Tiam1-mediated maladaptive plasticity underlying morphine tolerance and hyperalgesia

Abstract

Opioid pain medications, such as morphine, remain the mainstay for treating severe and chronic pain. Prolonged morphine use, however, triggers analgesic tolerance and hyperalgesia (OIH), which can last for a long period after morphine withdrawal. How morphine induces these detrimental side effects remains unclear. Here, we show that morphine tolerance and OIH are mediated by Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network.

Tiam1 is a Rac1 GTPase guanine nucleotide exchange factor that promotes excitatory synaptogenesis by modulating actin cytoskeletal dynamics. We found that prolonged morphine treatment activated Tiam1 in the spinal dorsal horn and Tiam1 ablation from spinal neurons eliminated morphine antinociceptive tolerance and OIH. At the same time, the pharmacological blockade of Tiam1-Rac1 signalling prevented the development and reserved the established tolerance and OIH. Prolonged morphine treatment increased dendritic spine density and synaptic NMDA receptor activity in spinal dorsal horn neurons, both of which required Tiam1. Furthermore, co-administration of the Tiam1 signalling inhibitor NSC23766 was sufficient to abrogate morphine tolerance in chronic pain management.

These findings identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause for the development and maintenance of morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

Yao et al. identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause of the development and maintenance of morphine tolerance and hyperalgesia. Inhibiting Tiam1 signalling reduced tolerance, suggesting potential to prolong morphine use in chronic pain management.

See Mangutov et al. (https://doi.org/10.1093/brain/awae170) for a scientific commentary on this article.

See Mangutov et al. (https://doi.org/10.1093/brain/awae170) for a scientific commentary on this article.

Introduction

Opioid pain medications, such as morphine, remain the mainstay for the treatment of severe and chronic pain caused by spinal cord injury, nerve injury, tissue injury and cancer.^1,2 However, over time morphine use can induce tolerance, where analgesic efficacy decreases progressively at fixed drug doses, and can paradoxically cause hyperalgesia (OIH), a sensitization process in which morphine increases pain sensitivity.³ Morphine tolerance and OIH leave patients with few treatment options and directly contribute to increased morbidity and mortality.^4,5 Understanding the mechanisms underlying tolerance and OIH is essential to enhance morphine’s utility in chronic pain management.

Morphine analgesic effects are predominately mediated by G-protein coupled µ-opioid receptors (MORs) that are expressed along pain-processing neural circuits.⁶ While extensive literature indicates that chronic morphine modifies the properties of MOR-expressing neurons at the level of dorsal root ganglion (DRG) nociceptors, spinal dorsal horn neurons, spinal microglia and brain,^7–18 it is the MOR function on primary afferent nociceptors in DRG neurons that have been proposed to initiate tolerance and OIH.^19,20 Morphine action at MORs expressed by nociceptors not only depresses synaptic transmission acutely between nociceptors and spinal dorsal horn neurons to alleviate pain but can also initiate maladaptive plasticity in downstream nociceptive networks in the CNS, which in turn results in tolerance and OIH.^8,20–24 Opioid tolerance and OIH may occur through similar mechanisms, as evidenced by animals receiving repeated morphine injections that show a progressive reduction in the pain threshold, this process occurs in parallel with the development of tolerance, and treatments that reduce OIH also attenuate tolerance.^22,25 However, the plasticity mechanisms in the CNS driving and maintaining tolerance and OIH remain unclear.

Rho-family small GTPases play fundamental roles in regulating excitatory synapse development, plasticity and function by modulating actin cytoskeleton dynamics.^26,27 In particular, Rac1 promotes the formation, growth and stabilization of dendritic spines and associated synapses by stimulating actin polymerization, whereas RhoA induces actin-myosin contractility, resulting in spine/synapse shrinkage and loss.^26,27 Rho GTPases cycle between an active GTP-bound state and an inactive GDP-bound state, which is tightly regulated by guanine nucleotide exchange factors (GEFs) that activate Rho GTPases and GTPase-activating proteins (GAPs), which inhibit them.^26,27 We and others previously identified the multifunctional Rac1-GEF Tiam1 as a critical regulator of the dendrite, spine and synapse development.^28–32 Notably, Tiam1 couples activated synaptic receptors such as N-methyl-D-aspartate receptors (NMDARs) and TrkBs to Rac1-dependent actin remodelling and spine morphogenesis in the developing brain.^{28,30,33–36} Here, we identify Tiam1-mediated synaptic structural and functional plasticity in the spinal nociceptive network as the maladaptive plasticity mechanisms underlying morphine tolerance and OIH.

