Nociceptive Behavior Research Articles

Upregulated spinal histone deacetylases induce nociceptive sensitization by inhibiting the GABA system in chronic constriction injury-induced neuropathy in rats.

Neuropathic pain (NP) affects countless people worldwide; however, few effective treatments are currently available. Histone deacetylases (HDACs) participate in epigenetic modifications in neuropathy-induced nociceptive sensitization. Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that can inhibit NP. The present study aimed to examine the role of spinal HDAC and its isoforms in neuropathy. Male Wistar Rat with chronic constriction injury (CCI)-induced peripheral neuropathy and HDAC inhibitor, panobinostat, was administrated intrathecally. We performed quantitative real-time polymerase chain reaction (RT-qPCR), western blot, and immunohistochemical analysis of lumbar spinal cord dorsal horn and nociceptive behaviors (thermal hyperalgesia and mechanical allodynia) measurements. Herein, RT-qPCR analysis revealed that spinal hdac3, hdac4, and hdac6 were upregulated in CCI rats. Western blotting and immunofluorescence staining further confirmed that HDAC3, HDAC4, and HDAC6 were significantly upregulated, whereas GABA and its synthesis key enzyme glutamic acid decarboxylase (GAD) 65 were dramatically downregulated. Intrathecal panobinostat attenuated nociceptive behavior and restored the downregulated spinal GAD65 and GABA expression in CCI rats. HDAC upregulation might induce nociception through GAD65 and GABA inhibition in CCI-induced neuropathy. These findings strongly suggest that HDACs negatively regulate inhibitory neurotransmitters, constituting a potential therapeutic strategy for an epigenetic approach to manage NP.

Cinnamaldehyde induces a TRPA1-mediated nociceptive behavior in planarians.

Nociception is defined as "the neural process of encoding noxious stimuli" by the International Association for the Study of Pain (IASP). Nociception relies on detecting noxious stimuli arising from a potentially or actually tissue-damaging event via specialized cells called nociceptors. In planarians, nociceptive behavior is often indicated by a 'scrunching' gait, in contrast to the usual gliding behavior displayed in normal conditions. The present study extends our previous study Reho et al. (2024) by testing a new potentially irritant molecule, Cinnamaldehyde (CA), which could induce scrunching gaits. We reproduced the nociceptive chemical tests from our previous study using CA instead of Allyl isothiocyanate (AITC) on Girardia dorotocephala (Gd) implementing an open field behavioral analysis. CA induced a dose-dependent increase in scrunching gait similar to the action of AITC and was expectedly partially suppressed by morphine and meloxicam. Knocking down the expression of the Gd-TRPA1 ion channel by RNA interference also suppressed the behavioral reaction to the molecule. In conclusion, we demonstrated that CA induced a nociceptive behavior in planarians through an action on the ion channel TRPA1. SIGNIFICANCE STATEMENT: In this article, we provide evidence that cinnamaldehyde induces a nociceptive behavior through a direct action in an invertebrate model (flatworm) much in the same way that in vertebrates.

Female specific interactions of serotonin and testosterone in the rostral ventromedial medulla after activity-induced muscle pain

