*2.2. Animals*

The Institutional Animal Care and Use Committee of Yale University approved all experiments (protocol code 2019-07942). Mice were kept under standard laboratory conditions with free access to standard chow food and water except during behavioral testing. Mice were generated by breeding C57BL/6J (n. 000664 Jackson Lab) with GHSR −/+ mice in order to obtain an F1 generation of heterozygous GHSR knock-out animals. These progenies were subsequently used to generate GHSR +/+ (WT) and GHSR −/− (KO) animals used in this study. All animals were generated, bred and weaned by our laboratory and housed in the same animal room. Further details can be obtained from our previous publication [39]. THC was administered to animals from 6 to 8 weeks of age and behavioral testing was performed at 12 to 13 weeks of age (Figure 1A). Animals were placed into 2 treatment groups (vehicle and 10 mg THC) for each genotype (wild type and knock-out). Since we did not have a strict hypothesis of what sort of difference we can expect, we used the "resource equation" method for defining our sample size, a commonly used way of establishing sample size in exploratory studies [40]. We did not control for the estrous cycle in female mice.

## *2.3. THC Administration*

Most adolescents smoke cannabis; therefore, we decided to mimic smoking as a method of administration of THC. We used commercially available vaporization equipment for marijuana administration, commonly used by marijuana consumers. Previously described experiments used the Volcano® Vapourization device (Storz and Bickel, GmbH and Co., Tuttlingen, Germany) to administer ethanol-dissolved THC to lab animals in a consistent and reproducible manner, and presented similar dose-dependent and timedependent changes for both pulmonary and parenteral administration [41,42]. We followed their procedure, except for the following modifications. In our study, the THC containing formulation and vehicle were vaporized at 175 ◦C in order to avoid excessive formation and vaporization of cannabinol (CBN, a psychoactive metabolite of THC which may confound results) and reaching the flashpoint of the vehicle (PEG400, 250 ◦C).

THC and vehicle were administered under a chemical hood to prevent cross-contamination due to leakage of vapor, as well as maintaining a consistent experimental environment (Figure 1B). Mice were placed, in groups of 2–4, inside a closed chamber (33 cm × 20.3 cm × 10.2 cm) with valves and tubing on two of the narrower sides. To further minimize vapor escaping, on one side the tubing led to an improvised activated charcoal trap, leading to an activated charcoal filter, leading to the vacuum line (Figure 1C,D). On the other side, tubing was open-ended with a Volcano Vaporizer mouthpiece fixed to it (Figure 1E). The mouthpiece was used to release the seal on the balloons, which were filled with a vapor containing THC (or vehicle; note that the content of the balloons is mostly air so that the animal was always normoxic while in the chamber). Parafilm was used to seal the connection making it airtight (Figure 1F). Animals were exposed to the vapor for 5 min, with half of the balloon being emptied at the beginning and the other half being emptied after 2 or 3 minutes of exposure. We separated the evacuation of the vapor-filled balloon into 2 parts to prevent excessive leaking of vapors caused by increasing pressure inside of the box which exacerbates cracks in the boxes' seal during balloon evacuation. The vacuum pump line was not efficient enough at maintaining a stable pressure to avoid side leakage and posed a danger to the animals inside the box if activated when they were still inside. The leakage was minimized to our satisfaction by splitting the balloon evacuation into 2 parts. After the exposure, animals were quickly removed from the chamber, and the vacuum line was turned on to remove any residual vapor. The inside of the box was cleaned with 70% ethanol between each group of animals.

## *2.4. Behavioral Assessments*

Open field and elevated zero maze were used to establish behavioral phenotypes induced by THC administration. Behavioral testing was performed during the light phase of the cycle from 1 pm to 7 pm.