Materials and methods

Animals

All animal protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and the University of Alabama at Birmingham and were conducted following the National Institutes of Health Guidelines. Global Tiam1 knockout (KO) mice, Tiam1 conditional knockout (cKO) mice from DRG neurons (Advillin-Tiam1 cKO) and Tiam1 cKO mice from postnatal forebrain excitatory neurons (CaMKIIα-Tiam1 cKO) were generated, as described.^32,37,38 Mice were housed in two to five per cage, under a 12-h light and 12-h dark-light cycle (lights on at 6:00 a.m. and off at 6:00 p.m.), with ad libitum access to food and water. All experiments used age-matched male and female mice. We did not find sex as a biological variable and pooled data from both sexes.

Behavioural assessments of nociception

The reflexive and affective-motivational nociceptive responses in mice were classified and performed based on previous reports.^20,37,39,40 Briefly, a cutaneous noxious stimulus can elicit several distinct behavioural responses: (i) withdrawal reflexes: rapid reflexive paw withdrawal that occurs in response to a noxious stimulus but ceases once the stimulus is removed; and (ii) affective-motivational responses: temporally delayed (relative to the noxious stimulation), directed licking and biting of the paw (termed ‘attending’), extended lifting or guarding of the paw and/or escape responses characterized by hyperlocomotion, jumping away from the noxious stimulus, or rearing.

Mechanical reflexive assays

To evaluate mechanical reflexive sensitivity, we used a series of calibrated von Frey filaments (Stoelting). These filaments were applied perpendicular to the plantar surface of the hind paw with sufficient force to bend the filament. Rapid withdrawal of the paw away from the stimulus within 4 s was characterized as a positive response. If there was no response, the filament of the next greater force was applied. After a response, the filament of the next lower force was applied. We calculated the tactile stimulus force that produced a 50% likelihood of a withdrawal response using the ‘up-down’ method.^37,38,41

Thermal nociception assays

To evaluate reflexive and affective-motivational responses evoked by thermal stimulation, we used the hotplate test.²⁰ Mice were placed on the 55°C plate. The latency to reflexively withdraw the hind paw was recorded as a positive nociceptive reflex response and the latency to the first appearance of an attending response (lick and/or bite at one or both hind paws) was recorded as a positive affective-motivational response. A maximal cut-off of 45 s was set to prevent tissue damage. To prevent behavioural sensitization that can result from multiple noxious exposures and then averaging those responses, only one exposure to the hotplate was applied during a given testing session.

Tolerance paradigm of cumulative dose-response curves

Cumulative dose-response curves were generated with repeated administration of cumulative doses of morphine in the hotplate test based on a previous study.⁴² Briefly, 1 day before a 7-day morphine treatment (Day 0), mice were injected with 3.75, 3.75, 15 and 30 mg/kg morphine at 30-min intervals, resulting in final cumulative doses of 3.75, 7.5, 22.5 and 52.5 mg/kg morphine. To induce tolerance, mice were treated with morphine at a fixed dose of 10 mg/kg/day for 7 days. On Day 8, mice were again treated using a repeated cumulative dosing regimen with morphine [22.5, 37.5, 60 mg/kg for wild-type (WT) mice at 30-min intervals resulting in final cumulative doses of 22.5, 60, 120 mg/kg and 7.5, 15, 37.5 mg/kg for Tiam1 KO mice at 30-min intervals resulting in final cumulative doses of 7.5, 22.5, 60 mg/kg]. On Day 15 (7 days withdrawal from chronic morphine treatment), mice were treated with repeated cumulative dosing of morphine again, same as Day 8. The antinociceptive response was measured with the 55°C hotplate assay with a 30-s cut-off before and 30 min after each morphine administration on Day 0, Day 8 and Day 15. The response was reported as per cent maximum possible effect (% MPE), calculated as follows: 100 × [(drug response latency − basal response latency) / (30 s − basal response latency)].

Inflammatory pain model

Complete Freund’s adjuvant (CFA)-induced inflammatory pain model was generated, as described.³⁷ Briefly, CFA (10 μl, Sigma-Aldrich) was injected into the plantar surface of the left hind paws of mice using an insulin syringe (29-gauge) under brief isoflurane anaesthesia to induce persistent inflammatory pain.

