Morphine-induced antinociception is potentiated and dopamine elevations are inhibited by the biased kappa opioid receptor agonist triazole 1.1

Emanuel F Lopes; Alyssa M West; Jason L Locke; Katherine Holleran; Leighelle A Adrian; Monica H Dawes; Alyson M Curry; Harlie A McKelvey; Thomas Martin; Sara R Jones

doi:10.1021/acschemneuro.5c00075

. Author manuscript; available in PMC: 2025 Oct 23.

Published in final edited form as: ACS Chem Neurosci. 2025 Mar 25;16(7):1377–1387. doi: 10.1021/acschemneuro.5c00075

Morphine-induced antinociception is potentiated and dopamine elevations are inhibited by the biased kappa opioid receptor agonist triazole 1.1

Emanuel F Lopes ^a,^‡, Alyssa M West ^a,^‡, Jason L Locke ^a, Katherine Holleran ^a, Leighelle A Adrian ^a, Monica H Dawes ^a, Alyson M Curry ^a, Harlie A McKelvey ^a, Thomas Martin ^b, Sara R Jones ^a,^*

PMCID: PMC12542968 NIHMSID: NIHMS2113534 PMID: 40129263

Abstract

Traditional analgesic opioid compounds, which act through μ opioid receptors (MORs), engender a high risk for misuse and dependence. κ opioid receptor (KOR) activation, a potential target for pain treatment, produces antinociception without euphoric side-effects but results in dysphoria and aversion. Triazole 1.1 is a KOR agonist biased towards G-protein coupled signaling, potentially promoting antinociception without dysphoria. We tested whether triazole 1.1 could provide antinociception, and its effects in combination with morphine. We employed a lactic acid abdominal pain model, which induced acute pain behaviors, decreased basal dopamine levels in the nucleus accumbens (NAc), and increased KOR function. We administered several interventions including triazole 1.1 (30 mg/kg) and morphine (12 mg/kg or 24 mg/kg), individually and in combination. Triazole 1.1 alone reduced the pain behavioral response and changes to KOR function but did not prevent the reduction in basal dopamine levels. Morphine dose-dependently prevented behavioral pain responses, but also elevated NAc dopamine and did not prevent the pain-induced increase in KOR function. However, combining low-dose morphine with triazole 1.1 prevents behavioral pain responses, changes to NAc dopamine levels, and changes to KOR function. Therefore, we present triazole 1.1 as a dose-sparing pain treatment to be used in combination with a lower dose of morphine, thus reducing the potential for opioid misuse.

Keywords: morphine, triazole 1.1, antinociception, kappa opioid receptor, dopamine, Nucleus accumbens

INTRODUCTION

The rate of opioid drug overdose deaths is very high in the United States, with approximately 82000 reported deaths in 2022, which account for over 75% of all overdose deaths that year.¹ A principal factor driving these opioid use and overdose statistics has been the increase in availability of prescription opioids to manage pain.^2,3 These compounds are highly efficacious in providing analgesia through their action on μ opioid receptors (MORs).^4–6 However, activation of MORs causes euphoria which, along with the subsequent aversive withdrawal, engenders a high risk for misuse and dependence.^7,8

The lack of alternative effective pain treatments highlights a need to explore other target mechanisms with an improved risk-benefit profile, or adjunct therapies that allow MOR ligands to achieve therapeutic doses while avoiding unwanted side effects. The κ opioid receptor (KOR) has emerged as a potential therapeutic target, because activating the KOR produces analgesic properties without euphoric side-effects.^9–14 Nalfurafine, the first centrally acting full KOR agonist and partial MOR agonist in clinical use, has been shown to mediate analgesic and antipruritic effects via the KOR.^15–17 However, KOR activation also results in dysphoria and aversion.^18–22 These dual effects have been suggested to be segregated to distinct KOR downstream signaling cascades,^23,24 with the G-protein-mediated pathway implicated in analgesia and the βarrestin2-mediated pathway in aversion and dysphoria.^25–29 Consequently, selective biased compounds have been developed to exploit these specific pathways with an aim to provide analgesia while limiting the contribution of the KOR system in subsequent negative affect.^30–33 It is worth noting that several studies using G-protein biased KOR agonists have identified unwanted side effects. For example, the nalfurafine analog 42B and the salvinorin analog RB-64 induce motor incoordination in mice, while collybolide induces aversive responses.^34–36 These findings suggest correlating signaling pathways with behavioral outcomes may not be as clear cut. Factors such as drug dose, off-target effects, and the choice of in vitro/vivo models and methods should be considered when interpreting these findings.