Classical preclinical studies show that serotonin (5-HT) injected into the rostral ventromedial medulla (RVM) produces analgesia that is blocked by 5-HT2 receptor antagonists. One key modulator of 5-HT activity is the serotonin transporter (SERT) which reduces serotonergic signaling through reuptake into the presynaptic terminal. In the activity-induced muscle pain model, females show widespread pain and increased SERT expression in the RVM whereas males show localized pain and no changes in SERT expression. Since prior studies show testosterone protects from the development of widespread pain, and females have widespread pain in the activity-induced pain model, we hypothesized that testosterone modulates serotonin signaling to enhance analgesia in female mice with widespread pain. We showed that testosterone reduced the enhanced SERT protein expression and increased 5-HT2A receptor mRNA expression in the RVM normally observed in the activity-induced pain model in females, but not males. Inhibition of SERT in the RVM was analgesic in both female and male mice; this analgesia was blocked by co-administration of 5-HT2A antagonist. Next, using in situ hybridization, we demonstrated co-expression of SERT, 5-HT2A receptor, and androgen receptor mRNA in cells within the RVM in female mice. Lastly, activation of androgen receptors using dihydrotestosterone reduced hyperalgesia in female mice. These data therefore show for the first time expression of androgen receptors in the RVM in female mice, that activation of androgen receptors reduces nociceptive behaviors, and endogenous testosterone modulates SERT and 5-HT2 receptor expression. Thus, we show a sex-specific role for how testosterone modulates analgesia in female mice. PerspectiveThis article presents novel mechanisms testosterone’s protection against muscle pain in female mice showing modulation of the serotonin system in the rostral ventromedial medulla. Understanding the relationship between testosterone and serotonin could lead to better treatment of individuals with muscle pain.

The role of neutrophils in pain: systematic review and meta-analysis of animal studies.

The peripheral inflammatory response is an attractive therapeutic target for pain treatment. Neutrophils are the first circulating inflammatory cells recruited to sites of injury, but their contribution to pain outcomes is unclear. We performed a systematic review and meta-analysis of original preclinical studies, which evaluated the effect of preemptive neutrophil depletion on pain outcomes (PROSPERO registration number: CRD42022364004). Literature search (PubMed, January 19, 2023) identified 49 articles, which were meta-analyzed using a random-effects model. The risk of bias was evaluated using SYRCLE's tool. The pooled effect considering all studies showed that neutrophil depletion induced a consistent pain reduction. Inflammatory, joint, neuropathic, and visceral pain showed significant pain alleviation by neutrophil depletion with medium-large effect sizes. However, muscle and postoperative pain were not significantly alleviated by neutrophil depletion. Further analysis showed a differential contribution of neutrophils to pain outcomes. Neutrophils had a higher impact on mechanical hyperalgesia, followed by nociceptive behaviors and mechanical allodynia, with a smaller contribution to thermal hyperalgesia. Interspecies (mice or rats) differences were not appreciated. Analyses regarding intervention unveiled a lower pain reduction for some commonly used methods for neutrophil depletion, such as injection of antineutrophil serum or an anti-Gr-1 antibody, than for other agents such as administration of an anti-Ly6G antibody, fucoidan, vinblastine, CXCR1/2 inhibitors, and etanercept. In conclusion, the contribution of neutrophils to pain depends on pain etiology (experimental model), pain outcome, and the neutrophil depletion strategy. Further research is needed to improve our understanding on the mechanisms of these differences.

Morphine-induced hyperalgesia impacts small extracellular vesicle miRNA composition and function.

Morphine and other synthetic opioids are widely prescribed to treat pain. Prolonged morphine exposure can paradoxically enhance pain sensitivity in humans and nociceptive behavior in rodents. To better understand the molecular mechanisms underlying opioid-induced hyperalgesia, we investigated changes in miRNA composition of small extracellular vesicles (sEVs) from the serum of mice after a morphine treatment paradigm that induces hyperalgesia. We observed significant differential expression of 18 miRNAs in sEVs from morphine-treated mice of both sexes compared to controls. Several of these miRNAs were bioinformatically predicted to regulate cyclic AMP response element binding protein (CREB), a well-characterized transcription factor implicated in pain and drug addiction. We confirmed the binding and repression of Creb mRNA by miR-155 and miR-10a. We tested if serum-derived sEVs from morphine-treated mice could elicit nociceptive behavior in naïve recipient mice. Intrathecal injection of 1 μg sEVs did not significantly impact basal mechanical and thermal threshold in naïve recipient mice. However, prophylactic 1 μg sEV administration in recipient mice resulted in faster resolution of complete Freund's adjuvant-induced mechanical and thermal inflammatory hypersensitivity. Other behaviors assayed following administration of these sEVs were not impacted including sEV conditioned place preference and locomotor sensitization. These results indicate that morphine regulation of serum sEV composition can contribute to analgesia and suggest a potential for sEVs to be a non-opioid therapeutic intervention strategy to treat pain.

