The bisphosphonate zoledronic acid produces analgesia by inhibiting the transient receptor potential vanilloid 1 (TRPV1) channel.

The bisphosphonate zoledronic acid produces analgesia by inhibiting the transient receptor potential vanilloid 1 (TRPV1) channel. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 20 May 2025 V1 Latest version Share on The bisphosphonate zoledronic acid produces analgesia by inhibiting the transient receptor potential vanilloid 1 (TRPV1) channel. Authors : Karina Carvajal-Zamorano , Cesar Amaya-Rodriguez 0000-0003-1916-573X , Ana Gomez del Campo , Angelina Palacios , Carlos Ancaten-Gonzalez , Rodrigo Meza , Wladimir Plaza-Briceño , … Show All … , Ignacio Segura , Rosa Scala , Domenico Tricarico 0000-0003-2726-0462 , Pablo R. Moya , Rodolfo Madrid , Andres Chavez , Ramon Latorre , and Karen Castillo 0000-0003-0353-7025 [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.174773025.57881971/v1 1491 views 850 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The transient receptor potential vanilloid 1 (TRPV1) channel, known as the heat and capsaicin (CAP) receptor, plays a crucial role in pain sensation. Although CAP and its analogs can induce topical analgesia, their use is limited by severe thermoregulatory side effects. Here, we provide robust evidence that the bone antiresorptive agent zoledronic acid (ZOL) exerts analgesic effects by inhibiting TRPV1. In vitro, ZOL suppresses CAP-evoked responses in dorsal root ganglion (DRG) neurons and reduces TRPV1 channel conductance in Xenopus laevis oocytes, inhibiting its activity over a broad concentration range by occluding the permeation pathway from both the extracellular and intracellular sides. In vivo, ZOL administration attenuates thermal nociceptive behaviors in mice and flies exposed to noxious heat. Moreover, ZOL prevents CAP- and TRPV1-mediated synaptic effects, mimicking the actions of the TRPV1 antagonist capsazepine. Collectively, these findings identify ZOL as a potent TRPV1 blocker and provide compelling mechanistic insight into its analgesic effects in vivo, positioning it as a promising scaffold for the development of novel pain-relief compounds. INTRODUCTION The transient receptor potential vanilloid type 1 (TRPV1) channel is a key integrator of pain and inflammatory signals, making it a promising target for pain management. First identified as the heat and capsaicin (CAP) receptor (Caterina et al. 1997), TRPV1 is expressed at the free nerve endings of somatosensory neurons that innervate the skin, where it responds to noxious stimuli including heat (≥42 °C), low pH (pH ≤6.0), and inflammatory mediators (Moriyama et al. 2005; Caterina et al. 1997; Tominaga et al. 1998; Kashio and Tominaga 2022). Activation of TRPV1 produces a burning sensation and contributes to itch, peripheral neuropathic pain, chronic cutaneous and visceral inflammation, and chronic pain. Prolonged exposure to CAP, the pungent compound in chili peppers, leads to TRPV1 desensitization, resulting in analgesia (Blair 2018). Several biomedical and pharmacological studies have aimed to develop CAP analogs that relieve pain via TRPV1 modulation. However, systemic administration of CAP induces significant thermoregulatory disruption and locomotor side effects (Inagaki et al. 2019), making it a two-edge sword in pain treatment. Pain is the most common reason for seeking medical care (Bigio et al. 2022; St Sauver et al. 2013; Dahlhamer et al. 2018). It is often disabling, diminishing quality of life, and carries substantial societal and economic costs (Cohen, Vase, and Hooten 2021). While opioids remain effective for managing severe pain, their prolonged use carries a high risk of dependence and abuse, highlighting the need for safer alternatives. Moreover, the current pharmacological options for chronic pain are outdated and frequently ineffective. Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate (BP) widely used to treat bone diseases such as osteoporosis, act by reducing bone loss, preserving microarchitecture, and lowering fracture risk (Scala et al. 2022). Interestingly, ZOL also alleviates pain associated with bone metastasis and other skeletal disorders, although the mechanisms underlying its analgesic effects remain poorly understood (Shea et al. 2022; Jansen et al. 2024; Lorange et al. 2023; Wang et al. 2013; Costa and Major 2009). TRPV1 plays a critical role in osteoblastogenesis (Idris, Landao-Bassonga, and Ralston 2010; Lieben and Carmeliet 2012; He et al. 2017) and is upregulated in several bone pathologies (Scala et al. 2022), raising the possibility that ZOL’s analgesic effects may involve modulation of TRPV1 activity. However, whether and how ZOL effects TRPV1 function remains unknown. Here, using a combination of in vitro and in vivo approaches, we demonstrate that ZOL is potent TRPV1 inhibitor that suppresses its activation and ion conductance, leading to reduced nociception in both mice and flies. We further show that ZOL acts by occluding the pore, thereby dramatically reducing ion flow through TRPV1. These findings provide mechanistic insight into the analgesic action of ZOL and offer a foundation for the development of new pain-relief therapies targeting TRPV1-related pathologies. RESULTS Zoledronic acid inhibits TRPV1-dependent capsaicin-evoked responses in primary sensory neurons form dorsal root ganglia . Transient receptor potential (TRP) channels, including TRPV1, are among the main transducers of painful stimuli in dorsal root ganglion (DRG) neurons, particularly in nociceptors, (Patapoutian, Tate, and Woolf 2009). Because capsaicin (CAP) activates TRPV1 and promotes calcium influx (Caterina et al. 1997), we evaluated the effect of zoledronic acid (ZOL) on CAP-induced calcium responses in dissociated mouse DRG neurons. To investigate this, we employed a double-pulse stimulation protocol using two consecutive applications of a subsaturating concentration of CAP (20 nM), as shown in Figure 1 . ZOL (10 µM) was applied three minutes prior to