Triazole 1.1 is a KOR agonist biased towards G-protein coupled signaling which promotes activation of the signaling pathway associated with antinociception without the dysphoria associated with recruitment of the βarrestin2-mediated pathway.³³ Our lab and others have produced evidence of an association between negative affect-like behaviors, including dysphoria, and a KOR-mediated decrease in dopamine (DA) in the nucleus accumbens (NAc) due to βarrestin2 recruitment.^33,37,38 Previous work has demonstrated that triazole 1.1 increases tail flick response latency in the warm water tail withdrawal acute pain assay and reduces itching in a non-histamine pruritis model; both behaviors are mediated by the G-protein pathway;³³ however, it does not decrease DA release when applied to ex vivo NAc brain slices, indicating a reduction or absence of βarrestin2 pathway engagement. This evidence highlights the potential of triazole 1.1 as a pain therapeutic that avoids the dysphoric effects of KOR activation and the euphoric effects of MOR, providing a foundational justification to further investigate the use of this compound in an acute pain model.

In this study we employed a lactic acid (LA)-induced abdominal pain model,^39–43 which we demonstrate induced acute pain behaviors, decreased in vivo basal DA levels in the NAc, and increased KOR function in NAc slices. We systemically administered a variety of pain interventions including triazole 1.1 (30 mg/kg) and morphine (12 mg/kg or 24 mg/kg), individually and in combination. Morphine prevented the behavioral pain responses only at the high dose, but also caused an increase in basal NAc DA and did not prevent the pain-induced increase in KOR function. Triazole 1.1 alone was not sufficient to fully reduce the pain behavioral response. However, in combination with a low dose of morphine it prevented LA-induced behavioral pain responses, changes to basal NAc DA, and changes to KOR function. Therefore, we present triazole 1.1 as a potential dose-sparing pain treatment to be used in combination with a lower dose of morphine, thus reducing the potential for drug misuse.

RESULTS AND DISCUSSION

Lactic acid induces a behavioral pain response, reduces basal NAc dopamine levels, and enhances KOR-mediated inhibition of dopamine release

We tested whether the LA model of abdominal pain can induce pain-related behavioral responses and cause alterations to extracellular DA levels and KOR-modulation of dopamine (DA) release. An intraperitoneal (i.p.) injection of LA (0.54%, 10 ml/kg) caused an increase in writhing and hunched posture, represented as an increase in the overall behavior pain z-score when compared to vehicle (10% DMSO, 10% Tween80, 80% saline; Figure 1A; all statistics can be found in the Figure legends).

We then tested whether LA could induce changes in extracellular DA levels in the NAc using microdialysis (Figure 1B,C). Mice injected with LA showed a significant decrease in DA levels compared to vehicle controls (Figure 1B); this can be further demonstrated by comparing the area under curve (AUC) from the DA vs Time plots between treatment groups (Figure 1C).

To further elucidate the mechanisms underlying the decrease in basal DA levels, we investigated whether there were changes in KOR function in the NAc. The decrease in basal DA plateaus approximately 45 minutes following LA injection, therefore, we chose this timepoint to sacrifice animals for ex vivo FSCV experiments. To test whether KOR control of DA release is altered following LA pretreatment, the KOR agonist U50,488 was superfused at increasing concentrations (10 nM - 1 μM, increased in half-log doses) over NAc brain slices of animals pretreated with i.p. LA or vehicle (Figure 1D-E). We found an enhanced reduction in evoked DA mediated by U50,488 in LA-treated animals (Figure 1D), suggesting an enhancement of KOR function. This is further demonstrated by a reduction in U50,488 IC50 (Figure 1E) and maximum decrease in DA release at the highest dose of U50,488 (Figure 1F). This finding suggests that LA treatment results in an increased response of NAc DA release to KOR agonists. Put together, these data suggest the LA model induces a behavioral pain response, reduces basal NAc DA levels, and produces an enhancement of KOR function within the NAc.