Viral injection

A new minimally invasive method for microinjection of the virus into the mouse spinal dorsal horn without laminectomy was conducted, as previously published.^38,43,44 Briefly, mice were anaesthetized with 2% isoflurane and placed in a stereotaxic frame (Kopf Instruments). A heating pad was used to maintain the core body temperature of the animals at 37°C. The skin was incised at Th12–L3 and custom-made clamps were attached to the rostral and caudal sites of the vertebral column. Paraspinal muscles around the left side of the interspace between Th13 and L1 vertebrae were removed, and the dura mater and the arachnoid membrane were carefully incised using the tip of a 27-gauge needle to make a small window to allow the glass micropipette insert directly into the spinal dorsal horn (500 μm lateral from the midline and 250 μm in depth from the surface of the dorsal root entry zone) via interspace between Th13 and L1 vertebrae. A volume of 0.5 μl rAAV8-hSyn-GFP (2.6 × 10¹² gc/ml, UNC Vector Core) or rAAV8-hSyn-Cre-GFP (2.4 × 10¹² gc/ml, UNC Vector Core) was injected using glass micropipette attached to a Hamilton microsyringe connected to an infusion pump at a rate of 200 nl/min. After injection, the glass micropipette remained in place for 5 min and the skin was sutured.

Intrathecal injection

The intrathecal injection by lumbar puncture in briefly anaesthetized mice was performed, as previously described.⁴⁵ Mice were anaesthetized with 2% isoflurane and placed in a prone position with a small tube under the abdomen to expose the lumbar vertebral space. Lumbar puncture was performed between L5 and L6 vertebrae with a 30.5-gauge needle connected to a 10 μl Hamilton syringe. Successful intrathecal injection (5 μl of 1 mg/ml NSC23766) was indicated by brisk tail movement.

Biochemical assays

Affinity-precipitation assay for Tiam1 GEF activity

The Tiam1 activity was measured using a previously described affinity precipitation assay.^37,38 Briefly, the spinal dorsal horn from saline or morphine-treated mice was isolated and homogenized in cold lysis buffer [25 mM HEPES, pH 7.4, 0.1 M NaCl, 1% NP-40 (a non-ionic detergent), 5 mM MgCl₂, 10% glycerol, 1 mM dithiothreitol (DTT), 10 μg/ml leupeptin, 10 μg/ml aprotinin and 1 mM Na₃VO₄], and centrifuged at 15 000g for 30 min. The supernatant was incubated with 30 μg of GST-Rac1G15A bound to GSH-agarose beads for 2 h at 4 °C and mixed gently on a rocking shaker. After washing with lysis buffer three times, beads were resuspended in Laemmli buffer. Samples were resolved by SDS-PAGE, transferred to the nitrocellulose membrane, blocked with 5% fat-free milk in 0.1% Tween-PBS and incubated with anti-Tiam1 antibody (1:1000). Active Tiam1 was determined by western blot analysis from the precipitated fraction and normalized to total protein (input).

F-actin to G-actin ratio

The F-actin to G-actin ratio was determined by western blot, as previously described.^37,46,47 Briefly, the two forms of actin differ in that F-actin is insoluble, whereas G-actin is soluble. The spinal dorsal horn from sham or morphine-treated mice was isolated and homogenized in cold lysis buffer (10 mM K₂HPO₄, 100 mM NaF, 50 mM KCl, 2 mM MgCl₂, 1 mM EGTA, 0.2 mM DTT, 0.5% Triton X-100, 1 mM sucrose, pH 7.0) and centrifuged at 15 000g for 30 min. Soluble actin (G-actin) was measured in the supernatant. The insoluble F-actin in the pellet was resuspended in lysis buffer plus an equal volume of Buffer 2 (1.5 mM guanidine hydrochloride, 1 mM sodium acetate, 1 mM CaCl₂, 1 mM ATP, 20 mM Tris-HCl, pH 7.5) and incubated on ice for 1 h to convert F-actin into soluble G-actin, with gentle mixing every 15 min. The samples were centrifuged at 15 000g for 30 min, and F-actin was measured in this supernatant. Samples from the supernatant (G-actin) and pellet (F-actin) fractions were proportionally loaded and analysed by western blotting.