and during the second CAP pulse ( Figure 1B) . We compared the responses to the first (CAP1) and second (CAP2) CAP applications in the same neuron. The CAP2/CAP1 ratio was significantly lower in neurons treated with ZOL than in control cells ( Figure 1C), with an average reduction in CAP-evoked responses 74% in the ZOL group, 15% in controls ( Figure 1D ). These findings suggest that ZOL negatively modulates capsaicin-evoked calcium signaling in primary somatosensory neurons, likely through functional inhibition of TRPV1 channels. Zoledronic acid inhibits heterologously expressed TRPV1 channels . To directly assess the effects of ZOL on TRPV1 activity, we performed electrophysiological recordings in Xenopus laevis oocytes expressing TRPV1 channels. Macroscopic currents and single-channel recordings were obtained from excised membrane patches under various concentrations of ZOL. As shown in Figure 2A-B , 1 nM ZOL inhibited CAP-evoked macroscopic TRPV1 currents (500 nM CAP) in a time-dependent manner during voltage steps from -60 to +260 mV. At the single-channel level, intracellular application of 10 nM ZOL caused a progressive and significant reduction in the channel conductance over time ( Figure 2C-D ), decreasing from ∼42 pS to ∼6 pS after 17 minutes. This inhibition was observed at both nanomolar and micromolar concentrations and was largely irreversible ( Supplementary Figure 1 ). Notably, washout with ZOL-free solution failed to restore activity, and only high concentration of CAP (≥10 µM) were able to partially recover TRPV1 currents after ZOL exposure. This inhibitory effect was also observed upon the extracellular application of ZOL ( Supplementary Figure 2 ). Remarkably, TRPV1 currents were reduced by ZOL intracellular (I/0, inside out) and extracellular (O/O, outside out) sides ( Figure 3A,D ). The time constants of current decay were 3.53 ± 0.94 min (intracellular) 2.07 ± 0.54 min (extracellular), respectively ( Figure 3B, E ), indicating that extracellular application of ZOL produces faster inhibition under our experimental conditions. Together, these results demonstrate that ZOL inhibits TRPV1 channel function from both sides of the membrane. Zoledronic acid blocks the TRPV1 permeation pathway. The observed inhibition from both membrane leaflets suggested that ZOL might act by occluding the ion permeation pathway of TRPV1. To test this, we performed macroscopic current recordings outside out patches under low ionic strength (symmetrical 10 mM NaCl; Figure 4A-B ). TRPV1 currents were evoked using voltage steps from -60 mV to +300 m and then returned to -60 mV. Under control conditions (trace i), TRPV1 activity was potentiated by 500 nM CAP (trace ii). Application of 100 nM ZOL markedly reduced current amplitude (trace iii), and this inhibition persisted after extensive washing (trace iv). Subsequent perfusion with a high ionic strength solution (140 mM NaCl; trace v) caused a notable increase in current amplitude, consistent with a “knock-off” of ZOL from the pore due to increased electrochemical driving force. Finally, switching back to 10 mM NaCl (trace vi) restored the current to near baseline, confirming that the increased conductance in high saline solution was due pore unblocking, not simply increased ion availability. Similar results were obtained in inside-out configuration ( Supplementary Figure 3 ). These data indicate that ZOL occludes the TRPV1 pore from both intracellular and extracellular sides . TRPV1 blockade by zoledronic acid is favored when the channel is open . Given ZOL’s apparent pore-blocking mechanism, we investigated whether its action depends on the functional state of TRPV1. Specifically, we tested whether inhibition is favored when the channel is in its open or closed state. To reduce channel opening, we implemented a voltage protocol that held the membrane potential -60 mV, a brief depolarizing pulse to +260 mV, and a return to -60 mV, repeated every three minutes ( Figure 4C ). ZOL (100 nM) was applied during this period. In the outside-out configuration, the inhibition of TRPV1 by ZOL was markedly slower under these conditions compared to protocols where membrane was held at o mV between pulses ( Figure 4E vs. Figures 2-4 ), supporting the idea that ZOL preferentially blocks TRPV1 in its open conformation. Moreover, inhibition from the extracellular side was consistently faster than from the intracellular side ( Supplementary Figure 4 ), regardless of the protocol, though still dependent on channel opening. Zoledronic acid reduces nociceptive behavior in mice The ability to detect environmental and internal temperature is essential for maintaining thermal homeostasis and protecting tissues from potentially harmful stimuli. Noxious thermal stimuli activate nociceptors, specialized peripheral sensory neurons expressing ion channels such as TRPV1, a key transducer of noxious heat (Caterina et al. 1997). Given the reported analgesic effects of ZOL, we first evaluated whether ZOL modulates thermal nociception in mice using the hot plate test (Deuis, Dvorakova, and Vetter 2017). Mice were intraperitoneally injected with vehicle (saline) or a clinically relevant dose of ZOL (Chen et al. 2002; Hughes et al. 2019), and then placed on a metal surface maintained at constant temperatures of 45°, 50° and 55°C. The latency to nocifensive behaviors (e.g., hind paw licking or jumping) was recorded as an indicator of thermal pain sensitivity ( Figure 5 ). ZOL administration significantly increased the latency to nociceptive behaviors, particularly at 50°C and 55°C, indicating reduced sensitivity to noxious heat stimuli. These results support the hypothesis that ZOL exerts analgesic effects in vivo . To rule out confounding effects on general locomotion activity, we performed an open field test using the same apparatus. No significant differences were observed in locomotor parameters such as exploration time or distance travel ( Supplementary Figure 5 ), confirming that ZOL’s effects on thermal nociception