Triazole 1.1 partially ameliorated pain response and prevented enhancement of KOR agonist effects on dopamine release

We proceeded to test whether the biased KOR agonist triazole 1.1 can prevent the previously described effects induced by LA. Triazole 1.1 (Tri; 30 mg/kg) was administered 15 minutes prior to LA injection, and pain related behaviors were assessed between vehicle, LA, and triazole 1.1 + LA conditions (Figure 2A). The overall behavior z-scores indicate that triazole 1.1 significantly reduced LA-induced pain behaviors (Figure 2A); however, this improvement did not restore the behavioral z-score back to the level of vehicle-treated animals (Figure 2A). These results suggest that triazole 1.1 can partially prevent the LA-induced behavioral phenotype.

In contrast, triazole 1.1 did not prevent the decrease in basal NAc DA levels seen in animals which received only LA (Figure 2B). This finding was confirmed by comparing the AUC from the microdialysis traces (Figure 2C). In acute NAc slices, an examination of KOR function in regulating NAc DA release revealed that triazole 1.1 prevented the enhanced KOR function caused by LA (Figure 2D). These results are reinforced by similar trends when comparing the directional effects in IC50 values (Figure 2E) and maximal %DA decrease (Figure 2F).

In summary, these data suggest that triazole 1.1 can ameliorate certain aspects of the LA-induced phenotype; triazole 1.1 may provide mild antinociception and normalizes KOR function in the NAc, but it does not block the LA-induced decrease in basal NAc DA.

Morphine prevented lactic acid-induced pain responses and dopamine reduction, but potentiated KOR function

We tested whether the classic opioid analgesic morphine impacts the previously identified lactic acid-induced effects. We tested two doses of morphine, a low dose (12 mg/kg – “ML”) or a high dose (24 mg/kg – “MH”). Mice treated with MH and LA resulted in significantly improved behavioral pain scores when compared to animals treated with LA alone (Figure 3A). This finding is further reinforced with the lack of significance when comparing vehicle-treated mice and animals treated with MH and LA. Similarly, mice treated with ML and LA resulted in significantly improved behavioral pain scores when compared to animals treated solely with LA. However, there was a significant difference when comparing vehicle-treated mice and animals treated with ML and LA (Figure 3A), indicating that pain had not been fully ameliorated by the ML treatment.

In mice that received i.p. LA, both doses of morphine caused a significant increase in basal DA compared to LA and vehicle controls (Figure 3B,C). In addition, this increase was greater in LA animals treated with MH when compared to LA animals treated with ML (Figure 3B,C).

In NAc slices, LA, LA+ML, and LA+MH show heightened effects of the KOR agonist U50,488 on NAc DA release. (Figure 3D). In terms of the U50,488 IC50 and maximal DA decrease, MH+LA show significant differences in comparison to vehicle controls, with other conditions showing trends that mirror the effects seen in the full U50,488 concentration-response curves (Figure 3E,F).

In summary, while MH provided relief of LA-induced pain, it also significantly boosted basal NAc DA levels beyond vehicle and LA-treated animals and did not prevent the LA-induced increase of NAc KOR effects on DA release. This dual effect on NAc DA function likely contributes to the addictive potential of morphine^7,8. Furthermore, ML did not fully reverse the pain phenotype and still caused a significant elevation of NAc DA levels and an augmented U50,488 response.

Triazole 1.1 combined with low doses of morphine prevents lactic acid-induced pain, changes to basal dopamine, and changes to KOR function

Finally, we tested whether combining ML with triazole 1.1 can improve on the behavioral and neurochemical changes induced in the lactic acid model. Combining triazole 1.1 (30 mg/kg) with ML (12 mg/kg) in LA-treated animals caused a significant improvement in the combined behavioral z-score compared to vehicle (Figure 4A). We note this is not significantly different from either ML + LA or triazole 1.1 + LA conditions, but the average z-score closely resembles vehicle controls. In conclusion, these data suggest this combination is sufficient to prevent LA-induced pain.

