The study of pain in animals raises ethical, philosophical and technical challenges. Ethically because the animal must be tested awake and is subject to discomfort; philosophically, pains as a subjective experience cannot be fully captured in non‐human organisms, given that its emotional dimension is uniquely human. In animal models, we can only evaluate the nociceptive response (Le Bars et al. 2001), the neural process that detects and reacts to potentially harmful stimuli. Some reviews have discussed the large number of animals used as models for chronic pain (Coderre and Laferrière 2020; Mogil 2009), mainly focusing on mammalian systems. Few reviews have addressed invertebrate models (Abboud et al. 2021; Burrell 2017).

In this study, we propose the para ^bss1 mutant as a promising genetic model for investigating nociceptive sensitization in Drosophila melanogaster . A comparative analysis of human sodium channels (Na_v1.7, Na_v1.8 and Na_v1.9) and the para gene in flies revealed a high degree of homology with Na_v1.7, which is strongly associated with human pain syndromes (Cox et al. 2006). Three‐dimensional structural modelling further demonstrated that the human Na_v1.7 R1150W mutation induces conformational changes, while no such structural alteration was observed in para ^bss1 , suggesting that its gain of function mechanism occurs through altered channel dynamics rather than major folding changes. Although our behaviour assays addressed acute responses, the persistent hyperexcitability of para ^bss1 closely parallels mechanisms underlying chronic nociception. Behaviorally, para ^bss1 larvae exhibited increased sensitivity to chemical stimuli but not thermal ones, compared to w ¹¹¹⁸ . When treated with CBZ, a sodium‐channel blocker, w ¹¹¹⁸ had an increased nociceptive latency after 1 and 6 h of treatment with both tested concentrations, and para^bsss1 larvae displayed a biphasic response: a mild increase in latency after 1 h at 3 mg/mL but not at 6 mg/mL, followed by the opposite pattern after 6 h, when only the higher dose produced a significant effect. This temporal inversion likely reflects the altered kinetics of sodium‐channel inactivation previously described for para ^bss1 (Parker et al. 2011). Similarly, CBD significantly prolonged latency in both strains, supporting its analgesic potential and further validating the model for the pharmacological screening of candidate analgesics.

Drosophila melanogaster is a well characterised model for nociception studies. The anatomical and physiological aspects of class I‐IV neurons are well described (Karim and Moore 2011) and these neurons are responsible for mediating nociception in larvae (Cheng et al. 2010; Xiang et al. 2010; Yan et al. 2013). The first gene related to nociception in D. melanogaster was named painless and characterised in 2003, which encodes a non‐selective cation channel in the TRPA family that is responsible for thermal and mechanical nociception (Tracey et al. 2003). Its human orthologue, TRPA1, is likewise a key pain mediator (Souza Monteiro De Araujo et al. 2020).

Another important class of channels related to pain is the Na⁺ channel family, and to our knowledge, there is no previous information about the modulation of nociception by Na⁺ channels in D. melanogaster , supporting the novelty of our findings.

Drosophila melanogaster has two genes encoding Na⁺ channels (Na_v1 and Na_v2) in its genome, each in single copy (Liebeskind et al. 2011). There is little information in the literature about Na_v2 in invertebrates. Thus, Na_v1 is the predominant sodium channel gene in invertebrates (para gene in D. melanogaster ), and the orthologous Na_v channel in vertebrates is Na_v1 (Zakon 2012).

In the human genome, 9 different genes encode Na_v1 channels. The genes SCN1A to SCNA5A encode Na_v1 to Na_v1.5, and the genes SCN8A to SCN11A encode Na_v1.6 to Na_v1.19, respectively (Catterall 2014). Of particular interest are Na_v1.7 (SCN9A), Na_v1.8 (SCN10A), and Na_v1.9 (SCN11A), which are predominantly expressed in peripheral sensory neurons and are directly associated with inherited human pain syndromes. Gain‐of‐function mutations in Na_v1.7 have been implicated in inherited erythromelalgia, causing severe burning pain in the extremities (Dib‐Hajj et al. 2005). Similarly, mutations in Na_v1.8 have been identified in patients with painful neuropathy and shown to increase sensory neuron excitability (Faber et al. 2012). Na_v1.9 mutations have been linked to familial episodic pain syndromes characterised by recurrent episodes of intense pain (Halbritter et al. 2013). The human Na_v1.7, Na_v1.8 and Na_v1.9 proteins were analysed in this work for comparison with para in D. melanogaster because variants in these genes have been associated with human Mendelian pain disease (Bennett et al. 2019).