are not due to impaired mobility. Zoledronic acid suppresses TRPV1-mediated nociceptive behavior in Drosophila melanogaster To further explore the analgesic properties of ZOL in a genetic model, we assessed nociceptive behavior in Drosophila melanogaster flies expressing human TRPV1 channels in sensory nociceptor neurons. TRPV1 expression was driven by the pain -GAL4 promoter, which targets multidendritic neurons innervating most of the body, including the mouthparts and antennal-maxillary complex (Neely et al. 2011; Jang et al. 2023). Control genotypes, pain -GAL4>w 1118 (Control A), UAS- hTRPV1 > w 1118 (Control B), and pain -GAL4>UAS- hTRPV1 (Control AB), displayed robust avoidance (~100%), indicating normal nociceptive sensitivity and that TRPV1 overexpression alone does not alter nociceptive behavior (Figure 6) . In contrast, pain -GAL4>UAS- hTRPV1 flies fed ZOL exhibited a clear dose-dependent reduction in avoidance responses, with the greatest suppression (~20–40%) observed at concentrations ≥150 μM, in both sexes. Flies fed with ZOL for 5 days These findings indicate that ZOL suppresses TRPV1-mediated thermal nociception in Drosophila, consistent with anesthetic-like effect likely mediated by TRPV1 channel inhibition. This aligns with previous studies showing that TRP channels are modulated by local anesthetics and play key roles in nociceptive sensory neurons (Leffler et al. 2008). Zoledronic acid abolishes TRPV1-mediated synaptic depression in hippocampal slices. Although best known for its peripheral functions, TRPV1 is also expressed in the central nervous system, including the hippocampus, where regulates synaptic transmission and plasticity (Kauer and Gibson 2009) (Meza et al. 2022). excitatory transmission and mediate a form of endocannabinoid-dependent long-term depression (LTD)(Chavez, Chiu, and Castillo 2010; Chavez et al. 2014). To assess whether ZOL modulates TRPV1-dependent plasticity in the brain, we recorded excitatory postsynaptic currents (EPSCs) in the medial perforant path- dentate granule cell (MPP-DGC) synapses of acute mouse hippocampal slices ( Supplementary Figure 6A ). ZOL application did not affect baseline EPSC amplitude or paired-pulse ratio (PPR), indicating no alteration in basal synaptic transmission or presynaptic release probability ( Supplementary Figure 6B ). Next, we examined whether ZOL interferes with TRPV1-mediated LTD. Capsaicin (1 μM) induced robust depression of AMPA receptor-mediated EPSCs in control slices, an effect that was abolished by co-application of either the TRPV1 antagonist capsazepine (10 μM) or ZOL (100 nM) ( Supplementary Figure 6C ). Additionally, low frequency stimulation (1Hz) elicited LTD at MPP-DGC synapse in control slices that was eliminated by both ZOL and capsazepine ( Supplementary Figure 6D ). Importantly, ZOL did not affect other forms of synaptic plasticity in which TRPV1 is not involved, such as LTD at inhibitory synapses (Chevaleyre and Castillo 2003) ( Supplementary Figure 6E ), suggesting selective inhibition of TRPV1-dependent mechanisms . Collectively, these findings demonstrate that ZOL abolishes TRPV1-mediated synaptic depression in the hippocampus, reinforcing its role as a functional TRPV1 antagonist in the central nervous system. DISCUSION TRPV1 channel activation occurs in response to various noxious stimulus, including heat, acidic pH, vanilloids, and inflammatory mediators, producing a burning sensation and pain. Its activation can lead to the release of neuropeptides such as CGRP and substance P, contributing to neurogenic inflammation and increased vascular permeability. Sensitization of TRPV1 lowers its activation threshold, enhancing pain perception and rendering this channel a central player in the development and persistence of inflammatory pain. While acute exposure to TRPV1 agonists typically evokes pain, repeated stimulation can lead to desensitization, reducing pain perception. This principle underlies the clinical use of topical CAP chronic pain (Blair 2018) and resiniferatoxin (RTX) in bladder overactivity and cancer-related pain (Szallasi and Sheta 2012; Brederson, Kym, and Szallasi 2013). However, due to their initial excitatory effects, TRPV1 agonists can exacerbate pain in sensitized state and are associated with adverse effects when administrated systemically (Hayes et al. 1984; Arnold, Bruce-Low, and Sammut 2016). Consequently, TRPV1 antagonists have emerged as promising alternatives to opioids for the treatment of chronic pain. Capsazepine (CPZ), the prototypical TRPV1 antagonist, showed efficacy in preclinical models but failed to translate clinically due to poor bioavailability and off-target effects (Bevan et al. 1992; Dickenson and Dray 1991). Since the cloning of TRPV1 (Caterina et al. 1997), numerous synthetic and natural compounds have been designed to target the channel through diverse mechanisms, some of which have progressed to clinical trials (Kort and Kym 2012; Lee et al. 2015; Aghazadeh Tabrizi et al. 2017). Zoledronic acid (ZOL), a third-generation bisphosphonate, is a potent anti-resorptive agent widely used to prevent skeletal-related events in cancer patients. Although its primary mechanism involves inhibition of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway (Soki et al. 2013), ZOL exhibits pleiotropic actions, including anti-angiogenic, pro-apoptotic, and anti-inflammatory effects (Singh et al. 2015; Rodan and Fleisch 1996) (Li, Sun, et al. 2017; Li, Yang, et al. 2017) (Singh et al. 2015; Morii et al. 2015) (Peng et al. 2007; Hiraga et al. 2004) (Gao et al. 2017) (Morii et al. 2015; Yamakawa et al. 2017). ZOL reduces bone pain in patients with osteoporosis and metastatic bone diseases (Sevcik et al. 2004; Iwamoto et al. 2004; Jimenez Andrade and Mantyh 2010), yet the molecular basis of its analgesic effects has remained poorly understood. (Tzschentke 2021). In this study, we provide evidence that ZOL inhibits TRPV1 activity and exerts analgesic effects in vivo. ZOL significantly reduced CAP-evoked