external ligands could lead to dysregulation in their associated neurotransmitter systems and functions.

Regarding the CNR2 gene, our findings also suggest that the polymorphism rs25014131 and cannabis use are associated with brain activity changes in patients with a first-episode psychosis. While brain CB2R has lower expression levels than CB1R [20], its expression appears to be altered under certain pathological conditions (e.g., addiction, inflammation, and anxiety), suggesting that these receptors are highly inducible (reviewed in [22]). In this regard, Ishiguro et al. [61] described an increased risk of schizophrenia among people with low CB2R function, measured through its expression. These authors also identified functional changes in CB2R attending to nucleotide variants. Thus, according to these data [61], it could be hypothesised that, in patients with psychotic disorders, CNR2 genetic variability influences CB2R activity and, finally, brain activity due to the regulation role of ECBS on other neural systems. Interestingly, we found opposite brain activation patterns for CNR1 and CNR2 according to cannabis use. While further data are needed, this result could be interpreted in line with the described possible opposite roles of CB1R and CB2R [23] in the reinforcement processes of substance abuse and dopamine-related behaviours.

Finally, when looking at the task execution, aligned with Løberg et al. [34] or Potvin et al. [40], we did not find differences in behavioural performance between CU and CNU. However, regarding the interaction effect between genetic variability and cannabis use on task performance, we report a significant impact of CNR2, but not of CNR1. Task performance in CU was similar for both genotypic CNR2 variants, while among the CNU, the genotype seems to play a differential modulating role. Interestingly, when analysing together the functional and behavioural data, we observe that the genotypic groups showing a larger modulatory effect in response to the increasing task difficulty also show a wider task performance drop between the 2-back vs. the 1-back task (Gcar -for CNU- and AA -for CU).

Our study should be interpreted in the context of some limitations. First, we identified cannabis use from self-reports and medical records, and we used cannabis use as a dichotomised no/yes category. While this classification has also been implemented in previous studies [34,42,50], more extensive cannabis intake data (% of THC, age at onset, frequency, consumption via, etc.) will be valuable in future studies. Second, the sample size of our study limits our conclusions, and that is why it should be considered as a pilot approach that warrants further research. However, our sample based on the first-episode of psychosis contrasts with the previous fMRI and cannabis use studies to date [40,41,42], based on patients with chronic schizophrenia. To avoid potential confounding effects of differences associated with cannabis use before and after the onset of the psychotic symptoms, studies focused on the first-episode are especially useful. In addition, while we are aware of the imbalanced sample size regarding gender (77.5% males) as recent studies have proven sex-dependent effects of cannabis [62] and the ECBS [63], from our sample size, we could not develop sex-specific analyses. In this sense, our analyses of the whole sample must be compared with previous studies using mixed samples and also showing a predominance of males [42,50]. Third, the unavailability of a control group restricts interpretations of the role of cannabis use and genetic make-up to early phases of psychotic disorders and precludes analyses in terms of health–disease status and vulnerability. Finally, to improve our understanding of causal interactions between relevant factors for psychosis, longitudinal designs would be more appropriate. For instance, future studies would benefit from follow-up studies of high-risk individuals and the analysis of cannabis use jointly with the genetic profiling of different endocannabinoid system components [45]. Additionally, based on recent data showing different epigenetic factors contributing to the regulation of cannabinoid receptors [64,65] and epigenetic changes linked to the conversion to psychosis [66], the combined analysis of genetic and epigenetic variants appears as a promising avenue for novel therapeutic targets and innovative treatment strategies.