Administration of Δ⁹-Tetrahydrocannabinol (THC) in Adolescent and Adult Mice

We studied the body's own cannabis, the so-called endocannabinoid system, and one of our main objectives is to understand how THC, the intoxicating component in cannabis, interacts with this system. One major challenge in working on cannabis is to design animal experiments that are relevant to the human condition. Factors like dose and route of administration are important.

Using mice we found that THC exposure during adolescence has pronounced effects on other physiology, even long after exposure is stopped. For example, it alters how the brain responds to social stress. This article describes a protocol for preparing THC for parenteral administration in assessing its acute pharmacodynamic effects in mice of both sexes at two developmental stages, adolescence and young adulthood.

Understanding how age, genetics, and health conditions affect THC responses can guide targeted prevention and treatment. Notably, adolescent cannabis use requires further study, as exposure during this critical neurodevelopmental phase may cause lasting negative effects. To begin, use a pipette to withdraw an appropriate volume of tetrahydrocannabinol, or THC, based on the dose in milligrams per kilogram and the body weight of the mice in grams, keeping the container on ice.

Carefully add the solution to the bottom of an 8 milliliter glass vial. After moving the vial to a chemical fume hood, gently evaporate the solvent to dryness using a gentle stream of nitrogen gas. Record the amount of THC used in the Controlled Substance 1 Usage log located in the Controlled Substance binder.

Once the solvent is completely dried, add the volume of Tween 80 required to achieve a final 5%concentration in saline. Warm the vial in a beaker containing hot water at or below 60 degrees Celsius. Vortex the vial to fully dissolve THC and repeat the process if needed.

Now, gradually add sterile saline in small increments. Vortex and heat the vial between each addition. Repeat these steps until the required volume of saline has been added to make a final THC solution in 5%Tween 80 and 95%saline, and the solution is fully dissolved.

Sonicate the vial in an ultrasonic water bath set to approximately 100%power at 40 kilohertz and 37 degrees Celsius for 5 minutes. Confirm that the solution is clear and free from any undissolved material. Next, weigh the mouse to calculate the appropriate volume of THC solution to inject based on 10 milliliters per kilogram per mouse.

Prepare a horizontal bar 7 centimeters in length and elevated 4 centimeters above the surface of a clean lab bench under ambient illumination of 160 luxe. Place an electronic timer within reach. Using clean latex gloves, remove the mouse from the cage of the testing lab.

Administer the calculated dose of THC or vehicle and return it to the cage. After approximately 45 minutes post-injection, gently place the mouse's front paws on the horizontal bar. Start the electronic timer to record the duration of immobility.

Stop the timer when the mouse removes one or both paws or when 10 seconds have passed. After repeating the test, use the average of the two immobility times measured in seconds as the indicator of catalepsy for final data analysis. For the open field test, use an arena equipped with video tracking equipment, ensuring it is clean and free of any odor cues.

Approximately 30 minutes after injecting THC, gently placed the mouse in the center of the arena, minimizing handling stress. Record a 10-minute session using a camcorder and behavioral tracking software. At the end of the test, return the mouse to its home cage and clean the open field arena thoroughly with a cleaning solution.

Calculate the total distance traveled, measured in meters, as the primary parameter to quantify locomotor activity. For the tail immersion test, prepare a water bath set to 54 degrees Celsius under ambient lighting of 160 luxe. Position an electronic timer nearby and ready for use.

Gently restrain the mouse in a soft tissue pocket made from pet training pads, leaving the tail accessible. Immerse the distal one third of the tail into the water bath and start the timer simultaneously. Record the latency to tail withdrawal, defined by a reflexive tail flick, with a cutoff time of 10 seconds to prevent tissue damage.

After repeating the test, average the two latency measurements, recorded in seconds, to calculate the primary outcome for evaluating antinociceptive effects. THC induced dose dependent catalepsy in both adolescent and adult male mice with stronger responses at 10 milligrams per kilogram. Female mice also showed catalepsy at 10 milligrams per kilogram, but adult females responded less than adolescents.

Adolescent females exhibited lower catalepsy than males, indicating a modest sex difference. In adults, males showed much higher catalepsy than females at 10 milligrams per kilogram. THC reduced locomotor activity in male mice, with adolescents being more sensitive than adults.

Female mice showed reduced movement at higher doses, with overall significant effects of both dose and age. THC increased tail flick latency in adolescent males and females at 5 and 10 milligrams per kilogram. In adults, antinociceptive effects were limited to the highest dose at 60 minutes post injection.

At 300 minutes only adolescent mice still showed antinociceptive effects.