Figure 4- — Triazole 1.1 in combination with low-dose morphine improves pain behaviors, normalizes NAc basal dopamine levels and prevents KOR sensitization. (A) Pain-related behavioral z-scores in vehicle treated (white), lactic acid treated (LA - brown), low-dose morphine and lactic acid (ML+LA, 12 mg/kg, light pink), triazole 1.1 and lactic acid (Tri+LA - blue), and co-treatment of low-dose morphine and triazole 1.1 followed by lactic acid (Tri+ML+LA, purple). Dotted line indicates mean z-score of pain behaviors in vehicle-treated animals. Low-dose morphine in combination with triazole 1.1 significantly reduce LA-induced pain related behaviors. F_(3,28)=19.18, p<0.0001, one-way ANOVA. Vehicle vs. LA - p<0.0001, Vehicle vs. Tri+LA - p=0.0170, LA vs. ML+LA - p=0.0014, LA vs. Tri+LA - p=0.0064, LA vs. Tri+ML+LA – p <0.0001, Tukey’s multiple comparisons test. N=8. (B) Time profile of basal NAc DA levels detected using microdialysis. The first arrow indicates triazole 1.1 and/or morphine treatment, and the second arrow indicates LA injection timepoint. F_(3,383)=20.46, p<0.0001, effect of treatment, two-way ANOVA. Vehicle vs. LA - p<0.0001, Vehicle vs. ML+LA – p=0.0104, Vehicle vs. Tri+LA – p=0.0021, LA vs. ML+LA – p<0.0001, LA vs. Tri+ML+LA – p=0.0004, ML+LA vs. Tri+LA – p=0,0114, ML+LA vs. Tri+ML+LA – p=0.0114, Tri+LA vs. Tri+ML+LA – p=0.0226. Tukey’s multiple comparisons test. N=11 Vehicle-treated animals, N=17 LA-treated animals, N=6 ML+LA treated animals, N=11 Tri+LA treated animals, N=7 Tri+ML+LA treated animals. (C) AUCs of basal DA levels, calculated from panel B. Low-dose morphine in combination with triazole 1.1 prevent the LA-induced reduction in basal NAc DA levels, and morphine-dependent increases in basal DA levels. F_(3,34)=33.97, p<0.0001, one-way ANOVA. Vehicle vs. LA - p<0.0001, Vehicle vs. ML+LA – p=0.0087, Vehicle vs. Tri+LA – p=0.0002, LA vs. ML+LA – p<0.0001, LA vs. Tri+ML+LA – p<0.0001, ML+LA vs. Tri +LA – p<0.0001, ML+LA vs. Tri+ML+LA – p=0.0056, Tri+LA vs. Tri+ML+LA – p=0.0088. Tukey’s multiple comparisons test. (D) Concentration-response curves of *ex vivo* electrically-evoked NAc dopamine release following an acute application of U50,488. Effects of U50,488 are potentiated in NAc from animals injected with LA and with low-dose morphine with LA, co-administration of triazole 1.1 and low-dose morphine prevents this sensitization. F_(3,150)=15.46, p<0.0001, effect of treatment, two-way ANOVA. Vehicle vs. LA - p<0.0001, Vehicle vs. ML+LA – p<0.0001, LA vs. Tri+LA – p=0.0031, LA vs. Tri+ML+LA – p=0.003, ML+LA vs. Tri+LA – p=0.004, ML+LA vs. Tri+ML+LA – p=0.0039, Tukey’s multiple comparisons test. N=6 Vehicle-treated animals, N=5 LA-treated animals, N=4 ML+LA-treated animals, N=4 Tri+LA treated animals, N=4 Tri+ML+LA-treated animals. (E) IC50 values obtained from U50,488 concentration-response curves in panel D. F_(3,30)=3.139, p=0.0398, one-way ANOVA. Vehicle vs. LA – p=0.077, Vehicle vs. ML+LA – p=0.941. (F) Maximum % dopamine decrease values obtained from U50,488 concentration-response curves in panel D. F_(3,31)=3.509, p=0.0267. Vehicle vs. LA – p=0.0318, Tukey’s multiple comparisons test. n.s. p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

We then tested basal NAc DA levels, and a combination of triazole 1.1 + ML following LA treatment normalizes basal DA levels similar to control animals (Figure 4B,C). Basal DA levels in co-treated animals are significantly higher than LA- and triazole 1.1- treated animals, and significantly lower than animals treated solely with ML following LA (Figure 4B). These significant effects are maintained when assessing the AUC of these time plots (Figure 4C), suggesting triazole 1.1 + ML normalizes basal NAc DA levels by preventing both the LA-induced reduction and the morphine-related increase in basal NAc DA levels.