Synaptosome preparation

Synaptosome preparation was performed as in our previous publications.^37,38 The spinal dorsal horn (L4 and partial L5 spinal cord segment) from saline or morphine-treated mice was homogenized using a glass Teflon homogenizer in 10 volumes of ice-cold HEPES-buffered sucrose (0.32 M sucrose, 1 mM EGTA, and 4 mM HEPES at pH 7.4) containing a protease inhibitor cocktail (Sigma-Aldrich). The homogenate was centrifuged at 1000g for 10 min at 4°C to remove the nuclei and large debris. The supernatant was centrifuged at 10 000g for 15 min to obtain the crude synaptosome fraction. The synaptosome pellet was lysed via hypo-osmotic shock in nine volumes of ice-cold HEPES buffer with the protease inhibitor cocktail for 30 min. The lysate was centrifuged at 25 000g for 20 min at 4°C to obtain the synaptosome membrane fraction for the following immunoblotting experiments.

Immunoblotting

The protein samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing (in mM) 50 Tris-HCl (pH 7.4), 1% NP-40, 0.1% SDS, 150 NaCl, 1 EDTA, 1 Na₃VO₄ and 1 NaF in the presence of a proteinase inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged at 18 000g for 30 min at 4°C. The supernatant was carefully collected and the protein concentration was measured using a DC Protein Assay Kit (Bio-Rad). Fifteen micrograms of the total proteins from each sample were loaded and separated using 4–15% Tris-HCl SDS-PAGE gels. The resolved proteins were transferred to an Immobilon-P membrane (Millipore). The membrane was treated with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at 25°C for 1 h and then incubated in TBST supplemented with 0.1% Triton X-100 and 1% bovine serum albumin (BSA) and primary antibodies overnight at 4°C. The membrane was washed three times and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 25°C. The protein band was revealed using enhanced chemiluminescence (ECL) Plus Detection Kit (Thermo Fisher Scientific) and the protein band density was quantified with the Odyssey Fc Imager (LI-COR Biosciences) and normalized to the control protein band on the same blot. Tiam1 was detected using rabbit anti-Tiam1 antibody (sc-872, 1:1000; Santa Cruz) or sheep anti-Tiam1 antibody (AF5038, 1:1000; R&D Systems); Actin was detected using mouse anti-Actin antibody (MAB1501, 1:10 000; Millipore); GluN1 was detected using rabbit anti-GluN1 antibody (G8913, 1:1000; Sigma-Aldrich) or rabbit anti-GluN1 antibody (AB52177, 1:1000; Abcam); GluN2A was detected using rabbit anti-GluN2A (PA5-35377, 1:1000; Thermo Fisher); GluN2B was detected using anti-mouse GluN2B (75-002, 1:1000; NeuroMab); GluA1 was detected using mouse anti-GluA1 antibody (75-327, 1:1000; NeuroMab); GluA2 was detected by using rabbit anti-GluA2 antibody (ab10529, 1:1000; Millipore); PSD-95 was detected by using rabbit anti-PSD-95 antibody (ab18258, 1:2000; Abcam).

Morphological analysis

To observe the effects of Tiam1 on spine remodelling in the spinal dorsal horn, rAAV8-hSyn-GFP (UNC vector core) was injected via intra-spinal dorsal horn microinjection and was used to label the neurons specifically. Spinal cord lumbar sections (40-μm thick) were collected from mice perfused with 4% paraformaldehyde and only dendritic spines on neurons labelled with green fluorescent protein (GFP) were selected for the spine analysis in a blinded manner, as previously described.³¹ Wide dynamic range (WDR) neurons are of particular interest in our study because they are the convergent points for all somatosensory inputs from Aα, Aβ, Aδ and C afferents from the neurons in other layers including spinal layer II neurons.^48,49 Furthermore, WDR neurons and other projection neurons relay pain information to the brain via the spinothalamic tract, a major pain pathway.⁵⁰ The morphology of WDR neurons (synaptic structural plasticity) has been extensively analysed for the long-term nature of different types of neuropathic pain.^38,50–52 All dendritic spine images of WDR neurons were captured using a laser scanning confocal microscope (LSCM, Zeiss LSM 880) with a 63× oil 210 immersion objective. Z-series were taken at an interval of 0.37 μm for each dendrite. Spine morphometric analysis was done in a blinded manner using Imaris software (Bitplane Scientific), as previously described.^37,38 Five criteria allowed us to sample and analyse whole cells with morphology similar to those observed for WDR neurons identified by previous studies^50,51,53: (i) neurons were located within lamina IV and V; (ii) GFP-positive neurons must have had dendrites and spines that were completely impregnated, appearing as a continuous length; (iii) at least one dendrite extended into an adjacent lamina relative to the origin of the cell body; (iv) at least one-half of the primary dendritic branches remained within the thickness of the tissue section, such that their endings were not cut and instead appeared to taper into an ending; and (v) the cell body diameter fell between 20 and 50 μm.

