**4. Discussion**

In this study, we demonstrated that administration of THC to adolescent mice does not cause anxiety-like behavior in adult mice nor affect basic locomotor activity. Further, we showed that impairment of the ghrelin signaling through the knockout of the GHRS does not confer an increased risk of developing THC induced anxiety in adult mice. Our results are consistent with previous reports in rodent models that concluded that prolonged adolescent THC exposure in mice does not have substantive negative impacts on several mPFC-mediated behaviors [47–50]. In particular, Chen et al. [49] treated 28-day-old C57BL6/J mice of both sexes for three weeks with 3 mg/kg THC (daily intraperitoneal injections i.p.). One week after recovery, they analyzed several cognitive behaviors and

detected little effect on anxiety-like behaviors. In another study, Zuo et al. [48] treated female and male mice with 10 mg/kg of THC in early adolescence (1 i.p. for 21 consecutive days during postnatal weeks 5–7) and assessed the impact on anxiety-like behaviors two weeks later. Their behavioral analysis demonstrated that adolescent exposure to THC in mice led to long-term impairments in object recognition, memory and social interaction, but not in anxiety-like behaviors. The experimental evidence on long-term effect of cannabis exposure during adolescence includes cannabidiol (CBD) as well, a non-intoxicating phytocannabinoid. Prolonged adolescent CBD exposure had no detrimental effects on locomotor activity in the open field and anxiety-like behaviors on the elevated plus maze in male and female C57BL/6J mice treated for 20 days mg/kg with two daily i.p. injections of CBD (20 mg/kg) [47].

Our exposure period starting at postnatal day (PND) 42 and ending at PND 55 represents the mouse brain development period similar to human adolescence [51]. Earlier findings identified this period as the critical time window for persistent detrimental effects of cannabis misuse [52,53]. Cannabis mainly acts on the developing cerebral cortex, especially the medial prefrontal cortex, a late-developing brain region whose volume decreases dramatically during adolescence as it undergoes synaptic refinement [54].

Interestingly, our results show that impairing ghrelin signaling through GHSR knockout does not affect the long-term outcome of the THC treatment. Since ghrelin and THC often act synergistically in many pathways [37], the results on GHRS KO mice further corroborate the lack of significant long-term alterations of anxiety-like behavior induced by THC in our experimental setting.

Preclinical studies collectively sugges<sup>t</sup> that ghrelin/GHSR reinforces the action of cannabinoids and CB1 agonists [55,56].

These studies imply the potential interaction of the ghrelin signaling with other neurotransmitter systems (the endocannabinoid, and GABA systems) within the NAC in the reinforcing effects of cannabinoids [57].

For example, the GHSR antagonist JMV2959 significantly reduced several parameters of cannabinoid reward and attenuated cannabinoid intake and drug-seeking behavior [56].

The ghrelin receptor can interact with the CB2 cannabinoid receptor in both heterologous cells and cells of the central nervous system [58].

Overall, the long-term outcomes of cannabis exposure during adolescence are complex and can result from multiple factors. Early life interferences such as maternal deprivation or immune system activation could increase the vulnerability to cannabis-related developmental insults [59,60]. Investigations into the neurodevelopmental exposure to THC in translational animal models could provide insights into various neural pathways and biomarkers involved in THC-related pathological outcomes, identifying potential molecular targets for novel pharmacotherapeutic approaches [61].

#### *Limitations and Future Studies*

In this study, we used the vaporization method to administer THC. Compared to i.p. injections, the pharmacodynamics of the THC following vaporization is less known. Future research should establish the dose and plasma level relationship for the vaporization method using the liquid chromatography–mass spectrometry (LC/MS) method, as performed before [41,42]. Moreover, we used a lower temperature for the vaporizations, thus we should assume that less of the material was vaporized compared to the original papers [41,42]. Most of our animals exposed to THC displayed instant changes in behavior, such as headshakes (similar to hallucinogen-induced headshakes), hyperactivity, or mild somnolence. Considering that the experiments on rats, from the original study, showed a hyperactivity phenotype at 1 mg and hypomobility/somnolence with 10 mg of vaporized THC, we assumed that in our experiments, active THC reached levels between these two reported doses.

Changing conditions such as dose, the time of THC exposure, concomitant stress exposure, and presence/lack of food could clarify if there are any relevant conditions under which THC can significantly alter long-term anxiety-like behaviors with or without an intact Ghrelin/GHRS signaling. Male and female mice might be affected differently by THC exposure since the literature suggests that females may be more vulnerable to THC's effect on anxiety [62,63]. To address the possible sex differences and ghrelin's role in them, THC exposure should be coupled with a variety of adjunct treatments, such as sex hormone inhibitors and ghrelin. Lastly, additional behavioral tests, such as pre-pulse inhibition, marble burying, and tail suspension, should be employed to investigate whether THC exposure in late adolescence affects behaviors related to sensory gating, compulsiveness, and mood regulation.

**Author Contributions:** T.L.H. developed the concept; M.C.M. supervised the work; M.C.M. and M.S.-P. developed the experimental strategy with input from T.L.H.; M.S. provided throughout technical support; M.C.M. and M.S.-P. conducted experiments and analyzed data; M.S.-P., M.C.M. and T.L.H. wrote the paper with input from all authors. All the experiment were performed in Tamas L. Horvath's laboratory, Yale University School of Medicine, CT-06520 New Haven USA. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partly funded by the Swiss National Science Foundation (Early Postdoc. Mobility P2BEP3\_172252 to M.C.M.).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Animal Are and Use Committee of Yale University (protocol code 2019-07942).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Acknowledgments:** We would like to thank Connecticut Pharmaceutical Solutions, LLC for providing us with the compounds used in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