calcium influx in cultured DRG neurons. Our electrophysiological results in excised patches expressing TRPV1 demonstrate that ZOL occludes the pore. Occlusion of the channel pore is released by increasing ionic strength suggesting a non-covalent and a reversible mode of interaction. Additionally, ZOL suppressed TRPV1-mediated excitatory synaptic transmission at central synapses similar to CPZ, confirming its function as a TRPV1 antagonist. Furthermore, both mice and flies subjected to ZOL showed delayed responses to noxious heat, strongly suggesting analgesic properties. Previous work by our group (Scala et al. 2019) reported that ZOL enhanced TRPV1 currents in Xenopus laevis oocytes. However, these results contrast with our present findings. We attribute these discrepancies to differences in experimental protocols and drug preparation. In particular, the previous study used a synthesized form of ZOL dissolved in PBS, while we employed a commercially available formulation dissolved in MilliQ water. These differences could alter ZOL’s protonation state and its interactions with TRPV1. Additionally, our experiments included time-resolved monitoring of current dynamics, revealing a progressive reduction in TRPV1 activity upon ZOL application, a detail not captured previously. Electrophysiological assays in excised patches demonstrated that ZOL blocks TRPV1 more efficiently when applied extracellularly, consistent with its hydrophilic nature and low membrane permeability. The magnitude and kinetics of inhibition resemble those observed with QX-314, a lidocaine derivative that also act as a state-dependent, permeant TRPV1 blocker (Michelino Puopolo 2013). Our findings suggest that ZOL preferentially inhibits the open state of the TRPV1 channel and does so more effectively from the extracellular side. Ion strength increases experiments further support the notion that ZOL occupies the pore, and that its displacement when driven force from one side is increased relieves the block. These findings indicate that ZOL acts as non-covalent, pore-targeting inhibitor of TRPV1. In the broader context of TRPV1-targeted therapeutics, several promising antagonists have been identified. AMG9810 and SB-366791 are cinnamide derivatives that block multiple TRPV1 activation modes and show efficacy in preclinical pain models (Gavva et al. 2005) (Neuberger et al. 2023). SAF312, a non-competitive inhibitor that binds the vanilloid pocket (VBP), has demonstrated clinical potential with minimal adverse effects (Mogi et al. 2023). From natural sources, chrexanthomycins from Streptomyces spp (Ye et al. 2023) and agatoxin-489 (Kitaguchi and Swartz 2005) inhibit TRPV1 through distinct structural mechanisms. Offering novel pharmacophores for drug development. Other inhibitors are from endogenous origin and lipidic nature. Indeed, it has been demonstrated that omega-9 fatty acids, such as oleic acid, act as endogenous inhibitors of TRPV1, attenuating both nociceptive and pruritic behaviors in mice (Morales-Lazaro et al. 2016), highlighting the capacity of lipid molecules to modulate TRPV1 function. Structural insights into TRPV1 inhibition continue to grow with the advent of cryo-EM, but the exact residues involved in ZOL binding remain unidentified. Given its lack of covalent interaction and preferential blockade of the open channel, ZOL may be bind within the pore region or interact with residues accessible in the activated state. Importantly, TRPV1 is also expressed in bone-resident cells, where it regulates osteoclastogenesis and osteoblast function (He et al. 2017; Idris, Landao-Bassonga, and Ralston 2010). Its expression increases in pathological bone conditions such as osteoarthritis, osteosarcoma, and osteoporosis (Kelly et al. 2015; Niiyama et al. 2007; Yoshino et al. 2014), and of its inhibition protects against bone loss (Idris, Landao-Bassonga, and Ralston 2010; Rossi et al. 2014). Thus, ZOL’s inhibition of TRPV1 could have dual benefits in bone pathologies: reducing nociceptive input and modulating bone remodeling. Our in vivo data confirms ZOL’s analgesic effect in both vertebrate and invertebrate models. In mice, ZOL increased thermal pain thresholds in the hot plate test. In Drosophila, which expresses mammalian TRPV1 under the control of the pain-GAL4 driver, ZOL suppressed noxious heat avoidance in a dose-dependent manner. Notably, while TRPV1 is the principal mediator of noxious heat, other thermoTRP channels such as TRPA1 and TRPM3 may also contribute (Vandewauw et al. 2018), and we cannot rule out their involvement in ZOL-induced analgesia. Moreover, TRPV1 expression in sensory nociceptor neurons of Drosophila are under the control of the pain-GAL4 driver (Jang et al. 2023), which specifically targets TRPA1-expressing neurons implicated in nociception processing. As a result, the potential involvement of endogenous TRPA1 channel to ZOL-induced analgesia in the fly cannot be ruled out. Moreover, we observed sex-dependent differences in Drosophila nociceptive behavior, suggesting hormonal regulation, neuronal plasticity, or sex-specific drug metabolism in TROV1 signaling. In conclusion, we demonstrate that ZOL is a reversible, non-covalent inhibitor of TRPV1 that acts primarily via the extracellular permeation pathway and preferentially targets the channel in its open state. These actions may underline ZOL’s analgesic properties in animal models and possibly in clinical settings. Our findings open new avenues for the development of TRPV1-targeted analgesics, particularly by repurposing existing drugs with favorable safety profiles such as ZOL. Future studies aimed at identifying the precise binding site of ZOL on TRPV1 and assessing its efficacy in chronic pain models will be crucial to harnessing its full therapeutic potential. EXPERIMENTAL PROCEDURES Animals All procedures were conducted according to animal protocols approved by the Institutional Animal Care and Use Committee at the University of Valparaiso and University of