In NAc slices, U50,488 concentration-response curves in animals that underwent LA injection and combination triazole 1.1 with ML were significantly shifted than those in slices from animals given solely LA, or from animals given LA and ML (Figure 4D). Additionally, concentration-response curves in slices from these animals were not significantly different than those from vehicle-treated animals (Figure 4D), and trends towards similar effects are seen when assessing U50,488 IC50 and maximal %DA release (Figure 4E,F). These findings suggest that the combination of triazole 1.1 + ML can prevent the up-regulation of NAc KOR function on DA release seen in NAc slices from animals treated with LA and LA + ML.

Potentiation of antinociception and disruption of dopamine effects of morphine via the G-protein biased kappa opioid receptor agonist, Triazole 1.1

Our primary objective for this study was to identify an effective alternative to classic opioids for pain management. To achieve this, we first employed and characterized a model for acute inflammatory pain, the lactic acid-induced abdominal pain model. In C57BL/6J mice, we observed writhing and hunched posture following lactic acid injection, which are commonly used indicators of pain in mice. To account for inter-animal variability, we calculated z-scores for each behavior and then combined them into a single composite z-score. We demonstrated that lactic acid induced significant pain behaviors when compared to vehicle controls. This pain response was completely prevented by a high dose of morphine, whereas a low dose of morphine only partially attenuated the pain. This is notable given the high concentration of lactic acid used in this study—a dose previously shown to limit the effectiveness of MOR agonists in preventing pain behaviors.⁴³ Triazole 1.1 alone did not fully prevent the pain behavior induced by lactic acid; however, when triazole 1.1 was combined with a low dose of morphine the pain response was reduced to a level comparable to that achieved with a high dose of morphine, and indistinguishable from vehicle controls. While not a statistically significant observation, it is worth noting that the mean z-score values for the vehicle or triazole 1.1 + ML + LA conditions are negative, unlike the triazole 1.1 + LA or ML + LA conditions, potentially indicative of an additive antinociceptive effect. This finding is promising as it suggests that combining these treatments could allow for lower morphine doses to be efficacious in preventing pain, likely reducing the potential for opioid abuse.

Beyond pain prevention, it was crucial to assess whether triazole 1.1 affects DA levels, as changes in DA are linked to the rewarding effects of drugs and their abuse potential. Morphine’s abuse liability is well-documented, largely due to its ability to acutely elevate extracellular DA levels in the NAc.^44–47 Conversely, non-biased KOR agonists like U50,488H typically decrease extracellular DA, resulting in dysphoria.^48–50 Previous studies have shown that decreases in basal NAc DA are associated with negative affect, which in turn plays a significant role in pain.^22,51 Addressing these shifts in basal DA, along with any underlying mechanisms, is an important consideration when treating pain. In our lactic acid pain model, we observed a decrease in basal DA levels in the NAc, reflecting the negative affective state often associated with pain. Published literature suggests that pain-induced reductions in extracellular DA are mediated by KOR activation and reversed with KOR antagonists highlighting that an elevated dynorphin/KOR system likely leads to the basal DA effects seen in this study.^22,39,51,52 Indeed, in related aversive states, we have shown that the KOR antagonist norBNI reversed stress and alcohol withdrawal-induced DA reductions measured by microdialysis.^37,53,54 Triazole 1.1 alone was insufficient to prevent this decrease. In support of this finding, triazole 1.1 has been previously shown to not alter NAc DA levels.³³ However, it is worth noting that full KOR agonists inhibit NAc DA levels, and triazole 1.1 did not exacerbate the lactic acid-induced reduction in DA levels.^55–57 In contrast, morphine not only prevented the reduction in basal DA but further dose-dependently drove elevated DA levels above baseline. This observation suggests that morphine-treated patients may experience not only a relief from pain-related dysphoric decreases in DA levels, they may also experience a rewarding DA-elevating effect of morphine, highlighting the risk of abuse inherent to classic opioid compounds. Remarkably, when given in combination with a low dose of morphine, triazole 1.1 restored basal DA levels to baseline and prevented both morphine-induced increases and lactic acid-induced decreases. It is worth noting that these data indicate Tri does not prevent the LA-mediated decrease in NAc DA but does prevent the morphine-related hyperdopaminergia. Consequently, the morphine appears to be the primary mediator in blocking the LA-induced reduction in basal DA, and that the beneficial effect of triazole 1.1 is to curtail this effect, resulting in normalized DA levels. This suggests that the combination treatment is not necessarily additive across all the measures we test, indeed, in this case triazole 1.1 and ML appear to act antagonistically to promote beneficial outcomes. This finding strongly suggests that a combination treatment can both prevent pain-related decreases in basal DA and reduce the abuse liability of morphine.