Glucosylceramide synthase inhibitor ameliorates chronic inflammatory pain

Gangliosides play pivotal roles in neuronal tissue processes, such as axonal elongation, synaptic transmission, and neuronal degeneration. Several studies have shown that mice injected with gangliosides synthesized from glucosylceramide exhibit mechanical allodynia. Thus, we hypothesized that glucosylceramide synthase inhibitors affect nociceptive behavior. We investigated the analgesic effect of intrathecal glucosylceramide inhibition on bilateral allodynia caused by prolonged unilateral hind paw inflammation in mice. Repeated administration of a glucosylceramide inhibitor reduced mechanical allodynia in both inflamed and non-inflamed hind paws. These results suggested that ganglioside reduction is critical for analgesia during inflammatory pain.

Sex differences in the orofacial antinociceptive effect of metformin and the role of transient receptor potential channels.

Metformin is classified as a biguanide and is used in the treatment of type 2 diabetes. It is used worldwide and has been investigated in drug repositioning. The present study aims to investigate whether there is sexual dimorphism in the orofacial antinociceptive effect of metformin and the participation of TRP channels. Acute nociceptive behavior was induced by administering cinnamaldehyde or capsaicin to the upper lip. Nociceptive behavior was assessed through orofacial rubbing, and the effects of pre-treatment with metformin (125 or 250mg/Kg) or vehicle (control) were tested on the behavior. Nociceptive behavior was also induced by formalin injected into the temporomandibular joint. The chronic pain model involved infraorbital nerve transection (IONX) was evaluated using Von Frey electronic filaments. Trpv1 gene expression was analyzed in the nerve ganglion. Docking experiments were performed. Metformin, but not the vehicle, produced antinociception (p < 0.0001) in all acute nociceptive behaviors in both sexes, and these effects were attenuated by the TRPV1 antagonist capsazepine and the TRPA1 antagonist HC-030031. In IONX with better (**p < 0.01, ****p < 0.0001 vs. control) results in females. TRPV1 gene expression was observed in the metformin treated group (*p < 0.05 vs. control). Docking experiments revealed that metformin may interact with TRPV1 and TRPA1 channels. Metformin promotes orofacial antinociception in both sexes in acute pain and is more effective in chronic pain in females than in males, through the modulation of TRPV1 and TRPA1 channels. These preclinical findings suggest a potential repositioning of metformin as an analgesic agent in acute and chronic orofacial pain states.

Statins ameliorate oxaliplatin- and paclitaxel-induced peripheral neuropathy via glutathione S-transferase

Some therapeutic agents have been found to have effects beyond their primary indications. Peripheral neuropathy, a common side effect of chemotherapy, remains inadequately treated. Identifying additional properties of existing medications could thus uncover novel therapeutic avenues. Previous studies have identified an additional effect of simvastatin in reducing neuropathy; however, the mechanism underlying this effect remains unclear. We investigated the novel effects of statins on chemotherapy-induced peripheral neuropathy in mice. Mice treated with oxaliplatin or paclitaxel did not show exacerbation or improvement in cold sensations upon acetone testing with statin administration. However, concurrent oral statin treatment mitigated the nociceptive response to mechanical stimuli induced by each anti-tumor agent. Co-administration of a glutathione S-transferase inhibitor, which modulates redox reactions, abolished the ameliorative effect of statins on mechanical nociceptive behavior. Additionally, the glutathione S-transferase inhibitor did not affect normal sensory perception or impair the anti-tumor effect of chemotherapy agents. A search for GST-associated molecules and pathways using artificial intelligence revealed that GST regulates inflammatory cytokines as a regulatory or causative gene. Our findings suggest that statins have class effects that ameliorate cytotoxic anti-cancer drug-induced mechanical allodynia via GST pathway activation.