Santiago, Chile. Electrophysiology in Xenopus laevis oocytes . Heterologous Expression System: Xenopus laevis oocytes were used to measure TRPV1 currents. mMESSAGE from Ambion (Waltham, MA, United States) was used for in vitro transcription of the cRNA of wild type rat TRPV1 (GenBank TM accession no. NM031982) and mutants. The oocytes were injected with 150 ng of cRNA and then incubated in ND96 solution (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl 2 , 5 HEPES, pH 7.4) at 18°C for 3–5 days before electrophysiological recordings. Employing the patch-clamp technique, macroscopic and single channel current recordings were made in the inside-out and outside-out configurations. Symmetrical recording solutions contained: 150 mM NaCl, 10 mM EGTA, 2 mM MgCl 2 , 10 mM HEPES, pH 7.4. Symmetrical recording solutions for pore unblock experiment contained: 10 mM NaCl, 140 mM NMDG, 10 mM EGTA, 2 mM MgCl 2 , 10 mM HEPES, pH 7.4. ZOL (Trocris Cat. No. 6111) 3 mM stock solution was made in water, CAP (Sigma-Aldrich, M2028) 50 mM stock solution was made in absolute ethanol. The dilutions for experiments were prepared in the corresponding recording solutions at the final concentrations indicated, and perfused into the recording chamber, exchanging at least 10-times the chamber volume. Data was acquired with an Axopatch 200B amplifier (Molecular Devices), and the Clampex 10.7 acquisition software (Molecular Devices). Both the voltage command and current output were recorded at 100 kHz and filtered at 20 kHz using an 8-pole Bessel low-pass filter (Frequency Devices) and sampled with a 16-bit A/D converter (Digidata 1550B; Molecular Devices). Borosilicate capillary glasses (1B150F-4, World Precision Instruments, Sarasota, FL, United States) were pulled in a horizontal pipette puller (Sutter Instrument, Novato, CA, United States) and fire-polished with a microforge (MF-830, Narishige, Tokyo, Japan). All experiments were performed at room temperature (18-20°C). Macroscopic current recordings were evoked by a square pulse from -60 to +260/+300, returning to -60 mV. For single channel recordings patch membranes were held either at constant membrane potential (from -100 to 60 mV) for two min. Hippocampal slice preparation and electrophysiological recordings . Acute hippocampal slices (350 µm thick) were prepared from postnatal day 20 (P20) to P35 mice as previously described (Chavez, Chiu, and Castillo 2010; Hernandez-Garcia and Rosenbaum 2014) (Chavez, Chiu et al. 2010, Chavez, Hernandez et al. 2014). Briefly, the brain was isolated, and hippocampal slices were obtained using a DTK-2000 vibrating microslicer (Dosaka EM Co., Ltd.) in a solution containing (in mM): 215 sucrose, 2.5 KCl, 26 NaHCO 3 , 1.6 NaH 2 PO 4 , 1 CaCl 2 , 4 MgCl 2 , 4 MgSO 4 and 20 glucose. Thirty minutes post-sectioning, the cutting solution was gradually switched to an extracellular artificial cerebrospinal (ACSF) recording solution containing, in mM: 124 NaCl, 2.5 KCl, 26 NaHCO 3 , 1 NaH 2 PO 4 , 2.5 CaCl 2 , 1.3 MgSO 4 , and 10 glucose. All solutions were equilibrated with 95% O 2 and 5% CO 2 (pH 7.4). Slices were incubated for at least 60 min in the ACSF solution prior to recordings. Electrophysiology . All experiments, except where indicated, were performed at 28 ± 1°C in a submersion-type recording chamber perfused at ~1–2 ml/min with ACSF supplemented with the GABA-A receptor antagonist picrotoxin (100 µM). Whole-cell patch-clamp recordings using a Multiclamp 700A amplifier (Molecular Devices) were made from dentate granular cells (DGCs) voltage clamped at −60 mV using patch-type pipette electrodes (~3–4 MΩ) containing (in mM): 131 Cs-Gluconate, 8 NaCl, 1 CaCl 2 , 10 EGTA, 10 glucose, 10 HEPES; pH 7.2, 285 mmol/kg. Series resistance (~10–30 MΩ) was monitored throughout all experiments with a −5 mV, 80 ms voltage step, and cells that exhibited significant change in series resistance (>20%) were excluded from analysis. To stimulate excitatory synaptic inputs, a stimulating patch-type pipettes were filled with ACSF and placed in the middle third of the molecular layer to stimulate medial perforant pathway (MPP) inputs. MPP-DGC synapses were activated at low stimulus intensity (2–5 V, 200 µs square-wave pulses). TRPV1-LTD was evoked using a previous pairing protocol consisting of a series of 2 stimuli (70 ms interval) paired with small postsynaptic depolarization (30 mV for 30 ms) repeated 900 times at 1 Hz (Chavez, Chiu, and Castillo 2010) (Chavez, Chiu et al. 2010). Pharmacological agents (i.e., capsaicin, capsazepine, zoledronic acid) were bath applied after the establishment of a stable baseline (~10–15 min), and their effects were measured after responses reached a new steady state (typically >15 min). Cells that exhibited significant rundown (>10% within the first 10 min) before drug application were discarded. Drugs were obtained from Tocris, prepared in stock in DMSO (Picrotoxin, CAP, CPZ,) and added to the ACSF as needed. Total DMSO in the ACSF was maintained at <0.1%. Excitatory postsynaptic currents (EPSCs) were elicited at 20 s intervals, filtered at 2.2 kHz, and acquired at 5 kHz using custom-made software written in Igor Pro 4.09A (Wavemetrics, Inc., Lake Oswego, OR, USA). Dorsal root ganglion primary sensory neurons dissociation, culture, and calcium measurements . Cell culture Primary sensory neurons from dorsal root ganglia were dissociated and cultured as in (Gonzalez et al. 2017; Cornejo et al. 2020)(Gonzalez, Ugarte et al. 2017, Cornejo, Gonzalez et al. 2020). In brief, mice were euthanized by exposure to isoflurane and decapitated. Dorsal root ganglia (DRG) were removed and incubated in an enzymatic mixture including collagenase type XI (650 UI/mL; C7657, Sigma-Aldrich, St. Louis, USA) and dispase (5 UI/mL; 17105-041 GIBCO-Thermo Fisher Scientific, Waltham, MA, USA), in a INC-mix solution (in mM: NaCl 155, K 2 HPO 4 1.5, HEPES 10, Glucose 5, pH: 7.4), during 40 minutes at 37ºC. DRG were mechanically dissociated using a polished glass pipette and neurons were plated on