To further elucidate the mechanisms underlying the decrease in basal DA levels, we investigated whether there were changes in KOR function in the NAc. Pain is known to trigger the release of dynorphin, which activates KORs, potentially explaining the observed decrease in basal DA in our lactic acid model.²² Indeed, in this study we show heightened KOR function in NAc slices following lactic acid exposure. This finding contrasts with the canonical understanding of GPCR receptor function, which would posit that greater agonist engagement with the receptor leads to a reduction in receptor activity through desensitization or sequestration/downregulation.⁵⁸ While these results seem counterintuitive, it is a remarkably consistent finding and heightened KOR function has been identified following cocaine treatment⁵⁹ , ethanol exposure^38,53,60,61, and stress, across rodents and primates.^37,62 All these exposures have been postulated to increase dynorphin release. Treatment with morphine did not normalize this receptor sensitization, consistent with prior studies that suggest morphine itself upregulates KOR function.^63–65 The mechanism underlying NAc KOR sensitization is currently not understood, but this process is thought to underlie the shift of drug seeking towards negative reinforcement, i.e. consuming the drug to prevent negative affect.⁶⁶ Triazole 1.1 inhibited the upregulation of KORs seen in the lactic acid model, despite its inability to restore basal DA levels. These findings suggest that, while it is likely that factors beyond KOR function are involved in modulating DA levels, triazole 1.1 is efficacious in preventing a mechanism that could drive dysphoria during morphine withdrawal.

Other studies have tested the effects of combining KOR and MOR agonists on antinociception. Kaski et al. showed the combined KOR/MOR agonist nalfurafine can potentiate morphine-induced antinociception and limit morphine induced conditioned place-preference.⁶⁷ Similarly, Zamarripa et al. demonstrated that co-administration of triazole 1.1 with the MOR agonist oxycodone reduces oxycodone’s reinforcing effects and enhances oxycodone-induced antinociception.⁶⁸ One other treatment strategy that would result in combining KOR and MOR activation would be to employ a single mixed KOR/MOR agonist biased towards G-protein signaling. MP1207 and MP1208 are partial but potent agonists at both KOR and MOR, with negligible βarrestin-2 recruitment.⁶⁹ These compounds mediate antinociception without causing place-preference or aversion. One advantage in administering separate KOR and MOR agonists could be that it allows for the titration of these dual agonist effects to better limit the negative side effects associated with KOR or MOR receptor activation. This may be particularly important, for example, in treating pain in individuals with substance use disorders which have been shown to be accompanied by alterations to the KOR/dynorphin system.^70,71

A principal limitation of our study is the exclusive use of male mice. While our data suggest a promising approach to limit morphine’s addictive profile, future studies should test the effects of this combinatorial approach in female animals. Several reports have identified sex differences in the impact of the MOR and KOR systems, in particular in regard to pain and addiction^72–76.

In conclusion, triazole 1.1 shows promise as an adjunct pain treatment option particularly as a dose-sparing treatment in combination with morphine. Furthermore, this combinational approach could provide a multifaceted treatment, not only impacting on several mechanisms and features intrinsic to pain but further normalizing maladaptive systems that drive the abuse potential of morphine.

MATERIALS AND METHODS

Animals

Male C57BL6/J mice were ordered from Jackson Laboratories at 7 weeks old and allowed to habituate for at least one week before being used for experiments. Mice were group housed, 12:12 light cycle all experiments performed in the light cycle, food ad libitum. The protocol was approved by Wake Forest University School of Medicine’s Institutional Animal Care and Use Committee. All methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines.