In our work, we found that the molecular 3D models proposed in this work show no difference between protein from wild type allele (para) compared to mutant para ^bss1 . We found similar results for human mutant Na_V 1.7 (L858F) compared to human wild type Na_V 1.7, though the same mutational change of L to F the position on the protein is different. This human mutation (dbSNP rs80356476) has an autosomal dominant pattern of inheritance which is a gain of function and it promotes a hyperpolarizing shift in channel activation and an increase in the amplitude of the response to slow, small depolarizations (Han et al. 2006) similar to para ^bss1 on electrophysiology (Parker et al. 2011) and 3D modelling.

The human mutant Na_V 1.7 (R1150W) is encoded by the SCN9A gene with mutation (dbSNP rs6746030) and it has an autosomal dominant pattern of inheritance with gain of function. This analysis was included in the present work due to its high frequency in the population (5.24% to 15.17%) according to PHARMAGKB (https://www.pharmgkb.org/, accessed in May 2021). This variation promotes a problem in the inactivation of Na_v 1.7v(R1150W) compared to the wild type and its mutation is related to more pain sensitivity in osteoarthritis, postamputation and sciatica (Reimann et al. 2010). Here, we demonstrate that the mutation R1150W in Na_v1.7 3D provokes a conformational change in the 3D model structure, and it could be related to the increase in pain sensitivity.

After predicting the 3D protein structures, we validate the reproducibility of the chemical nociception assay using w ¹¹¹⁸ , the reference strain most widely used in D. melanogaster research (Ferreiro et al. 2018). The responses of third instar larvae exposed to HCl solution were consistent with those previously reported (Lopez‐Bellido et al. 2019). However, responses of para ^bss1 larvae displayed markedly enhanced sensitivity to the same chemical stimulus compared to w ¹¹¹⁸ , which carries the wild type of para allele. This hypersensitive phenotype is consistent with the well‐characterised gain of function mutation in the para sodium channel (L1699F) (Parker et al. 2011). The para ^bss1 mutation (L1699F) was originally characterised as a gain of function variant that enhances neuronal excitability and lowers the seizure threshold (Kroll et al. 2015; Loughney et al. 1989). Parker (Parker et al. 2011) demonstrated that flies carrying the Na_V1–1L1699F transgene exhibited increased recovery time (from 38 to 49 s) and reduced seizure threshold (from 19.1 to 12.1 V) compared with controls, confirming that the L1699F substitution drives the hyperexcitable para ^bss1 phenotype.

It is important to note that, although the para ^bss1 mutation is best known for producing seizure‐like activity and bang‐sensitive paralysis in adult flies (Parker et al. 2011; Song and Tanouye 2006), such phenotypes have not been observed in larvae under standard laboratory conditions. In our assays, para ^bss1 larvae exhibited normal locomotion prior to stimulation and consistently performed the characteristic nocifensive rolling behaviour in response to chemical stimuli, a rapid, coordinated 360° rotation mediated by class IV multidendritic nociceptors (Babcock et al. 2009; Tracey et al. 2003). This rolling response is highly stereotyped (Boivin et al. 2023; Sulkowski et al. 2011) and distinct from the spasmodic convulsions and transient paralysis reported in adult bang‐sensitive mutants, indicating that the behavioural responses described here reflect nociceptive activation rather than seizure activity.

The resulting neuronal hyperexcitability likely underlies the increased responsiveness of para ^bss1 larvae to noxious chemical stimuli. Although the para ^bss1 mutation is located at a different site from human Na_v1.7 variants (Cox et al. 2006; Dib‐Hajj et al. 2005; Faber et al. 2012), these mutations share a common outcome: increased sodium channel activity and heightened sensory excitability. The para gene, orthologous to mammalian SCN1A–SCN9A genes, has been implicated in both seizure and nociceptive pathways, reinforcing its relevance as a model for sodium channel‐driven sensory dysfunction. Our approach thus represents a mutant‐based, rather than humanised model, that captures the shared pathophysiological principle of ion‐channel hyperexcitability. Future genetic engineering targeting residues homologous to Na_v1.7 L858 or R1150 could further refine this system into a more precise model of human pain‐related sodium channelopathies.

