Recent evidence indicates that opioid and cannabinoid receptors may interact functionally within the central nervous system (CNS). These interactions can occur directly, such as through receptor heteromerization, or indirectly via signaling convergence, in which activation of one system influences endogenous ligand release or downstream signaling in another. Such receptor interplay may underlie various behavioral effects associated with drug use, including acute antinociception [1]. The opioid receptor system includes three main subtypes—mu (MOR), delta (DOR), and kappa (KOR)—which are activated by endogenous peptides such as β-endorphin, enkephalin, and dynorphin [2]. In parallel, the endocannabinoid system (ECS) consists of two classical cannabinoid receptors (CB1Rs and CB2Rs), endogenous cannabinoids (eCBs), and the enzymes that synthesize and degrade them. The discovery of additional lipid mediators, enzymes, and receptors has broadened this system into what is now termed the endocannabinoidome (eCBome) [3].

CB2Rs are predominantly expressed in peripheral tissues, particularly within organs involved in immune function [4, 5]. They are also present in central nervous system cells, including microglia and neurons in the hippocampus, striatum, and brainstem [6]. Although the functional presence of CB2Rs in neurons has been debated, mounting evidence—including our own research—has confirmed their expression and regulatory role in response to drugs of abuse [7–11]. Opioid and cannabinoid receptors share similar signaling characteristics. As G protein-coupled receptors (GPCRs), they inhibit cyclic AMP production via Giα coupling, activate MAP kinase pathways through secondary messengers, and suppress neurotransmitter release by inhibiting calcium influx and promoting potassium efflux [12, 13]. Opioid and cannabinoid receptors share similar Gi-coupled signaling properties, and their effects may converge at the level of intracellular pathways or neuroimmune modulation; however, the present study does not test direct receptor–receptor interactions [14].

There is growing evidence that the opioid and endocannabinoid systems interact across molecular and behavioral domains, influencing processes such as pain perception, reward mechanisms, and addiction susceptibility [15, 16]. Recognizing this interplay may inform the development of novel therapeutics with reduced risk of abuse. Strategically targeting both systems holds promise for advancing treatment approaches for pain and substance use disorders. In behavioral studies involving mice, the endocannabinoid system—particularly CB2Rs—has been implicated in regulating inflammation and motor activity. CB2Rs primarily exert their effects through immune cells and CNS microglia. Their activation has been shown to lower pro-inflammatory cytokine levels and limit immune cell infiltration into affected tissues [3, 17] These receptors also modulate neuroinflammation-related motor function. For instance, in a Parkinson’s disease model, CB2R activation reduced alpha-synuclein aggregation and protected dopaminergic neurons in the substantia nigra, indicating a neuroprotective capacity [17].

Mu-opioid receptors (MORs), the principal targets of endogenous β-endorphin and analgesics such as morphine and fentanyl, play a central role in pain modulation and motor control [18]. MOR activation suppresses neurotransmitter release and induces neuronal hyperpolarization, thereby dampening pain signals. Opioid drugs can also impact locomotor activity in mice—typically enhancing it at low doses via MOR activation. However, higher doses or specific opioid types may instead cause sedation and reduced mobility. These effects can vary based on factors such as drug type, dosage, mouse strain, and co-administered substances [19].

Despite evidence that opioid and cannabinoid systems interact at molecular and behavioral levels, it remains unclear how MOR deletion compares with cell-type-specific CB2 receptor loss in shaping nociceptive behavior and neuroimmune signaling. In particular, the distinct contributions of microglial versus dopaminergic CB2 receptors to pain processing have not been systematically examined. Therefore, the present study aimed to determine how MOR KO, DAT-Cnr2 cKO, and CX3CR1-Cnr2 cKO mice differ in nociceptive responses and cytokine expression across the prefrontal cortex and cerebellum. We hypothesized that MOR deletion would produce a pro-inflammatory cytokine profile and altered nociception, whereas microglial and dopaminergic CB2 deletion would yield distinct behavioral and neuroimmune phenotypes reflecting their divergent cellular roles.

Cannabinoid CB2 and MORs play distinct yet complementary roles in pain modulation through neuroimmune and neuronal mechanisms. CB2 receptors have emerged as promising targets for analgesia without the psychotropic side effects associated with CB1 activation. In chronic pain states, CB2 expression is upregulated in activated microglia, where it modulates inflammatory responses and dampens nociceptive signaling within the spinal cord. Selective CB2 agonists have demonstrated efficacy in reducing pain and inflammation in preclinical models, suggesting their therapeutic potential for neuropathic pain [28, 29]. MORs are widely distributed throughout the central and peripheral nervous systems and are the principal mediators of opioid-induced analgesia. Activation of MORs inhibits nociceptive transmission by suppressing neurotransmitter release and hyperpolarizing neurons via G-protein-coupled mechanisms [30–32]. Interestingly, endogenous opioids and cannabinoids may interact synergistically, with evidence indicating that CB2 activation can modulate opioid receptor signaling and vice versa, offering a potential avenue for combination therapies that enhance analgesia while minimizing adverse effects [33]. Together, these receptor systems represent critical nodes in the pain circuitry and are central to the development of next-generation analgesics. Although prior studies have reported synergistic behavioral effects when MOR and CB2 pathways are co-activated, the present study does not investigate molecular interactions between these receptors.

