The mammalian amygdala is composed of more than a dozen nuclei which have distinctive connections [4,23]. The BLA consists of three main nuclei, the lateral nucleus, the basal nucleus, and the accessory basal nucleus, each of which has several subdivisions [4]. The lateral nucleus has a cortical-like organization and contains two main neuronal types. These include spiny glutamatergic pyramidal projection neurons and spine-sparse or spine-free GABAergic nonpyramidal neurons [24,25,26]. The lateral nucleus is a key integrative site for emotional processing and pain modulation, making it a strategic target for investigating the distribution of cannabinoid and cannabinoid-related receptors in the horse.

In general, cannabinoid receptors are located densely in the animal and human brain areas [27] as in other central and peripheral nervous system areas of the horse, such as trigeminal ganglion, dorsal root ganglia, and also in the synovial membrane [28,29,30,31].

Relatively few studies have explored the distribution and expression of cannabinoid receptors in the amygdaloid complex across species to date. This is the first study to document the presence, both at molecular and cellular level of CB1, CB2, TRPV1, and PPARγ receptors in the lateral nucleus of the amygdaloid complex in horses. Most of the available literature has focused on CB1 receptors, particularly in rodent models. In rats and mice, CB1R has been shown to be strongly expressed in the basolateral amygdala (BLA), particularly in the lateral and basal nuclei. Katona et al. [32] reported that CB1R is predominantly localized presynaptically on the terminals of a specific subset of GABAergic interneurons that co-express cholecystokinin (CCK). Using electron microscopy, Fitzgerald et al. [33] confirmed that CB1R is concentrated in large, morphologically complex presynaptic boutons in the BLA and quantified a considerable number of CB1-positive axon terminals. Similarly, McDonald and Mascagni [21] identified CB1R immunoreactivity in weakly stained pyramidal neurons and intensely labelled multipolar aspiny interneurons; the latter correspond to CCK-positive GABAergic cells. Although less extensive, some evidence is also available in humans. Glass et al. [34] used quantitative autoradiography with the radioligand [³H]CP55,940 to demonstrate high binding site density for cannabinoid receptors in the human amygdaloid complex. Furthermore, Wang et al. [35] reported strong CB1R mRNA expression in the basal nuclear group of the amygdala and more moderate levels in the cortical and medial subdivisions using in situ hybridization in fetal human brains. Limited morphological and molecular data on cannabinoid receptors are also available in the marmoset amygdaloid complex [36]. In the dog, Freundt-Revilla et al. [37] investigated the central nervous system (CNS) but did not specifically examine the amygdala. Together, these findings support the idea that the distribution of CB1Rs in the amygdala is a conserved feature across mammalian species, and they emphasize the importance of our data in a broader comparative neuroanatomical context.

In contrast to CB1R, far fewer studies have investigated the distribution of CB2, TRPV1, and PPARγ receptors in the amygdaloid complex, and most of the available data come from rodent models. Regarding CB2R, immunohistochemical evidence in the rat brain revealed widespread expression of this receptor, including in the amygdala, where CB2 immunoreactivity was detected in putative neuronal cell bodies, glial processes, and neuronal fibers [38]. As for TRPV1, autoradiographic studies using [³H]Resiniferatoxin demonstrated its presence throughout the CNS of the rat, including the amygdala [39], and a subsequent immunohistochemical investigation confirmed the expression of VR1 (TRPV1) in neurons of the central amygdaloid nucleus [40]. PPARγ expression has also been documented in the mouse amygdala, where immunohistochemical analyses revealed that this receptor is present in approximately 60% of GABAergic and 40% of glutamatergic neurons [41]. A previous study further confirmed the high expression of PPARγ in the basolateral amygdala and its substantial colocalization with GABAergic neurons, suggesting a potential role in modulating inhibitory neurotransmission [42].

Regarding the gene expression of Cnr1, Cnr2, TRPV1, and PPARγ receptors in the amygdaloid complex on animal model, the data are relatively scarce. The transcripts of Cnr1 and Cnr2 were detected in rats [43,44] and in the bovine [45] amygdaloid complex while TRPV1 and PPARγ were detected only in rodents [41,43].

Typically, enhanced CB1R activation reduces anxiety, while its inhibition produces anxiogenic effects [46,47]. For this reason, compounds that modulate cannabinoid receptor activity may help to reduce anxiety and represent promising targets for future anxiolytic therapies in equine medicine. Since cannabidiol (CBD) does not act directly on CB1R [48], but rather as a hydrolyzer of fatty acid amide hydrolase (FAAH) which is the membrane enzyme hydrolyzing AEA [49], it could potentially reduce anxiety [27].