Spinal cord slice preparation and electrophysiology

The spinal lamina II outer zone neurons in the lumbar section were selected for recording NMDAR activity, as we did previously.^38,54,55 The reason we selected spinal lamina II outer zone neurons for electrophysiological recordings is these neurons predominantly and directly receive nociceptive inputs from C-fibres and modify the output of projection neurons located in both layer I and deeper layers of the dorsal horn.⁵⁶ The changes in electrophysiological properties of spinal lamina II neurons (synaptic functional plasticity) have been established to contribute to pain hypersensitivity and opioid tolerance/OIH.^{38,54,55,57–61} Mice were anaesthetized with 3% isoflurane and removed lumbar spinal cords via laminectomy. The spinal cords at the L4–L5 level were placed in ice-cold sucrose artificial CSF containing (in mM) 234 sucrose, 3.6 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 1.2 NaH₂PO₄, 12.0 glucose and 25.0 NaHCO₃, pre-saturated with 95% O₂ and 5% CO₂. The spinal cord tissue was glued onto the stage of a vibratome and transverse slices (400 μm) of spinal cords were cut in ice-cold sucrose artificial CSF and then preincubated in Krebs solution oxygenated with 95% O₂ and 5% CO₂ at 34°C for at least 1 h before being transferred to the recording chamber. The Krebs solution contained (in mM) 117.0 NaCl, 3.6 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 1.2 NaH₂PO₄, 11.0 glucose and 25.0 NaHCO₃ (pH 7.4). The spinal cord slices were placed in a glass-bottom chamber and continuously perfused with Krebs solution at 5.0 ml/min at 34°C maintained by an inline solution heater and a temperature controller. Recordings were performed using 5–10 MΩ glass electrodes filled with internal solution (pipettes have resistance between 5 and 10 MΩ when filled with internal solution), which comprised the following (in mM): 135.0 potassium gluconate, 5.0 tetraethyl-ammonium (TEA), 2.0 MgCl₂, 0.5 CaCl₂, 5.0 HEPES, 5.0 EGTA, 5.0 Mg-ATP, 0.5 Na-GTP and 10 lidocaine N-ethyl bromide (QX314) (adjusted to pH 7.2–7.4 with 1 M KOH; 290–300 mOsmol/l). QX314 was used to block voltage-gated Na⁺ channels to suppress action potentials.

Whole-cell voltage-clamp recordings were performed, as we previously described.^38,54,55,62 Briefly, miniature excitatory postsynaptic currents (mEPSCs) were recorded at a holding potential of −60 mV in the presence of 1 μM strychnine, 10 μM bicuculline and 1 μM tetrodotoxin, and presynaptic NMDAR-mediated glutamate release was tested by bath application of 50 μM Dl-2-amino-5-phosphonopentanoic acid (AP5). Once the cell membrane was ruptured and whole-cell recording was generated, we will wait at least 5 min for the internal solution and cytoplasm to reach osmotic equilibrium. Then, mEPSC was monitored for 3 min to calculate the amplitude and frequency. To distinguish mEPSC from the noise, the minimum peak amplitude was 10 pA. The input resistance was monitored and the recording was abandoned if the input resistance changed by >15%. All signals were recorded using an amplifier (MultiClamp700B; Axon Instruments Inc), filtered at 1–2 kHz, digitized at 10 kHz and stored for offline analysis. To record evoked monosynaptic EPSCs, electrical stimulation (0.6 mA, 0.3 ms and 0.2 Hz) of the ipsilateral dorsal root was carried out using a glass pipette suction electrode (A-M Systems). The monosynaptic inputs of unmyelinated C-fibre evoked EPSCs were identified by one-on-one response and no failures in the latency to high-frequency stimulation at 20 Hz.^63–65 Postsynaptic NMDAR currents were elicited by puff application of 100 μM NMDA to the recorded neuron using a positive pressure system (4 psi, 15 ms; Toohey Company) and puff application of the vehicle produced no currents. The tip of the puff pipette was placed 150 μm away from the recorded neuron. To minimize the Mg²⁺ block of NMDARs, the puff NMDA currents were recorded in an extracellular solution containing no Mg²⁺, 10 μM glycine and 1 μM tetrodotoxin at a holding potential of −60 mV.