The observed increase in tail flick latency in CX3CR1-Cnr2 mice underscores the critical role of CB2 receptors in microglia in modulating nociceptive responses. CB2 receptors are known to exert anti-inflammatory and neuroprotective effects, and their deletion has been shown to enhance pain sensitivity by disrupting microglial homeostasis [3, 20, 27, 34]. The significant elevation in latency compared to both wild-type C57BL/6 and DAT-Cnr2 mice suggests that microglial CB2 signaling plays a more pivotal role in thermal nociception than CB2 expression in dopaminergic neurons [35, 36]. Although CX3CR1-Cnr2 mice displayed significantly increased tail-flick latency, this finding should not be interpreted exclusively as enhanced antinociception. Tail-flick behavior is a spinally mediated reflex that can be modulated by factors beyond nociceptive threshold, including motor responsiveness, stress reactivity, and microglial influences on sensorimotor integration. Given that CB2 receptors regulate microglial activation and cytokine signaling, the altered latency may reflect broader neuroimmune effects rather than a pure analgesic phenotype.

Interestingly, MOR KO mice also exhibited elevated latency, albeit less pronounced. This aligns with previous studies indicating that MORs are central to opioid-mediated analgesia, and their absence leads to altered pain thresholds [19, 37]. However, the differential magnitude of latency between MOR KO and CX3CR1-Cnr2 mice may reflect distinct mechanisms - with MOR deletion affecting opioid signaling pathways, and CB2 deletion influencing neuroimmune interactions. These findings support the hypothesis that microglial CB2 receptors and MORs independently contribute to nociceptive regulation, and their disruption leads to enhanced antinociceptive responses, possibly via compensatory mechanisms involving cytokine signaling or altered neurotransmission.

The cytokine profile reveals a complex interplay between genotype and brain region in regulating neuroinflammation. Extensive evidence supports the involvement of both the prefrontal cortex and cerebellum in pain modulation, in addition to their roles in other behavioral processes [38–42]. Meta-analysis of studies employing experimental pain stimuli indicates the following brain areas to be positively associated with pain: primary and secondary somatosensory cortices, insular cortex, ACC, PFC, and thalamus [43]. The cerebellum plays a significant role in pain processing, particularly in inhibiting pain sensation Activation in lobules I–VI, VIII, and the vermis has been linked to nociceptive signalling, fear conditioning, and sensory integration [44]. The significant upregulation of IL-1β and IL-6 in MOR KO mice compared to C57BL/6 suggests a pro-inflammatory shift, consistent with prior reports that MOR deletion leads to enhanced glial activation and cytokine release [45, 46]. IL-1β and IL-6 are key mediators of neuroimmune responses, and their elevation may reflect compensatory immune activation in the absence of MOR-mediated inhibitory signaling.

Moreover, the regional differences in cytokine expression - particularly IL-1α, IFN-γ, IL-10, and CXCL-10 between the cerebellum and PFC - highlight the spatial heterogeneity of neuroimmune regulation. CXCL-10, an IFN-γ-inducible chemokine, is known to recruit T cells and modulate neuroinflammation, and its differential expression may indicate region-specific immune surveillance or vulnerability [47]. The increase in IL-10, an anti-inflammatory cytokine, in specific regions may represent a homeostatic counterbalance to elevated pro-inflammatory signals. This dynamic is supported by literature showing that cytokine expression in the brain is shaped by neural activity and regional microglial phenotypes [48]. Although the current study did not include a formal correlation analysis between cytokine levels and behavioral outcomes, the cytokine profile of MOR KO mice—characterized by elevated IL-1β and IL-6—suggests a pro-inflammatory shift that may contribute to altered nociceptive processing. In contrast, CX3CR1-Cnr2 mice exhibited increased tail-flick latency without a parallel increase in these cytokines, indicating that microglial CB2 deletion may influence nociception through mechanisms other than classical pro-inflammatory cytokine upregulation. Because the Brief Communication format restricts the amount of data that can be presented, only cytokines with significant effects are discussed here; however, all measured cytokines were analyzed across genotypes and brain regions.

This study has several strengths, including the use of multiple genetically modified mouse lines that allow for cell-type-specific dissection of CB2 receptor function, as well as direct comparison with MOR deletion. By integrating behavioral measures with cytokine profiling across two brain regions, we provide a multidimensional view of how MOR and CB2 signaling influence nociceptive and neuroimmune processes. However, nociception was assessed using only the tail-flick test, which primarily measures spinal reflexive withdrawal; additional assays such as hot-plate, von Frey, or Hargreaves would help determine whether the observed effects generalize to supraspinal or mechanical modalities. The cytokine analyses were conducted using small group sizes, which increases the risk of both Type I and Type II errors. Although these sample sizes are consistent with prior neuroimmune studies using mouse brain tissue, the findings should be interpreted cautiously and considered preliminary given the small sample size and limitations of normality testing. Larger, adequately powered studies will be necessary to confirm the genotype-specific immune patterns observed here.

In conclusion, the current findings provide compelling evidence that both cannabinoid CB2 and mu opioid receptors contribute to distinct behavioral and neuroimmune phenotypes. The significant increase in tail flick latency observed in CX3CR1-Cnr2 and MOR KO mice underscores their critical role in antinociceptive regulation, with CB2 deletion from microglia exerting a more pronounced effect. Concurrently, the cytokine data highlight genotype- and region-specific immune dynamics, with MOR KO mice showing elevated pro-inflammatory cytokine levels and the cerebellum and PFC exhibiting differential immune profiles. Together, the finding deepens our understanding of how neuroimmune interactions contribute to pain physiology and support the therapeutic potential of targeting these receptors to develop more effective and selective analgesics.