Astrocytic P2X7 receptor in retrosplenial cortex drives electroacupuncture analgesia.

P2X7 receptor (P2X7R) has been found to contribute to the peripheral mechanism of acupuncture analgesia (AA). However, whether it plays an important role in central mechanism remains unknown. In this study, we aimed to reveal the role of astrocytic P2X7R in retrosplenial cortex (RSC) in AA and provide new evidence for underlying the central mechanism of AA. We applied the chemogenetic receptors hM3Dq to stimulate or hM4Di to inhibit astrocytes ligand clozapine-N-oxide (CNO) following injection of adeno-associated virus (AAV) into the bilateral RSC, or pharmacologically intervened in the activity of the purinergic receptor P2X7R. Current data indicated that chemogenetic inhibition of astrocytes or injection of P2X7R agonist Bz-ATP in the bilateral RSC significantly reverses the analgesic effect of electroacupuncture (EA) in formalin tests while the bilateral injection of the P2X7R antagonist A438079 alleviated formalin-induced nociceptive behavior. Additionally, chemogenetic suppression of astrocytic P2X7R by injection of AAV in the bilateral RSC decreased hind paw flinches induced by formalin in the mice. These findings indicate the participation of both astrocytes and P2X7R in the RSC in EA analgesic. Moreover, P2X7R on astrocytes in the RSC appears to play a critical role in the ability of EA to attenuate formalin-induced pain responses in mice.

Prevention and reversal of neuropathic pain by near-infrared photobiomodulation therapy in male and female rats

Pathological nociception arising from peripheral nerve injury impacts quality of life. Current therapeutics are generally ineffective. However, photobiomodulation therapy (PBMT) has shown promise in addressing this issue. We aimed to assess the potential anti-allodynic effects of 2 p.m. protocols, each applied transcutaneously over the peripheral nerve injury. In addition to evaluating nociceptive behavior, we also conducted morphological analysis using electron microscopy (EM) to investigate potential ultrastructural changes at the cellular level. We sought to determine, using the chronic constriction injury (CCI) model, whether our parameters could alleviate established allodynia and/or dampen allodynia development. Adult male and female rats with CCI or sham were treated with PBMT (850-nm wavelength) for 2 min, 3 times a week over three or four weeks across three studies, where PBMT began either before or after CCI. Allodynia was assessed prior to surgery and across weeks and, at the conclusion of the third study, sciatic nerve was processed for EM and histomorphometrically evaluated. The results showed that PBMT before versus after CCI injury yielded similar behaviors, effectively decreasing allodynia. Interestingly, these positive effects of PBMT do not appear to be accounted by protection of the sciatic injury site, based on EM. CCI reliably decreased axon size and the number of myelinated axons present in both PBMT and control groups. While PBMT reduced the number of C-fibers in CCI samples, no improvement in any measure was observed in response to PBMT.

Comparison of pain modulatory effect of the LPGi estragon receptor on inflammatory pain between pro-estrus and estrus phases and OVX rats.

The present study has investigated whether circulating estrogen level variations in the pro-estrus and estrus phases of the intact rats and estrogen depletion in the ovariectomized animals (OVX) adjust the formalin-induced nociceptive behaviors. During the pro-estrus and estrus phases of rats' estrus cycle and in the OVX rats, 17β-estradiol and ICI 182,780 (estrogen receptor antagonist) were administered into the right paragigantocellularis lateralis (LPGi) nucleus. Then, the formalin-induced flexing and licking responses were recorded for 60min. The findings of this study revealed that intra-LPGi administration of 17β-estradiol (0.8μmol) reduced the formalin-induced flexing and licking duration in pro-estrus and estrus rats (P < 0.001), suggesting an analgesic effect. 17β-Estradiol injection into the LPGi nucleus of OVX rats increased the flexing duration (P < 0.05) while decreasing the licking duration (P < 0.05) of the formalin test. The pain modulatory effect of 17β-estradiol on the flexing response was reversed by ICI 182,780 (15nmol) in the pro-estrus (P < 0.001) and estrus rats (P < 0.001) but not in the OVX rats. Also, pretreatment of LPGi nucleus with ICI 182,780 reversed the analgesic effect of 17β-estradiol on the licking response in the pro-estrus (P < 0.05), estrus (P < 0.001), and OVX rats (P < 0.001). These results suggest that the pain threshold in intact female rats is modulated independently of the estrus state. Still, the basal level of plasma estrogen and the activation of its receptors are necessary for pain modulation.