Statistical analysis

All the behavioural, electrophysiological, biochemical and morphological data were obtained by counterbalancing experimental conditions with controls and analysed in a blinded fashion. Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications.^54,66 For electrophysiological data, three to five animals were used for each recording protocol and only one neuron was recorded in each spinal cord slice. The frequency and amplitude of mEPSCs were analysed using the MiniAnalysis program (Synaptosoft Inc., Fort Lee, NJ, USA). The 100 randomly selected mEPSC events were used to plot cumulative curves. The difference between the curves in cumulative plots was determined by the Kolmogorov-Smirnov distribution test. The amplitude of evoked EPSCs and puff NMDA currents was quantified by averaging six consecutive traces using Clampfit 11 software (Molecular Devices, San Jose, CA, USA). For effective dose (ED₅₀) values of dose-response curves, the best-fit line was generated following non-linear regression analysis based on the % MPE. The normality test was performed by the Shapiro–Wilk test. We used a Mann-Whitney U-test or two-tailed Student’s t-test to compare two groups and a one-way or two-way ANOVA (followed by Dunnett’s or Tukey’s post hoc tests) to compare more than two groups. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was accepted when P < 0.05.

Results

Prolonged morphine treatment increases Tiam1 activity in the spinal dorsal horn

Prolonged morphine treatment can induce tolerance and OIH. Indeed, 7 days of morphine treatment [10 mg/kg/day, subcutaneous (s.c.)] produced significant antinociceptive tolerance and OIH in wild-type mice (Fig. 1A–E). Notably, we found that the tolerance and OIH lasted at least 1 week after morphine withdrawal (Fig. 1A–E), suggesting that prolonged morphine treatment not only triggers functional plasticity but also promotes structural plasticity, which accounts for the long-term nature of tolerance and OIH. Accumulating evidence supports the idea of similar molecular mechanisms underlying morphine tolerance/OIH and neuropathic pain at the level of DRG nociceptors, spinal dorsal horn and brain.^7,13,67–69 Our recent study identified Tiam1-coordinated synaptic structural and functional plasticity in spinal dorsal horn neurons underlying neuropathic pain,³⁸ so we hypothesized that Tiam1-mediated maladaptive plasticity contributes to morphine tolerance and OIH.

Figure 1.

Prolonged morphine treatment increases Tiam1 activity in the spinal dorsal horn. (A) Drug use schedules for morphine tolerance and analgesia. Mice were treated with morphine once daily (10 mg/kg, s.c.) for 7 days. On Day 14, mice were treated with 10 mg/kg morphine subcutaneously (s.c.) to measure the long-term nature of tolerance and opioid-induced hyperalgesia (OIH) after 7 days of morphine withdrawal. The mechanical threshold to von Frey filament stimuli and latency to attending with hotplate (55°C) were measured before and 30 min after morphine treatment on Day 1, Day 7 and Day 14. (B and C) Morphine antinociceptive tolerance. The post-morphine antinociceptive behaviours were measured on Day 1, Day 7 and Day 14, 30 min after morphine treatment (B, reflexive antinociception, von Frey; C, affective-motivational behaviour, 55°C hotplate). (D and E) OIH. The pre-morphine baseline (BL) nociceptive behaviours were measured on Day 1, Day 7 and Day 14 before morphine treatment (D, reflexive antinociception, von Frey; E, affective-motivational behaviour, 55°C hotplate). Data are mean ± standard error of the mean (SEM) (n = 6 mice in each group). Multiple comparisons one-way ANOVA followed by Tukey’s post-test. **P < 0.01, ****P < 0.0001. (F) Timeline showing the experimental design for Tiam1 activity measurement. Mice were treated with morphine (10 mg/kg) or saline for 7 days, followed by 7-day withdrawal. Tiam1 activity was detected on Day 7 and Day 14. (G) Affinity-precipitation assay. Mutant Rac1 (GST-Rac1G15A) preferentially binds to activated guanine nucleotide exchange factors (GEFs). (H) Tiam1 activity was precipitated from lysates prepared from mouse spinal dorsal horns after 7-day treatment with saline (S) or morphine (M). (I) Tiam1 was precipitated from lysates prepared from saline- or morphine-treated mouse spinal dorsal horns after 7-day withdrawal. Total Tiam1 levels are also shown. Data are mean ± SEM (n = 6). Two-tailed Student’s t-test. *P < 0.05.