poly-L-lysine-coated 6 mm #0 glass coverslips (Menzel-Gläser, Braunschweig, Germany) and maintained in MEM media (Earle’s salts, 111095080, GIBCO-Thermo Fisher Scientific, Waltham, MA, USA) supplemented with MEM-vit (11120052, GIBCO-Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS (SH30910.03, Hyclone, General Electric Healthcare Life Science, UT, USA), 200 µg/mL streptomycin, and 125 µg/mL penicillin (15140-122, GIBCO-Thermo Fisher Scientific, Waltham, MA, USA). Neurons were used 6 to 12 hrs after plating for Ca 2+ imaging experiments. Ca 2+ imaging Ratiometric Ca 2+ imaging experiments in primary sensory neurons were made using a double-pulse stimulation protocol modified from (Rivera et al. 2021) (Rivera, Moreno et al. 2021). In brief, DRG neurons were incubated with 5 µM Fura-2 AM (F1221, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) dissolved in standard extracellular solution supplemented with 0.02% Pluronic (P6867, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) during 50 min at 37ºC in darkness. Fluorescence measurements were made using an inverted Nikon Ti microscope fitted with a 12-bit cooled ORCA C8484-03G02 CCD camera (Hamamatsu, Hamamatsu City, Japan). Fura-2 was excited at 340 nm and 380 nm at a frequency of 0.5 Hz with a Polychrome V monochromator (Till Photonics), and exposure times no longer than 40 ms. The emitted fluorescence was filtered with a 510 nm long-pass filter. Calibrated ratios were displayed online using the HCImage v2 software (Hamamatsu, Hamamatsu City, Japan). Bath temperature (see below for details) was sampled simultaneously with the fluorescence recording using a Physitemp BAT-12 microprobe thermometer (Physitemp Instruments, Clifton, NJ, USA) connected to an IT-18 T-thermocouple, and using Clampex 10 software (Molecular Devices, Sunnyvale, CA, USA). The signals were digitized with an Axon Digidata 1440A AD converter (Molecular Devices, Sunnyvale, CA, USA). Chemical stimulation and temperature control Coverslips with cultured DRG neurons were placed in a microchamber and continuously perfused with extracellular solution warmed at a basal temperature of 34 ± 1°C. The TRPV1 channel activator capsaicin was applied using a time-controlled perfusion system, with the outlet located over the field of neurons, allowing us to apply the compounds directly on these cells while keeping precise control of the temperature and compound exposure time. The temperature of the bath was adjusted with a computer-operated CS-1 Peltier device (Cool Solutions Research Devices, Carrigaline, Ireland), placed directly on the neurons and controlled by a feedback device. Reagents and drugs Capsaicin (M2028) was purchased from Sigma-Aldrich (St. Louis, MO, USA), Zoledronic acid (Tocris). Effects of acute administration of zoledronic acid on nociceptive behaviors in mice. Animals were male and female C57BL/6J wild type mice. The animals were experimentally naïve and 2 – 3 months old (20 – 25 g). Animals were weaned at 21 days of age, and group-housed (3 – 5 animals per cage) in an animal facility room with controlled temperature (21 ± 2°C) and humidity (40-70%), with ad libitum food and water, in a 12 h light:dark cycle (lights on at 8:00 AM). Mice were randomly assigned to a control group and treatment groups. The control group received an acute intraperitoneal injection of sterile saline (0.9% NaCl) at a volume of 5 ml/kg body weight, while the treatment groups received an acute intraperitoneal injection of the zoledronic acid at a dose of 100 µg/kg body weight. The injections were administered 1 hour before the hot plate test. Animals were transferred to the behavioral room in their home cages, acclimated for at least 1 h and tested between 10:00 and 16:00 h (during the light cycle) (Bevan et al. 1992; Caterina et al. 1997; Ahern 2003) (Bevan, Hothi et al. 1992, Caterina, Schumacher et al. 1997, Ahern 2003). Hot plate test The hot plate apparatus consisted of a metal surface with integrated temperature control. A transparent glass cylinder (13 cm in diameter and 18 cm high) was placed on the plate to prevent the mice from escaping. First, all individuals received an acclimatization trial on the hot plate apparatus at a non-nociceptive temperature (25°C) 30 minutes prior to testing. The hot plate test was conducted at 45°, 50° or 55°C and the latency to pain behaviors (licking of the hind paw or jumping) was recorded. A cut-off time of 90 s, 60 s or 40 s respectively were used to prevent tissue damage. The latency to pain behaviors represents the mean of three trials for each individual, with a 5-minute intertrial period. Fly rearing and stocks Drosophila strains were raised on standard cornmeal media and maintained at room temperature (20-22 0 C) on a 12 h light:12 h dark (12 L:12D) light: dark (LD) cycle. We used wild type white 1118 (w 1118 ) flies of 10-15 days old as control genotypes. The pain-GAL4 driver (stock BL27894) was obtained from the Bloomington Drosophila Stock Center. UAS-hTRPV1 line was kindly provided by Changsoo Kim (School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea) from the Korea Drosophila Resource Center (KDRC) (Jang et al. 2023). Thermal avoidance assay We used a thermal avoidance assay previously described for assessing nociceptive behavior in adult Drosophila (Neely et al. 2011) with a minor modification. Briefly, male and female flies were collected upon eclosion and maintained in groups of 20 per vial on standard food until the day of the experiment. For drug (ZOL) treatment, flies were transferred to vials containing apple juice agar supplemented with 100-300μM zoledronic acid (Tocris) for five days prior to testing. For testing, 10–15-day old flies were transferred to a behavior chamber (35mm x 10mm Petri dish) and sealed with tape. Chambers were allowed to rest at room temperature for at least 30min before being placed in a thermostatically controlled water bath maintained at 46 0 C for 4 min. The bottom of the chamber reached 46 0 C, while the sub-noxious zone