Microdialysis

Mice (N=17) were anesthetized with ketamine (i.p. 100mg/kg) and xylazine (i.p. 10mg/kg) and underwent stereotaxic surgery to implant a microdialysis guide cannulae (BAS, Bioanalytical Systems Inc, West Lafayette, IN) above the NAc (AP +1.1 mm, lateral +0.80 mm, ventral −3.0 mm relative to bregma, midline, and dura) in mice. Mice recovered for 5–7 days before the start of experimentation. Concentric microdialysis probes (1 mm membrane length; BAS) were inserted prior to the beginning of sample collection and continuously perfused at 0.6 μL/min overnight and then increased to 1.0 μL/min with artificial cerebrospinal fluid (aCSF; pH 7.4; 148 mM NaCl , 2.7 mM KCl , 1.2 mM CaCl₂ , 0.85 mM MgCl₂). Samples were collected every 15 minutes for 4 h and stored at −80°C until HPLC analysis. Each animal underwent two consecutive days of microdialysis collections during which an hour and a half of baseline samples were collected prior to drug injection. Collection following the last injection was for a total of two hours. The following drugs were administered alone or in combination: triazole (s.c. 30 mg/kg), morphine (i.p. 12 or 24 mg/kg), lactic acid (i.p. 0.54%, 10 ml/kg). The vehicle used for all drugs was saline, except triazole 1.1 which was made up in 10% DMSO, 10% Tween80, 80% saline. The order and combination that animals received drugs on days 1 and 2 was semi randomized with the condition that no animal received lactic acid for two consecutive days. Dialysate was analyzed for dopamine using an HPLC ESA Coulochem III detector running Chromeleon analysis software (ThermoFisher Scientific, Grand Island, NY).

Fast Scan Cyclic Voltammetry

Mice (N=23) receiving a pretreatment were given lactic acid injections 45 minutes prior to sacrificing, drugs that preceded lactic acid were administered 15 minutes before the lactic acid, and drugs given independently were injected one hour before sacrificing. Animals were deeply anesthetized using isoflurane gas prior to being rapidly decapitated. The brain was removed and placed into ice cold, pre-oxygenated artificial cerebrospinal fluid (aCSF; 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 2.4 mM CaCl₂, 1.2 mM MgCl₂, 25 mM NaHCO₃, 11.0 mM glucose, 0.4 mM L-ascorbic acid). A vibratome (Leica Biosystems, Buffalo Grove IL, USA) was used to prepare coronal brain slices (mouse: 300 μm thick,) containing the NAc. All slices were hemisected, transferred to recording chambers, and incubated at 32°C in oxygenated aCSF for at least one hour prior to experiment.

Carbon fiber microelectrodes (CFMs) were prepared in house using glass capillaries (1.2 mm x .68 mm, A-M Systems, Sequim, WA) and carbon fibers (7 μm diameter, cut to 100–150 μm length, Goodfellow Corp., Berwyn, PA). CFMs were placed on the surface of the slice near a bipolar stimulating electrode (Plastics One, Roanoke, VA). The dopamine waveform (−0.4 to +1.2 and back to −0.4 V, 400 Vs⁻¹, vs Ag/AgCl) was applied to the electrode and cycled at a frequency of 60 Hz for 5 minutes followed by 10 Hz for 5 minutes prior to baseline collections to precondition the electrode and allow it to reach equilibrium. Endogenous dopamine release was evoked by single electrical pulse stimulation (monophasic+, 4 ms, 7.5 mA) applied to the tissue every 3 minutes. After reaching a stable baseline (defined as no apparent trend of growth or decline of the signal across 5 consecutive files), U50,488H was bath applied to the aCSF washing over the slice to generate a concentration response curve (10 nM - 1 μM, increased in half-log doses). Each dose was applied until stability across 3 consecutive files was observed, or a maximum of 18 files was collected. All files were collected and analyzed with Demon Voltammetry and Analysis software.

Electrodes were calibrated using a multiple linear regression model which used the background current of each electrode to determine its sensitivity ⁷⁷. A unique set of regression coefficients were generated for the model from over 75 electrodes made within the lab and calibrated using the traditional in vitro method of injecting a known concentration of DA using a flow cell apparatus.