The restraint stress-induced antinociceptive effects decreased by antagonism of both orexin receptors within the CA1 region of the hippocampus

Studies have indicated that stress-related symptoms can lead to hormonal and neural changes, affecting the pain threshold and nociceptive behaviors. The precise role of orexin receptors (OX1r and OX2r) in stress-induced analgesia (SIA) remains an inquiry yet to be comprehensively elucidated. The current investigation aimed to assess the impact of acute immobilization restraint stress on pain-related behavioral responses after administering antagonists targeting OX1r and OX2r in a rat model using the tail-flick test. After a period of five to seven days post-stereotaxic surgery in CA1, the baseline tail-flick latency (TFL) was recorded for each animal. Subsequently, rats were unilaterally administered varying doses of the OX1r antagonist (SB334867; 1, 3, 10, and 30 nmol), the OX2r antagonist (TCS OX2 29; 1, 3, 10, and 30 nmol), or a vehicle (0.5 μl solution containing 12% DMSO) through an implanted cannula. Following a 5-min interval, the animals were subjected to a restraint stress (RS) lasting for 3 h. The tail-flick test was conducted after the stress exposure, and the TFLs were assessed at 60-min intervals. The findings of this study revealed that RS elicits antinociceptive responses in the tail-flick test. Microinjection of OX1r and OX2r antagonists into the CA1 attenuated RS-induced analgesia during the tail-flick test. Furthermore, the results underscored the preeminent role of OX2 receptors in modulating SIA. In conclusion, the orexin system localized within the hippocampal CA1 region may, in part, contribute to the manifestation of SIA in the context of acute pain.

Perceiving the Spectrum of Pain: Wavelength-Sensitive Visual Nociceptive Behaviors in Monolayer MoS2-Based Optical Synaptic Devices

Perceiving the Spectrum of Pain: Wavelength-Sensitive Visual Nociceptive Behaviors in Monolayer MoS₂-Based Optical Synaptic Devices

Neuroinflammation evoked mechanisms for neuropathic itch in the spared nerve injury mouse model of neuropathic pain

A large portion of neuropathic pain suffering patients may also concurrently experience neuropathic itch, with a negative impact on the quality of life. The limited understanding of neuropathic itch and the low efficacy of current anti-itch therapies dictate the urgent need of a better comprehension of molecular mechanisms involved and development of relevant animal models. This study was aimed to characterize the itching phenotype in a model of trauma-induced peripheral neuropathy, the spared nerve injury (SNI), and the molecular events underlying the overlap with the nociceptive behavior. SNI mice developed hyperknesis and spontaneous itch 7–14 days after surgery that was prevented by gabapentin treatment. Itch was associated with pain hypersensitivity, loss of intraepidermal nerve fiber (IENF) density and increased epidermal thickness. In coincidence with the peak of scratching behavior, SNI mice showed a spinal overexpression of IBA1 and GFAP, microglia and astrocyte markers respectively. An increase of the itch neuropeptide B-type natriuretic peptide (BNP) in NeuN+ cells, of its downstream effector interleukin 17 (IL17) along with increased pERK1/2 levels occurred in the spinal cord dorsal horn and DRG. A raise in BNP and IL17 was also detected at skin level. Stimulation of HaCat cells with conditioned medium from BV2-stimulated SH-SY5Y cells produced a dramatic reduction of HaCat cell viability. This study showed that SNI mice might represent a model for neuropathic itch and pain. Collectively, our finding suggest that neuropathic itch might initiate at spinal level, then affecting skin epidermis events, through a glia-mediated neuroinflammation-evoked BNP/IL17 mechanism.