at the top and middle of the chamber were measured at 31 0 C and 33 0 C, respectively at the end of the test. The temperature of the chamber was monitored using an electronic thermometer. To control potential phototactic behavior, a light source was placed at the bottom of the water bath during the assay, allowing us to distinguish between climbing due to avoidance of noxious heat and climbing due to phototaxis. After exposure, flies that resisted the heat were counted. The total number of flies was recorded to calculate the percentage of flies that avoided the noxious stimulus. Avoidance was defined as any behavior resulting in avoidance of the heated floor surface. Statistical analysis Statistical analysis was performed using Origin 9.0 software (OriginLab Corp). Data are expressed as mean values ± standard error of the mean (SEM). All data were analyzed to determine if they fitted to normal distribution using the Shapiro-Wilk test. For normally distributed data, Two-sample Student’s t-test was performed. Probability (p) < 0.05 was considered statistically significant in all tests. ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health Award R01GM030376 (R.L.); Fondo Nacional de Desarrollo Cientıfico y Tecnologico (FONDECYT) Grant # 1230267 (R.L.); FONDECYT grant #1201848 (A.E.C) and by ANID Millennium Science Initiative Program (P09-022F to A.E.C, R.L). R.C.M is supported by SIA grant #85240137. CA-G was supported by PhD fellowship from ANID #21201603. OAS Academic Scholarship Program Graduate Studies or Graduate Research (0000165325, CA-R), Beca de Doctorado Nacional ANID Chile (21241240/2024, CA-R) Beca de Doctorado Nacional ANID Chile (2139/2023, KC-Z), Beca FIB-UV (REXE Nº 2204, KC-Z). VRIIC-DICYT USACH 022443MM and Millennium Nucleus for the Study of Pain MiNuSPain (R.M). Proyecto PFE UVA22991 ”Fortalecimiento del Posgrado e Investigación, Innovación y transferencia de conocimiento de la Universidad de Valparaíso, para una mayor inclusión, diversidad e internacionalización de la Universidad de Valparaíso” and eLife Ben Barres Spotlight Award eLife Community (K.C). FONDECYT grant #1231012 (P.M); Beca Doctorado Nacional ANID N°21220829 (WP). We thank Changsoo Kim (School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea), the Korea Drosophila Resource Center (KDRC), and the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/). Programa de Doctorado en Ciencias Biológicas Mención Neurociencia, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile. Programa de Doctorado en Ciencias Mención Biofísica y Biología Computacional, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile. FIGURE LEYENDS Figure 1. Zoledronic acid reduces capsaicin-evoked calcium responses in primary sensory neurons from dorsal root ganglia (DRG) . A-B. Representative traces of intracellular calcium concentration ([Ca 2+ ] i ) measured by Fura-2 ratiometric imaging, in cultured DRG neurons responsive to capsaicin. Responses to two consecutive applications of 20 nM capsaicin are show under control conditions ( A ) and after treatment with 10 µM zoledronic acid ( B ). Bath temperature was recorded simultaneously. Arrowheads indicate the peak response to each capsaicin stimulus. C. Bar plot with individual data points showing the reduction of capsaicin -evoked response (CAP2/CAP1) in control and zoledronic acid-treated neurons. Data are presented as mean ± S.E. D. Summary plot showing the reduction of capsaicin-induced Ca 2+ influx in DRGs neurons following zoledronic acid treatment. Statistical comparisons were made using a two-tailed Mann-Whitney test (**, p = 0.0074; control, n= 14, zoledronic acid, n= 18 neurons). Figure 2 . Zoledronic acid reduces capsaicin- and voltage-activated TRPV1 currents in excised membrane patches from Xenopus laevis oocytes . A. Representative macroscopic TRPV1 currents recorded in inside-out patches in response to a voltage step from -60 mV up to +260 mV and back to -60 mV (protocol shown below traces). Black trace: basal TRPV1 current under control conditions. Red trace: capsaicin (500 nM CAP) potentiates the current via TRPV1 sensitization. Light blue trace: application of zoledronic acid (1 nM ZOL) indices a progressive reduction in current amplitude. B. Bar plot showing normalized TRPV1 current activity (Rel. Activity) under control, CAP, and ZOL conditions. Data are representative of three independent experiments. C. Representative single-channel recordings of TRPV1 activity at +100 mV in the inside-out configuration under control condition and following the application of 10 nM ZOL. Traces show channel activity at 5, 10 and 17 minutes post-ZOL application. “c” and “o” indicates closed and open states. D. Bar plot of unitary current amplitudes under control and ZOL-treated conditions. Each circle represents data from separate membrane patches (n= 5-7 independent experiments). Mean unitary conductance values (± SEM) are indicated to the left of each bar: control (A ∼42 pS); 5 min ZOL (∼21 pS);, 10 min ZOL (∼7 pS); 17 min ZOL, ∼5.8 pS). Bottom panels show zoomed-in segments of traces at the indicated time points. Figure 3 . Zoledronic acid inhibits TRPV1 from both intracellular and extracellular sides of the membrane . A , D. Representative macroscopic current recordings of wild-type TRPV1 channels in inside-out (I/O, A ) and outside-out (O/O, D ) patch configurations in response to a voltage step protocol from -60 mV to +260 mV returning to +60 mV (protocol shown below). Control recordings are shown in black. After the application of 500 nM zoledronic acid (ZOL) to the intracellular ( A ) or extracellular ( D ) bath solution, a time-dependent inhibition of TRPV1 current was observed (pink traces). Only traces at 6 min in I/O and 10 min in O/O post-ZOL application are shown (dark green traces). Data are representative of 4 (I/O) and 5 (O/O) independent experiments. B . Time course of TRPV1 current amplitude in the presence of ZOL, showing progressive inhibition in both configurations. The red arrow marks the