Behavior

Male mice (n=8) were singly housed and recorded in their home cages for one hour each week following a series of injections. The following drugs were administered and combined as follows: lactic acid (i.p. 0.54% 10 mL/kg), triazole (s.c. 30 mg/kg) then lactic acid (i.p. 0.54% 10 mL/kg), morphine (i.p. 24 mg/kg) then lactic acid (i.p. 0.54% 10 mL/kg), morphine (i.p. 12 mg/kg) then lactic acid (i.p. 0.54% 10 mL/kg), saline (i.p. 10mL/kg) then lactic acid (i.p. 0.54% 10 mL/kg), triazole (s.c. 30 mg/kg) and morphine (i.p. 12 mg/kg) then lactic acid (i.p. 0.54% 10 mL/kg). The behaviors scored included writhing and time spent with hunched posture. A z-score was calculated (See Supporting Figure 1) for each behavior in which the mean and standard error were taken with respect to all treatments for an individual animal using the following formula.

z = \frac{x - μ}{σ}

Where $x$ is the writhing or hunched posture value, $μ$ is the mean of all collected data for the behavioral modality, and $σ$ is the standard deviation of all collected data for the behavioral modality. Following this, an overall pain z-score was then calculated by averaging the writhing z-score and the hunched z-score.

Data Analysis

Microdialysis was analyzed using Chromeleon analysis software (ThermoFisher Scientific, Grand Island, NY). FSCV data was analyzed using the Demon Voltammetry and Analysis software using the peak and decay function, and the following Michaelis-Menten kinetics model was used in to model the DA baseline peak.

\frac{d [D A]}{d t} = \frac{f [D A_{p}] - V_{m a x}}{(\frac{K m}{[D A]} + 1)}

In the above formula, [DA] is the extracellular concentration of DA released, f is the stimulation frequency, [DA_p] is the release rate constant (concentration of DA released per stimulus pulse), V_max is the maximal uptake rate, and K_m is the apparent affinity.

All data was plotted, and statistical analysis performed using GraphPad Prism (Graph Pad Software, La Jolla, CA). Data is reported as mean ± standard error, and the significance level was set at p < 0.05. For behavioral data, a one-way analysis of variance test (ANOVA), with Tukey’s multiple comparisons test was to compare between all treatment groups. For microdialysis data and FSCV U50,488 concentration response curves, a two-way ANOVA was used to assess for significance, with time as the within-subject factor and treatment as the between-subjects factor. ANOVAs were followed by targeted pairwise comparisons between groups using Tukey’s multiple comparisons test. For microdialysis area-under-curves (AUCs), FSCV U50,488 concentration-response curve IC50s and FSCV U50,488 maximal DA decrease, a one-way ANOVA with post hoc Fisher’s Least Significant Difference test was used to compare between treatment groups.

Supplementary Material

NIHMS2113534-supplement-Supplementary_Material.docx^{(178.2KB, docx)}

ASSOCIATED CONTENT

Supporting Information: Supporting Figure 1 - Raw and z-scored behavioral data. (PDF)

ACKNOWLEDGMENTS

Research reported in this publication was supported by the National Institute of Drug Addiction of the National Institutes of Health under award number R01 DA048490.

Funding Sources

R01DA048490

ABBREVIATIONS

DA: dopamine
KOR: κ opioid receptor
LA: lactic acid
MH: high-dose morphine
ML: low-dose morphine
MOR: μ opioid receptor
NAc: nucleus accumbens
Tri: triazole 1.1

Footnotes

Notes

The authors declare no competing financial interest.

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PERMALINK

Morphine-induced antinociception is potentiated and dopamine elevations are inhibited by the biased kappa opioid receptor agonist triazole 1.1

Emanuel F Lopes

Alyssa M West

Jason L Locke

Katherine Holleran

Leighelle A Adrian

Monica H Dawes

Alyson M Curry

Harlie A McKelvey

Thomas Martin

Sara R Jones

Abstract

INTRODUCTION

RESULTS AND DISCUSSION

Lactic acid induces a behavioral pain response, reduces basal NAc dopamine levels, and enhances KOR-mediated inhibition of dopamine release

Figure 1-.

Triazole 1.1 partially ameliorated pain response and prevented enhancement of KOR agonist effects on dopamine release

Figure 2-.

Morphine prevented lactic acid-induced pain responses and dopamine reduction, but potentiated KOR function

Figure 3-.

Triazole 1.1 combined with low doses of morphine prevents lactic acid-induced pain, changes to basal dopamine, and changes to KOR function

Figure 4-.

Potentiation of antinociception and disruption of dopamine effects of morphine via the G-protein biased kappa opioid receptor agonist, Triazole 1.1

MATERIALS AND METHODS

Animals

Microdialysis

Fast Scan Cyclic Voltammetry

Behavior

Data Analysis

Supplementary Material

ACKNOWLEDGMENTS

Funding Sources

ABBREVIATIONS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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