Fear extinction is a type of inhibitory learning. In this process, new associations compete with previously established fearful ones, suppressing fear responses and modulating fear-related neural circuits [50]. In rodents, fear extinction activates the ECS through projections from the prefrontal cortex to the BLA [51]. Glutamate released from prefrontal neurons stimulates cannabinoid production in the BLA, which then acts presynaptically on CB1 receptors. This suppresses further glutamate release and promotes the extinction of the conditioned fear response [52]. During stress, the production of endocannabinoids may modulate emotional responses by altering amygdalar neurotransmission [53]. Typically, anxiety elevates endocannabinoid tone, and endocannabinoids in turn reduce anxiety, as demonstrated by Marsicano et al. [54]. In addition, endocannabinoids appear to be crucial for the extinction of aversive memories and this process is likely to be mediated by the amygdala [55]. In addition to its role in fear extinction, the ECS has also been implicated in fear-conditioned analgesia, a phenomenon whereby pain perception is suppressed in the presence of a fear-related context. In rodents, Rea et al. [56] showed that CB1R activation in the BLA is required for fear-induced analgesia. Administration of a CB1R antagonist directly into the BLA abolished the antinociceptive effect triggered by contextual fear. Rubino et al. [57] showed that treatment with the phytocannabinoid ∆9-THC, an agonist of CB1R, significantly decreased c-Fos levels in the amygdala, and that this effect was reversed by the CB1 inverse agonist AM251. Taken together, these findings underscore the central role of CB1R in modulating emotional and nociceptive processes through the amygdaloid circuitry. The identification of CB1R protein expression within the lateral nucleus of the equine amygdala in the present study supports the hypothesis that similar regulatory mechanisms of fear, anxiety, and pain may be conserved in horses, providing a neuroanatomical foundation for the potential therapeutic use of cannabinoid-based compounds in equine behavioral medicine.

Although traditionally considered a peripheral receptor mainly involved in immune function, CB2R expression has been increasingly reported in the CNS, including neurons and glial cells, challenging the early notion of its absence from the brain [58,59]. In the present study, CB2R-IR was observed in varicosities within the lateral nucleus of the amygdala, and Cnr2 gene expression was detected by RT-PCR, supporting the presence of this receptor at both protein and transcript levels. These findings are in line with previous studies showing CB2R localization in the neuropil and dendritic compartments of several brain regions, including the amygdala, hippocampus, and substantia nigra of rodents [60,61]. Ultrastructural studies have shown that CB2R can be found postsynaptically, often on dendritic membranes and the rough endoplasmic reticulum. This suggests endogenous synthesis and potential integration into synaptic signaling pathways [60]. Although CB2R-IR expression is lower than that of CB1R, CB2R in the CNS appears to play a modulatory role, particularly under pathological conditions, such as neuroinflammation, stress, and neuropsychiatric disorders [61,62]. Functionally, CB2R activation in the brain has been associated with the regulation of microglial activity, immune response modulation, and neuroprotection [59]. Moreover, behavioral studies in animal models have suggested that CB2R agonists, such as β-caryophyllene, exert anxiolytic and antidepressant effects, reinforcing the potential involvement of CB2R in the modulation of emotional states [63]. Several lines of evidence support a functional role for CB2R within the amygdala. Its expression has been documented under both physiological and stress-related conditions [64,65], and CB2R activation in the central nucleus has been shown to mediate cannabinoid-induced antinociceptive effects [66]. In the medial amygdala, early-life stimulation of CB2R influences neuronal morphology and contributes to the development of social behaviors [67]. Although most of the research has focused on CB1R within the BLA, recent findings have suggested that CB2R activation in this region may also modulate stress-induced alterations in synaptic plasticity and affective responses. Specifically, intra-BLA administration of the CB1R/CB2R agonist WIN55,212-2 reversed stress-impaired hippocampal–accumbens long-term potentiation, suggesting that CB2R could contribute to the amygdaloid regulation of stress-related neural circuits [68]. While direct evidence of CB2R expression in the BLA, and especially in the lateral nucleus, remains limited, the present findings aligned with the broader observations of CB2R functional relevance across the amygdaloid complex [58]. These data support the hypothesis that CB2R may also modulate emotional and sensory processing in these subregions.

The transient receptor potential vanilloid type 1 receptor is a non-selective cation channel known for its role in nociception and thermoregulation. However, increasing evidence has revealed its expression in the brain areas involved in emotional regulation, including the amygdala [69]. In particular, TRPV1 has been identified in the lateral nucleus of the amygdala, where it modulates synaptic plasticity and long-term potentiation, thereby influencing fear memory formation in mice [69]. Although the precise physiological role of TRPV1 in the amygdala remains unclear, functional studies in TRPV1 knockout mice demonstrated reduced anxiety-like behavior, and diminished fear conditioning and stress-sensitization, additionally implicating this receptor in emotional processing circuits [70,71]. Transient receptor potential vanilloid type 1 signaling intersects with the ECS since it can be activated by anandamide, a key endocannabinoid molecule [72]. This convergence has prompted the development of dual FAAH/TRPV1 inhibitors which have shown dose-dependent antidepressant-like effects in rodent models of depression [73], underscoring the therapeutic relevance of this pathway.

