Cannabinoid Pain Relief in Mice Reveals Sex Differences and a Surprise Serotonin Link

Scientists at Texas Tech University Health Sciences Center have reported that two synthetic cannabinoid compounds, one targeting the CB1 receptor and the other the CB2 receptor, significantly reduce pain-related behavior in a mouse model of inflammatory pain, and that the relief produced by the CB2 agonist depends on an unexpected partner: the serotonin 1A receptor. The study, published in Frontiers in Pain Research, also uncovered pronounced sex differences in the effectiveness of the CB1 agonist, with male mice responding at doses that did nothing for females. The findings add to a growing body of preclinical evidence that the endocannabinoid system, the body’s own network of cannabinoid receptors and lipid signaling molecules, could be harnessed for analgesia, while hinting that cannabinoid drugs may work through more intertwined neural pathways than previously appreciated.

The research team, led by Josée Guindon, used the formalin test, a classic laboratory model of inflammatory pain in which a dilute solution of formaldehyde is injected into a mouse’s hind paw. The injection triggers two distinct waves of pain behavior: an acute first phase lasting roughly the first fifteen minutes, driven by peripheral nociceptors expressing the TRPA1 channel, and a longer inflammatory second phase from fifteen to sixty minutes, driven by peripheral and central sensitization. Observers, blind to treatment, scored each animal’s behavior at five-minute intervals, assigning weighted composite pain scores that distinguish simple favoring of the injured paw from lifting it entirely or vigorous licking, shaking, and biting. The researchers then calculated the area under the pain-score curve for each phase to quantify the overall analgesic effect of each treatment.

For the CB1 arm of the study, the team used ACEA, a selective agonist of the cannabinoid receptor 1, the same receptor through which the psychoactive component of cannabis, delta-9-tetrahydrocannabinol, exerts most of its effects. Female and male C57BL/6J mice received intraperitoneal injections of ACEA at doses ranging from 0.1 to 2.5 milligrams per kilogram. In female mice, the 0.5 milligram per kilogram dose significantly reduced pain behavior during the inflammatory phase, while in males both the 0.5 and 0.75 milligram per kilogram doses were effective, and the 0.75 dose also significantly reduced pain in the acute phase. Notably, the analgesic effect was biphasic: higher doses lost their benefit, a pattern the authors suggest may reflect off-target activation of TRPV1 receptors, which are associated with inflammation and heightened pain sensitivity, counteracting the CB1-mediated relief.

The sex differences were striking. When the researchers directly compared males and females, male mice showed significantly greater antinociceptive responses to the 0.75 milligram per kilogram dose in both the acute and inflammatory phases, a dose that produced no significant benefit in females at all. The authors point to several possible explanations grounded in prior literature. Like the endocannabinoid anandamide, ACEA is metabolized by the enzyme fatty acid amide hydrolase, whose expression and activity are regulated by the ovarian hormones estradiol and progesterone, potentially shifting drug levels between the sexes. Previous work has also documented sex differences in CB1 receptor density across brain regions and in receptor affinity for cannabinoid drugs, including a recent report attributing sex-dependent ACEA analgesia in neuropathic pain to dimorphic CB1 expression on specific neurons in the medial prefrontal cortex.

The CB2 arm of the study told a different story. AM1241, a selective agonist of cannabinoid receptor 2, which is largely free of the psychoactive effects associated with CB1, produced significant, dose-dependent pain relief in both sexes at 1, 3, and 10 milligrams per kilogram, with the highest dose showing the strongest effect in the inflammatory phase. Unlike ACEA, AM1241’s efficacy showed no statistically significant sex differences in either phase. Because CB2 activation avoids the psychoactive liabilities of CB1 and does not appear to induce tolerance with chronic use, the authors argue that CB2-selective analgesics deserve intensified investigation as potential clinical candidates for inflammatory and chronic pain conditions.