Carbamazepine (CBZ) is a Na⁺ channel blocker (Kennebäck et al. 1995) widely used in the treatment of epilepsy (Beydoun et al. 2020) and chronic pain (Szok et al. 2019). As expected, CBZ modulated nociception in the w ¹¹¹⁸ strain in 1 h of treatment. However, CBZ was already tested in the para ^bss1 strain at the concentrations of 0.002–0.1 mg/mL and showed no results in altering the seizure phenotype (Reynolds et al. 2004), and therefore, para ^bss1 was treated as a refractory treatment strain. Because of this, we tested doses higher than those previously reported in a short time (1 and 6 h) to avoid toxicity. The para ^bss1 larvae responded to the lower concentration after 1 h of treatment, and after 6 h, only the highest concentration promoted some significant effect in increasing the latency, however, the delayed and dose‐dependent response to carbamazepine in para ^bss1 does not undermine the model's validity; rather, it mirrors the pharmacoresistance observed in human sodium‐channel gain‐of‐function disorders such as SCN9A‐linked erythromelalgia and neuropathic pain (Baker and Nassar 2020; Dib‐Hajj et al. 2013; Fischer et al. 2009). We propose the use of this compound as a positive control in experiments with para ^bss1 strain as a chronic nociception model for screening of new drugs.

Cannabidiol (CBD) has recently gained significant attention in pain management, leading to a growing body of research focused on cannabis‐based medical treatments. It is known that CBD promotes inactivation of the Na_v1.7 channels (human) (Huang et al. 2023). Oral administration of CBD oil significantly increased response latency in both D. melanogaster models evaluated, indicating a reduction in nociceptive sensitivity. These findings suggest that CBD may modulate nociceptive pathways involving para channels, supporting the suitability and efficacy of the fruit fly models employed in this study for investigating CBD‐mediated pain mechanisms.

Although Drosophila melanogaster lacks clear orthologs of the canonical cannabinoid receptors CB1 and CB2 found in mammals, several studies suggest that phyto cannabinoids such as cannabidiol (CBD) can modulate neuronal activity through alternative mechanisms (Candib et al. 2024; Clarke et al. 2021). In the absence of classical endocannabinoid signalling, CBD may exert its effects by interacting with other molecular targets, including voltage‐gated sodium channels, transient receptor potential (TRP) channels, and regulators of oxidative or inflammatory homeostasis. Notably, para encodes the sole voltage‐gated sodium channel in Drosophila, which shares functional and pharmacological similarities with mammalian Na_v channels (BSC1A family) (Ravenscroft et al. 2023; Tapia et al. 2021). Thus, it is plausible that CBD's antinociceptive effects in para ^bss1 mutants arise from its capacity to modulate sodium channel activity, either directly or indirectly, rather than through canonical cannabinoid receptors pathways. Consistent with this view, endocannabinoid‐related compounds have been reported to influence sodium channel function and excitability in Drosophila and other models (Jacobs and Sehgal 2020), supporting a noncanonical route through which CBD may regulate nociceptive responses.

In this work, we provide functional and pharmacological evidence supporting the use of the D. melanogaster para ^bss1 mutant as a model for chronic nociception. Building on previous electrophysiological characterizations of the para channel (Parker et al. 2011), our results expand its application beyond seizure susceptibility, demonstrating that the same gain of function mutation confers enhanced chemical nociceptive sensitivity. The increased responsiveness to noxious stimuli, combined with the delayed effect of carbamazepine and the modulation by cannabidiol, highlight the translational potential of this model for exploring sodium channel related pain mechanisms.

Despite the relevance of these findings, this study has limitations. Our comparative and structural analyses revealed a strong sequence and domain conservation between para and human Na_v1.7. However, we acknowledge that functional electrophysiological validation was not performed in this study. Demonstrating that human Na_v1.7 can rescue para mutant phenotypes would indeed provide direct evidence of functional equivalence. Future work incorporating electrophysiological and genetic rescue approaches will be critical to further validate para mutants as translational models for human pain‐related sodium channelopathies.

Second, the focus on a single nociceptive assay may limit the generalizability of the model to other pain modalities. The effects of CBD are promising; however, the mechanism by which it modulates the para channel in flies requires further investigation. Although our study focused on the behavioural manifestations of para hyperactivity in nociceptive contexts, employing targeted RNAi in pan‐neuronal or sensory‐neuron drivers (e.g., elav‐Gal4, ppk‐Gal4) will be instrumental in refining circuit‐specific contributions to nociceptive hypersensitivity in para ^bss1 larvae. Finally, even though para ^bss1 serves as an effective model for gain‐of‐function channelopathy, future studies employing site‐directed mutagenesis (e.g., via CRISPR/Cas9) to create mutations in Drosophila that directly correspond to residues associated with human pain, such as L858F and R1150W in Na_v1.7, would represent a powerful next step in creating even more accurate, translatable models of specific human pain syndromes.

In conclusion, our data supports the use of para ^bss1 as a genetic model for chronic nociception in D. melanogaster . The model is sensitive to chemical stimuli, resistant to conventional sodium channel blockers and responsive to cannabidiol, offering a robust in vivo platform for screening novel analgesics targeting sodium channel dysfunction.