TRPV1 is not only involved in emotional regulation but also plays a key role in pain perception and neurogenic inflammation. When activated on peripheral sensory terminals, TRPV1 allows a rapid influx of calcium and sodium ions. This leads to the release of neuropeptides, including substance P, neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) [74]. In addition to peripheral effects, TRPV1 activation in primary sensory neurons also modulates central nociceptive processing, supporting its role in the higher-order processing of nociceptive signals [74]. At the molecular level, TRPV1 protein has been shown to localize to the plasma membrane, cytoplasmic vesicles, and membranes of the endoplasmic reticulum in neurons, compartments which support its involvement in both calcium influx and intracellular calcium release [75] with some studies also describing its presynaptic localization in amygdaloid circuits [76]. These data are in line with the immunofluorescence results in the present study which revealed TRPV1 expression predominantly in the neuronal somata and neuropil within the lateral nucleus of the equine amygdala, with notable cytoplasmatic distribution patterns. The effect of TRPV1 activation on synaptic transmission varies depending on the brain region. According to Edwards et al. [77], TRPV1 can enhance or inhibit glutamatergic signaling, depending on the specific neural context. Altogether, these findings support a multifaceted role for TRPV1 as a molecular integrator of nociceptive and emotional signals and highlight its conserved expression and functional relevance within the amygdaloid circuitry across mammalian species, including the horse, as demonstrated by the present study.

Peroxisome proliferator-activated receptor gamma is a nuclear receptor traditionally associated with metabolic regulation and anti-inflammatory signaling; however, it has also been implicated in CNS functions, including emotion, stress, and pain modulation. It has been shown to be expressed in rat, mouse, monkey, and human neurons, astrocytes, and rat microglial and oligodendrocyte cell cultures where it contributes to mitochondrial protection, redox balance, and modulation of neuroinflammatory responses [78,79,80,81,82,83,84]. In the present study, PPARγ-IR was detected in the lateral nucleus of the equine amygdala, localized to the nuclear compartment of both neurons and glial fibrillary acidic protein (GFAP)-positive glial cells. This distribution is consistent with its role as a ligand-activated transcription factor. When activated by selective agonists like rosiglitazone or pioglitazone, PPARγ forms a heterodimer with the retinoid X receptor. This complex then binds to peroxisome proliferator response elements. It has been shown to reduce anxiety and depression-like behavior, attenuate fear expression, and alleviate mechanical allodynia and thermal hyperalgesia by means of mechanisms involving both central and peripheral pathways [85,86]. Taken together, these findings reinforce the view that PPARγ plays a modulatory role in emotional and nociceptive processing. The authors’ identification of PPARγ in both neurons and GFAP-positive glial cells of the lateral amygdaloid nucleus in the horse adds novel comparative neuroanatomical evidence to this growing body of the literature and supports a conserved role for PPARγ in the regulation of limbic functions across mammalian species.

Although the effects of cannabinoids on emotion and memory are well documented, little is known about how they influence neuronal networks in the equine brain, particularly within the amygdala. In the present experiment, the authors focused on the lateral nucleus which is the best-described region of the mammalian amygdala. The present investigation demonstrated the presence of the CB1, CB2, TRPV1, and PPARγ receptors in the equine amygdaloid complex for the first time. Furthermore, the present investigation showed that there was a very high density of CB1R axon terminals forming pericellular baskets presumably contacting the pyramidal somata, suggesting that cannabinoids may reduce tonic GABAergic inhibitory control over pyramidal cells in the lateral nucleus, as demonstrated by previous immunohistochemical and electrophysiological findings [32]. The distribution of other cannabinoid receptors in neuropilar and somatal structures supports strong cannabinoid modulations on amygdala activities. Consistent with the present findings, the BLA of primates [22] and rodents [21] contains many CCK-IR axon terminals containing the CB1R. These axons form a pericellular basket which contacts the somata of putative pyramidal cells and may be critical for fear expression and extinction [21,22]. Regarding the transcripts of the genes of interest, gene expression for Cnr1 and Cnr2, consistent with the protein (CB1 and CB2) data was detected. In contrast, TRPV1 and PPARγ transcripts were not detected in the fixed amygdala samples. However, since the authors were able to detect these transcripts in non-fixed samples (preliminary data) and in line with the protein expression data, they suggested that the absence of TRPV1 and PPARγ transcripts in the fixed samples described in this manuscript was due to the known negative effect of formaldehyde fixation on RNA integrity, as has been well documented in the literature [87,88].

Despite the valuable insights provided, some limitations of the present study must be acknowledged. The sample size (n = 6), although reflective of the inherent difficulty in obtaining well-preserved equine brain specimens, limits the generalizability of the findings. Additionally, the wide age range of the animals included (3–25 years) could influence the anatomical and molecular features of the CNS. Although this variability mirrors the heterogeneity encountered in real-world conditions, future studies should include larger, more stratified cohorts, by both age and sex, to clarify whether these factors impact receptor expression or localization within the amygdala. Expanding the sample base would also help identify potential interindividual variability in the ECS. Comparative analyses across species could further elucidate the evolutionary conservation and functional roles of cannabinoid and cannabinoid-related receptors in limbic circuits. Moreover, the adoption of advanced techniques such as in situ hybridization, electron microscopy, or single-cell transcriptomics may refine our understanding of their cellular and subcellular distribution. In particular, it would be of great interest to investigate receptor expression in horses exhibiting stress- or anxiety-related behavioral disorders and in individuals experiencing pain, to explore their potential involvement in pathological states and their value as therapeutic targets.