The most novel result emerged when the researchers blocked the serotonin 1A receptor with WAY-100635, a so-called silent antagonist that binds the receptor without triggering any cellular response while preventing other ligands from doing so. Pretreating mice with WAY-100635 before AM1241 completely abolished the CB2 agonist’s antinociceptive effect in the acute phase of the formalin test, in both males and females. In the inflammatory phase, however, AM1241 retained its pain-relieving effect even after 5-HT1A blockade, suggesting the serotonin receptor is specifically required for the early, acute component of CB2-mediated relief. The authors note that the absence of an inflammatory-phase effect could reflect the relatively rapid clearance of WAY-100635, and they call for follow-up experiments using higher antagonist doses or shorter injection intervals.

Crucially, the same blockade left CB1-mediated analgesia untouched. When mice received ACEA after WAY-100635 pretreatment, the compound’s pain-relieving effect in the inflammatory phase was indistinguishable from that of ACEA alone, in both sexes. This dissociation is significant because recent research has identified functional connections between 5-HT1A and CB2 receptors, and cannabidiol has been found to act as a positive allosteric modulator at 5-HT1A, contributing to its analgesic and antidepressant-like effects. The new data extend that story by showing that the CB2-5-HT1A link is functionally relevant to cannabinoid analgesia in vivo, while indicating that CB1 operates through a separate, serotonin-independent pathway.

To rule out trivial explanations for the pain relief, the team ran the non-nociceptive components of the cannabinoid tetrad, the battery of behaviors classically produced by CB1 activation in rodents: reduced locomotion, hypothermia, catalepsy, and analgesia. Neither ACEA at 0.5 milligrams per kilogram nor AM1241 at 10 milligrams per kilogram, with or without WAY-100635 pretreatment, significantly altered open-field locomotion, time spent in the center of the arena, rotarod motor coordination, or rectal temperature across testing sessions at thirty minutes, ninety minutes, six hours, and twenty-four hours after injection. The authors argue that the preserved center-time preference indicates the animals were not experiencing stress, which itself can produce analgesia, and the unchanged body temperature rules out hypothermia-driven artifacts, lending confidence that the observed pain relief was genuine.

The study sits within a broader context of frustration with current pain management. Chronic rheumatic disease alone affects roughly one in five adults in the United States, and in about twenty-one percent of cases the condition is refractory to all available treatments. Clinical guidelines have moved away from opioids toward multimodal approaches combining nonsteroidal anti-inflammatory drugs, anticonvulsants, and antidepressants, but even this strategy faces headwinds: recent work has associated prolonged gabapentinoid use for pain with an increased risk of Alzheimer’s disease. Meanwhile, most pain research has historically focused on males even though female sex is repeatedly identified as a risk factor for developing chronic pain, making the sex differences documented here particularly relevant to translating cannabinoid pharmacology to the clinic.

The authors are careful to frame their conclusions as preclinical. All experiments were conducted in mice, the compounds were synthetic research ligands rather than approved medicines, and the mechanisms underlying the CB2-5-HT1A connection remain unknown. They emphasize that future work should determine whether the functional link extends to the non-analgesic effects of CB2 activation, whether it exists in humans, whether tolerance develops to CB1- and CB2-based analgesics over long-term use, and whether the ACEA sex differences seen in acute inflammatory pain carry over to models of chronic inflammation. If those questions are answered favorably, combinations of cannabinoid compounds with serotonin-targeting drugs, including antidepressants and the 5-HT1A partial agonist buspirone, could open a promising avenue in the search for safer, more effective treatments for the millions of patients whose pain remains inadequately managed.

Subject of Research: CB1 and CB2 cannabinoid receptor agonist antinociception, sex differences, and 5-HT1A-dependent mechanisms in a mouse model of inflammatory pain

Article Title: Assessing CB1 and CB2 receptor agonist antinociception while exploring sex differences and 5-HT1A-dependent mechanisms

Article References: Barnes, R. C., Quick, H., Robison, D., Logsdon, A. F., & Guindon, J. (2026). Assessing CB1 and CB2 receptor agonist antinociception while exploring sex differences and 5-HT1A-dependent mechanisms. Frontiers in Pain Research, 7, Article 2005000. https://doi.org/10.3389/fpain.2026.2005000

Image Credits: AI Generated

DOI: 10.3389/fpain.2026.2005000

Keywords: cannabinoid receptors, CB1, CB2, 5-HT1A, inflammatory pain, formalin test, sex differences, antinociception, ACEA, AM1241, preclinical study, endocannabinoid system