Antinociceptive and Anti-Inflammatory Activities of Acetonic Extract from Bougainvillea x buttiana (var. Rose).

Background:Bougainvillea x buttiana is an ornamental plant with antioxidant, anti-inflammatory, and cytotoxic activities, which has been traditionally used to treat respiratory diseases. This study aimed to investigate whether the acetonic extract of Bougainvillea x buttiana var. Rose (BxbRAE-100%) has analgesic and anti-inflammatory properties and its potential action mechanisms. Methods: Analgesic and anti-inflammatory activities were evaluated using three murine pain models and two acute inflammation models. In vitro, the ability of the extract to inhibit proteolytic activity and the activities of the enzymes phospholipase A2 (PLA2) and cyclooxygenase (COX) were evaluated. In silico analysis was performed to predict the physicochemical and Absorption, distribution, metabolism, and excretion (ADME) profiles of the compounds previously identified in BxbRAE-100%. Results: In vivo BxbRAE-100% decreased the nociceptive behaviors in the writhing model, the tail immersion, and the formalin test, suggesting that the extract has the potential to relieve pain at peripheral and central levels. Additionally, topical or oral BxbRAE-100% treatment reduced dose-dependent 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced ear inflammation and carrageenan-induced paw edema, respectively. In vitro, BxbRAE-100% significantly inhibited proteolytic activity and PLA2, COX-1 and COX-2 activities. In silico, the compounds previously identified in BxbRAE-100% met Lipinski's rule of five and showed adequate ADME properties. Conclusions: These results support the use of B. x buttiana in Traditional Mexican Medicine and highlight its potential for the development of new treatments for pain and inflammation.

TiN/TiOx/WOx/Pt heterojunction memristor for sensory and neuromorphic computing

The advancement of artificial intelligence (AI) has spurred increasing demands for efficiency, as AI technologies require rapid processing and large-scale data handling to perform complex tasks and enable real-time decision-making. By incorporating biological nociceptor functions, memristors have become instrumental in E-Skin applications, aiding robotics in assessing potential danger. Moreover, in pursuit of energy-efficient data processing, researchers have turned their attention to neuromorphic computing, mimicking the operations of the human brain. In this context, the multifunctional capabilities of individual memristors are pivotal for their utility across diverse conditions. To enhance efficiency, this study investigated the observation of both synaptic and nociceptive behaviors within a heterojunction memristor comprising a TiN/TiOx/WOx/Pt stack. By employing a controlled pulse scheme, advanced sensing capabilities were discovered within our memristor, mimicking key functions of nociceptors such as threshold detection, lack of adaptation, relaxation, and sensitization. Additionally, the processes of potentiation and depression were leveraged to demonstrate the linear adjustment of synaptic weights based on applied pulse schemes, exhibiting various synapse-like behaviors, and enabling advanced computing functions. Consequently, both sensing and computing functionalities of the memristor were achieved using a singular TiN/TiOx/WOx/Pt memristor.

Insulin enhances acid-sensing ion channel currents in rat primary sensory neurons

Insulin has been shown to modulate neuronal processes through insulin receptors. The ion channels located on neurons may be important targets for insulin/insulin receptor signaling. Both insulin receptors and acid-sensing ion channels (ASICs) are expressed in dorsal root ganglia (DRG) neurons. However, it is still unclear whether there is an interaction between them. Therefore, the purpose of this investigation was to determine the effects of insulin on the functional activity of ASICs. A 5 min application of insulin rapidly enhanced acid-evoked ASIC currents in rat DRG neurons in a concentration-dependent manner. Insulin shifted the concentration–response plot for ASIC currents upward, with an increase of 46.2 ± 7.6% in the maximal current response. The insulin-induced increase in ASIC currents was eliminated by the insulin receptor antagonist GSK1838705, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol-3 kinase antagonist wortmannin. Moreover, insulin increased the number of acid-triggered action potentials by activating insulin receptors. Finally, local administration of insulin exacerbated the spontaneous nociceptive behaviors induced by intraplantar acid injection and the mechanical hyperalgesia induced by intramuscular acid injections through peripheral insulin receptors. These results suggested that insulin/insulin receptor signaling enhanced the functional activity of ASICs via tyrosine kinase and phosphatidylinositol-3 kinase pathways. Our findings revealed that ASICs were targets in primary sensory neurons for insulin receptor signaling, which may underlie insulin modulation of pain.

