Chloride-dependent mechanisms of multimodal sensory discrimination and neuropathic sensitization in Drosophila

Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval Class III (CIII) neurons are peripheral noxious cold nociceptors and gentle touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. Here, we show that the TMEM16/anoctamins subdued and white walker (wwk; CG15270) are required for cold-evoked CT, but not for touch-associated behavior, indicating a conserved role for anoctamins in nociception. We also evidence that CIII neurons make use of atypical depolarizing chloride currents to encode cold, and that overexpression of ncc69—a fly homologue of NKCC1—results in phenotypes consistent with neuropathic sensitization, including behavioral hypersensitization and spontaneous nociceptor activity, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

The evolution of cold nociception in drosophilid larvae and identification of a neural basis for cold acclimation

Cold temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. While there is a growing body of knowledge concerning the mechanisms underlying cold tolerance, relatively little is known concerning how insects sense noxious cold (cold nociception), or how cold nociception might function in cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform highly stereotyped, cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors) innervating the barrier epidermis. In the present study, we first sought to describe cold-nociceptive behavior among 11 drosophilid species with differing cold tolerances and from differing climates. Behavioral analyses revealed that the predominant cold-evoked response among drosophilid larvae is a head-to-tail contraction (CT) behavior, which is likely inherited from a common ancestor. However, despite lack of phylogenetic signal (suggesting trait lability), the CT behavior was transient and there was no clear evidence that cold sensitivity was related to thermal environment; collectively this suggests that the behavior might not be adaptive. We therefore sought to uncover an alternative way that cold nociception might be protective. Using a combination of cold-shock assays, optogenetics, electrophysiology, and methods to genetically disrupt neural transmission, we demonstrate that cold sensing neurons in Drosophila melanogaster (Class III nociceptors) are sensitized by and critical to cold acclimation. Moreover, we demonstrate that cold acclimation can be optogenetically-evoked, sans cold. Collectively, these findings reveal that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.Significance StatementMany insects adapt to cold in response to developmental exposure to cool temperatures. While there is a growing body of knowledge concerning the mechanisms underlying cold tolerance, it is unknown how sensory neurons might contribute. Here, we show that noxious cold sensing (cold nociception) is widely present among drosophilid larvae, and that cold-sensing neurons (Class III cold nociceptors) are necessary and sufficient drivers of cold acclimation. This suggests that cold acclimation has, at least in part, a neural basis.

Peripheral kappa opioid receptor activation drives cold hypersensitivity in mice

Noxious cold sensation is commonly associated with peripheral neuropathies, however, there has been limited progress in understanding the mechanism of cold pain. Transient receptor potential (TRP) A1 channels facilitate the perception of noxious cold at the level of dorsal root ganglia (DRG), where kappa opioid receptors (KOR) are also expressed but have not previously been implicated in cold sensation. Here we identify a new role for KOR in enhancing cold hypersensitivity. First, we show that systemic KOR agonism (U50,488, KOR agonist), significantly potentiates the latency to jump and the number of jumps on the cold plate compared controls at 3°C. Importantly, NorBNI (KOR antagonist) attenuates U50,488-induced cold hypersensitivity. However, the central administration of NorBNI does not block U50,488-induced cold hypersensitivity suggesting that peripheral KOR likely modulate this effect. Furthermore, the peripherally-restricted KOR agonist, ff(nle)r-NH2 also induces cold hypersensitivity. Using fluorescent in situ hybridization, we show that KOR mRNA colocalizes with the transcripts for the cold-activated TRPA1 and TRPM8 channels in DRG. Finally, using calcium imaging in DRG, we show that intracellular calcium release is potentiated during the simultaneous application of a TRPA1 agonist, mustard oil (MO), and a KOR agonist (U50,488), when compared to MO alone. This potentiated calcium response is absent in TRPA1 KO mice. Together our data suggest that KOR-induces cold hypersensitivity through modulation of peripheral TRPA1 channels. These findings indicate that whether activation of peripheral KOR is protective or not may be dependent on the pain modality.

The effect of pain conditioning on experimentally evoked cough: evidence of impaired endogenous inhibitory control mechanisms in refractory chronic cough

The pathophysiology of refractory chronic cough (RCC) is unclear. We hypothesised that endogenous inhibitory control mechanisms, such as those activated by noxious stimuli inducing pain (conditioned pain modulation) may be capable of inhibiting coughing and urge to cough evoked by inhaled capsaicin. Furthermore, these mechanisms may be impaired in patients with RCC.The objective was to investigate the effects of pain on cough and urge to cough in healthy volunteers and RCC patients. Healthy volunteers and RCC patients underwent a randomised, controlled, four-way crossover study comparing the effect of four interventions on capsaicin-evoked coughing and urge to cough. The interventions comprised immersing a hand in 1) noxious cold water; 2) warm water; 3) warm water, but subjects were instructed to voluntarily supress coughing; and 4) no intervention. The co-primary outcomes were numbers of evoked coughs and urge to cough scores.20 healthy volunteers (mean±sd age 50.1±14.2 years, male:female 10:10) and 20 RCC patients (age 60.1±7.9 years, male:female 9:11) participated. Overall, noxious cold water reduced capsaicin-evoked urge-to-cough scores and cough numbers compared with warm water (1.6 (95% CI 1.3–2.0) versus 2.2 (1.8–2.6), p<0.001 and 4.8 (3.7–6.2) coughs versus 7.9 (6.7–9.5) coughs, p<0.001, respectively). Healthy volunteers and RCC patients demonstrated similar reductions in the urge to cough during noxious cold-water immersion, but noxious cold water and voluntary suppression interventions were less effective at reducing capsaicin-evoked cough in RCC patients than in healthy volunteers (p=0.041).Endogenous inhibitory control mechanisms, specifically those activated by pain, can reduce both coughing and the urge to cough. Impairment of endogenous inhibitory control mechanisms may contribute to excessive coughing in RCC.

Human and Mouse TRPA1 Are Heat and Cold Sensors Differentially Tuned by Voltage

Transient receptor potential ankyrin 1 channel (TRPA1) serves as a key sensor for reactive electrophilic compounds across all species. Its sensitivity to temperature, however, differs among species, a variability that has been attributed to an evolutionary divergence. Mouse TRPA1 was implicated in noxious cold detection but was later also identified as one of the prime noxious heat sensors. Moreover, human TRPA1, originally considered to be temperature-insensitive, turned out to act as an intrinsic bidirectional thermosensor that is capable of sensing both cold and heat. Using electrophysiology and modeling, we compare the properties of human and mouse TRPA1, and we demonstrate that both orthologues are activated by heat, and their kinetically distinct components of voltage-dependent gating are differentially modulated by heat and cold. Furthermore, we show that both orthologues can be strongly activated by cold after the concurrent application of voltage and heat. We propose an allosteric mechanism that could account for the variability in TRPA1 temperature responsiveness.

Is TRPA1 Burning Down TRPV1 as Druggable Target for the Treatment of Chronic Pain?

Over the last decades, a great array of molecular mediators have been identified as potential targets for the treatment of chronic pain. Among these mediators, transient receptor potential (TRP) channel superfamily members have been thoroughly studied. Namely, the nonselective cationic channel, transient receptor potential ankyrin subtype 1 (TRPA1), has been described as a chemical nocisensor involved in noxious cold and mechanical sensation and as rivalling TRPV1, which traditionally has been considered as the most important TRP channel involved in nociceptive transduction. However, few TRPA1-related drugs have succeeded in clinical trials. In the present review, we attempt to discuss the latest data on the topic and future directions for pharmacological intervention.