To test our hypothesis, we first determined whether prolonged morphine alters Tiam1 activity in the spinal dorsal horn (Fig. 1F). We carried out an active GEF affinity-precipitation assay using glutathione S-transferase (GST)-Rac1G15A, a guanine nucleotide-free mutant form of Rac1 that preferentially binds to activated GEFs, to assess Tiam1 activation (Fig. 1G).⁷⁰ We incubated GST-Rac1G15A or GST (control) with spinal dorsal horn homogenates from mice treated with saline or morphine (10 mg/kg/day, s.c.) for 7 days. We found that the levels of Tiam1 that precipitated with Rac1G15A from morphine-treated mice were markedly increased compared to saline-treated controls (Fig. 1H), indicating that Tiam1 is activated in the spinal dorsal horn of prolonged morphine-treated mice. Furthermore, our affinity-precipitation assay showed that Tiam1 remained activated after 7-day morphine withdrawal (Fig. 1I). Together, these data suggest that the potential Tiam1 may function in the development and/or maintenance of opioid tolerance and OIH.

Global Tiam1 deletion prevents the development of morphine tolerance and OIH

To investigate whether activated Tiam1 in the spinal dorsal horn contributes to morphine antinociceptive tolerance and OIH, we utilized global Tiam1 KO mice, as in previous studies.^32,38Tiam1 KO mice perform as well as wild-type littermates on an accelerating rotarod, suggesting that they do not have deficits in motor coordination, motor learning or balance.³² We first determined whether global deletion of Tiam1 alters acute morphine antinociception. Clinically, morphine provides substantive relief of both sensory and affective dimensions of the pain experience.²⁰ We evaluated basal nociception and morphine antinociception in Tiam1 KO and wild-type mice, monitoring both nociceptive sensory reflexive and affective-motivational behaviours (latency to attending response to 55°C hotplate).²⁰Tiam1 KO mice exhibited similar basal nociceptive reflexes and affective-motivational responses to mechanical and thermal stimuli as their wild-type littermates (Supplementary Fig. 1A and B). Likewise, subcutaneous administration of morphine (10 mg/kg) produced comparable reflexive and affective-motivational antinociception in Tiam1 KO and wild-type mice (Supplementary Fig. 1C and D). These data indicate that global Tiam1 loss does not alter nociceptive behaviour or acute morphine antinociception.

We next evaluated the development of morphine antinociceptive tolerance and OIH in Tiam1 KO mice. Mice were treated with systemic morphine (10 mg/kg, s.c.) once daily for 7 days. We measured thermal and mechanical nociceptive thresholds and affective-motivational response to noxious thermal stimuli before and 30 min after each daily injection to evaluate OIH and tolerance, respectively. Notably, we found that morphine antinociception progressively diminished in wild-type mice, whereas morphine retained nearly full antinociceptive efficacy across all days in Tiam1 KO mice (Fig. 2A–D). Moreover, Tiam1 KO mice developed significantly less OIH than wild-type mice for thermal and mechanical stimuli (Fig. 2E–H). To further confirm Tiam1 functions in the development of morphine antinociceptive tolerance, mice were given repeated cumulative doses of morphine to generate antinociceptive dose-response curves to determine the changes in efficacy and potency. The antinociceptive response was measured with the 55°C hotplate assay with a 30-s cut-off before and 30 min after morphine administration using a cumulative dosing regimen on Day 0 (before chronic morphine treatment), Day 8 (after chronic morphine treatment at a fixed dose of 10 mg/kg/day for 7 days) and Day 15 (7 days after withdrawal from chronic morphine treatment) (Supplementary Fig. 2A). Before chronic morphine treatment, there were no significant differences in the doses for reaching the maximum possible antinociceptive effect or the 50% ED₅₀ values in wild-type mice and Tiam1 KO mice (Supplementary Fig. 2B–D), indicating Tiam1 deletion does not alter acute morphine antinociception with a cumulative dosing regimen. After chronic morphine treatment, wild-type mice showed a marked rightward shift in potency, whereas Tiam1 KO mice did not exhibit a significant shift in their sensitivity to morphine (Supplementary Fig. 2B and D), suggesting that antinociceptive tolerance was abrogated. Notably, there were no significant changes in morphine efficacy and potency in either wild-type mice or Tiam1 KO mice 7 days after morphine withdrawal from chronic treatment (Supplementary Fig. 2C and D), which highlights the Tiam1 functions in the long-term nature of morphine tolerance. Together, these findings suggest that Tiam1 is required for the development of morphine antinociceptive tolerance and OIH.