Nociception in Drosophila Larvae.

Nociception is the sensory modality by which animals sense stimuli associated with injury or potential tissue damage. When Drosophila larvae encounter a noxious thermal, chemical, or mechanical stimulus, they perform a stereotyped rolling behavior. These noxious stimuli are detected by polymodal nociceptor neurons that tile the larval epidermis. Although several types of sensory neurons feed into the nociceptive behavioral output, the highly branched class IV multidendritic arborization neurons are the most critical. At the molecular level, Drosophila nociception shares many conserved features with vertebrate nociception, making it a useful organism for medically relevant research in this area. Here, we review three larval assays for nociceptive behavior using mechanical stimuli, optogenetic activation, and the naturalistic stimuli of parasitoid wasp attacks. Together, the assays described have been successfully used by many laboratories in studies of the molecular, cellular, and circuit mechanisms of nociception. In addition, the simple nature of the assays we describe can be useful in teaching laboratories for undergraduate students.

Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae.

In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have been identified based on the expression patterns of genes that are known to be required for nociception responses. The synaptic output of these neurons, known as class IV multidendritic sensory neurons, is required for behavioral responses to thermal, mechanical, and chemical triggers of the rolling escape locomotion. Importantly, optogenetic stimulation of the class IV multidendritic neurons has also shown that the activation of those cells is sufficient to trigger nociceptive rolling. Optogenetics uses light-activated ion channels expressed in neurons of interest to bypass the normal physiological transduction machinery so that the cell may be activated in response to light that is applied by the investigator. This protocol describes an optogenetic technique that uses channelrhodopsin-2 (ChR2) to activate larval nociceptors and trigger nociceptive rolling. First, we explain how to set up the necessary genetic crosses and culture the larval progeny. Next, we describe how to perform the optogenetic nociception assay on third-instar larvae.

Assaying Nociception Behaviors in Drosophila Larvae During Parasitoid Wasp Attacks.

Nociception in fruit fly (Drosophila melanogaster) larvae is characterized by a stereotyped escape behavior. When a larva encounters a noxious (potentially harmful) stimulus, it responds by curving its body into a c-shape and rolling in a corkscrew-like manner around its long-body axis. This rolling behavior may serve to quickly remove the larva from the source of the noxious stimulus, and is particularly adaptive to escape from a common natural predator of fruit fly larvae: parasitoid wasps (Leptopilina boulardi). L. boulardi completes its life cycle by using fruit fly larvae as hosts for its offspring. Female wasps sting fly larvae with an ovipositor and lay an egg within the larva. The wasp offspring hatches inside the fly larva, consumes the fly tissues during pupation, and eventually emerges from the pupal case as an adult wasp. Fruit fly larvae respond to oviposition attacks by rolling, which causes the long flexible ovipositor to be wound around the larval body like a spool. This dislodges the wasp and allows the larva to attempt to escape by crawling. Rolling behavior is triggered by the activation of sensory neurons (nociceptors) whose function can inform our understanding of the mechanisms of nociception. In this protocol, we describe a simple behavioral assay to test and measure nociceptive responses in Drosophila larvae during oviposition attacks by female parasitoid wasps. First, we discuss parasitoid wasp husbandry and culturing methods in the laboratory. We then describe how to perform the wasp nociception assay on third-instar fruit fly larvae.