time of ZOL application, while blue arrows indicate the time point corresponding to the representative traces shown in panel A and D . Exponential fitting yielded a time constant (τ) of 3.53 ±0.94 min for intracellular application ( B ), and 2.07±0.54 min for extracellular application ( E ). C, F. Bar plots summarizing the relative TRPV1 activity under control (white bars) and ZOL-treated conditions (pink and dark green bars) in I/O ( C ) and O/O ( F ) configuration. Individual data points (black circles) represent independent experiments (n = 4 for I/O, n = 5 for O/O). Data are represented as mean ± SEM. Statistical analysis was performed using the two-tailed Mann-Whitney test. Figure 4 . Zoledronic acid blocks the TRPV1 channel pore preferentially from the open state . A . Schematic step-by-step representation of the experimental protocol corresponding to the recordings shown in panel B . B. Representative macroscopic current recordings of TRPV1 in response to the voltage protocol showing at the bottom. The control trace is shown in black. Application of 500 nM capsaicin (CAP) activated TRPV1 currents (red trace). Subsequent application of 100 nM ZOL resulted in a marked reduction of current amplitude (blue trace). Perfusion with 10 nM NaCl (low ionic strength) had minimal effect (light blue trace). Increasing extracellular ionic strength with 140 mM NaCl led to a significant enhancement of current amplitude (dark green trace), which was then reversed by perfusion with 10 mM NaCl, restoring current amplitude to near baseline level (black vi trace). Data are representative of 5 independent experiments. C. Voltage protocol used for the recordings shown in panel D . Membrane patches were held -at -60 mV, depolarized briefly (5 ms), followed by a prolonged step to -60 mV (3 min) during which 100 nM ZOL was applied. The protocol ended with another brief depolarizing pulse and returned to -60 mV. D. Macroscopic current recordings obtained under the conditions illustrated in panel C . Control recording is shown in black followed by ZOL application (pink trace), which induced a time-dependent reduction in current amplitude. Data are representative of 4 independent experiments. E. Time course of current amplitude during the experiment shown in panel D , demonstrating progressive inhibition of TRPV1 by ZOL. Figure 5. Zoledronic acid administration induces analgesia in vivo in a mouse model of thermal nociception . A-C. Acute systemic administration of zoledronic acid (ZOL) produces significant analgesic effects in the hot plate assay at three different noxious temperatures: 45°C ( A ), 50°C ( B ) and 55°C ( C ). Mice treated with ZOL displayed increased latency to nocifensive behaviors compared to controls. Data are represented as mean ± SEM, with individual data points shown. Statistical significance was determined using an unpaired two-tailed test (**p < 0.01). Forup sizes: saline-treated, n = 8; ZOL-treated, n = 8, per temperature condition. Figure 6. Z oledronic acid suppresses nociceptive avoidance behavior in Drosophila expressing human TRPV1 in sensory neurons . A. Female Drosophila melanogaster expressing human TRPV1 in sensory nociceptor neurons exhibited reduced avoidance responses to noxious heat following dietary exposure to zoledronic acid (ZOL). Bar plots display the percentage of avoidance (% Avoidance) in response to a 46°C thermal stimulus across experimental conditions. Control groups pain -GAL4> w 1118 (Control A) and UAS- hTRPV1 > w 111 8 (Control B) show robust avoidance behaviors (~100%), indicating preserved nociceptive responses. Flies expressing human TRPV1 under the pain-GAL4 driver ( pain -GAL4>UAS- hTRPV 1, Control AB) similarly exhibit normal avoidance, confirming that TRPV1 expression alone does not alter behavior. However, flies fed with ZOL for 5 days display a dose-dependent reduction in avoidance, with the most significant effects observed at concentrations ≥150 μM. B. 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Keywords anaesthesia drug discovery/target validation electrophysiology hippocampus imaging in vivo ion channels pain repurposing trp Authors Affiliations Karina Carvajal-Zamorano Centro Interdisciplinario De Neurociencia De Valparaíso View all articles by this author Cesar Amaya-Rodriguez 0000-0003-1916-573X Centro Interdisciplinario De Neurociencia De Valparaiso View all articles by this author Ana Gomez del Campo Universidad de Santiago de Chile View all articles by this author Angelina Palacios Centro Interdisciplinario De Neurociencia De Valparaiso View all articles by this author Carlos Ancaten-Gonzalez Centro Interdisciplinario De Neurociencia De Valparaiso View all articles by this author Rodrigo Meza Universidad de Valparaiso View all articles by this author Wladimir Plaza-Briceño Centro Interdisciplinario De Neurociencia De Valparaiso View all articles by this author Ignacio Segura Centro Interdisciplinario De Neurociencia De Valparaíso View all articles by this author Rosa Scala Washington University in St Louis View all articles by this author Domenico Tricarico 0000-0003-2726-0462 University of Bari View all articles by this author Pablo R. Moya Universidad de Valparaiso View all articles by this author Rodolfo Madrid Universidad de Santiago de Chile View all articles by this author Andres Chavez Centro Interdisciplinario De Neurociencia De Valparaiso View all articles by this author Ramon Latorre Facultad de Ciencias, Universidad de Valparaiso View all articles by this author Karen Castillo 0000-0003-0353-7025 [email protected] Universidad de Valparaíso View all articles by this author Metrics & Citations Metrics Article Usage 1491 views 850 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Karina Carvajal-Zamorano, Cesar Amaya-Rodriguez, Ana Gomez del Campo, et al. The bisphosphonate zoledronic acid produces analgesia by inhibiting the transient receptor potential vanilloid 1 (TRPV1) channel.. Authorea . 20 May 2025. 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