Figure 2.

Global Tiam1 deletion prevents the onset of morphine antinociceptive tolerance and OIH. (A–D) Morphine antinociceptive tolerance. (A and B) Daily morphine antinociception throughout a 7-day chronic morphine schedule (10 mg/kg, s.c., once daily, post-morphine +30 min time points only). Nociceptive reflex antinociception to von Frey filament mechanical stimulation of the hind paw (A) and affective-motivational behaviour to 55°C hotplate (B). (C and D) Antinociceptive tolerance. Left: Per cent maximal possible effect (% MPE) for morphine antinociception from the first administration (Day 1: +30 min) compared with the last administration (Day 7: +30 min). von Frey (C) and hotplate (D). Right: The per cent change for each subject. (E–H) Opioid-induced hyperalgesia (OIH). (E and F) Daily nociceptive behaviour throughout a 7-day chronic morphine schedule [pre-morphine baseline (BL) time points only]. Nociceptive reflex hypersensitivity to von Frey filament mechanical stimulation of the hind paw (E) and affective-motivational behaviour to 55°C hotplate. (G and H) Hyperalgesia. Left: The pre-morphine baseline nociceptive behaviours change before the first administration (Day 1: baseline, BL) compared to the last day (Day 7: BL). Right: The per cent change for each subject. Data are mean ± standard error of the mean (SEM) [wild-type (WT), n = 8 mice; Tiam1 knockout (KO), n = 8 mice]. Two-tailed Student’s t-test (right panels of C, D, G and H). Repeated measures two-way ANOVA followed by Tukey’s post-test. (A, B, E, F and left panels of C, D, G, H). *P < 0.05, **P < 0.01, ***P < 0.001.

Tiam1 expression in spinal dorsal horn neurons and dorsal root ganglion neurons determines morphine tolerance and OIH

Morphine action at MORs expressed by nociceptors initiates maladaptive alterations in nociceptive networks at the peripheral, spinal or brain levels that may result in tolerance and OIH.²⁰ We next sought to identify the specific nociceptive network where Tiam1 functions in the onset of morphine antinociceptive tolerance and OIH by using mouse genetic engineering and vial tools to delete Tiam1 specifically in spinal dorsal horn neurons, DRG neurons or forebrain excitatory neurons.

To determine the role of spinal Tiam1 in the development of morphine antinociceptive tolerance and OIH, we specifically deleted Tiam1 in spinal dorsal horn neurons by performing intra-spinal dorsal horn microinjections in Tiam1^fl/fl mice with a recombinant adeno-associated virus 8 (rAAV8) vector expressing a Cre-GFP under the control of the neuron-specific promotor human synapsin (hSyn). An rAAV8 vector expressing GFP alone served as a negative control (Fig. 3A). Two weeks after virus injection, mice were treated with morphine (10 mg/kg, s.c.) for 7 days. Following 7 days of morphine administration, Cre-GFP virus-injected Tiam1^fl/fl mice developed significantly less antinociceptive tolerance and OIH on both reflexes and affective-motivational behaviours in response to mechanical and thermal stimuli than the control GFP virus-injected Tiam1^fl/fl mice (Fig. 3B–E and Supplementary Fig. 3). To test whether genetic deletion of Tiam1 from nociceptors affects the development of tolerance and OIH, we crossed Tiam1^fl/fl mice with Advillin-Cre to delete Tiam1 from DRG neurons³⁸ (Fig. 3F). We found that Tiam1 deletion from DRG neurons partially prevented the development of morphine antinociceptive tolerance and OIH in contrast to control mice (Fig. 3G and H and Supplementary Fig. 4), which suggests that Tiam1-mediated synaptic plasticity in both DRG neurons and spinal dorsal horn neurons contributes to morphine tolerance and OIH.

Figure 3.

Tiam1 deletion from spinal dorsal horn neurons or DRG neurons reduces morphine antinociceptive tolerance and OIH. (A) Infection of spinal dorsal horn neurons. Expression of the green fluorescent protein (GFP, green) in the left spinal dorsal cord of the L4 section 2 weeks after the intra-spinal dorsal horn injection of 0.5 µl rAAV8-GFP (10⁹ virus molecules/µl) (top, scale bar = 1 mm). Representative immunoblots and quantification analysis of Tiam1 from the spinal dorsal horn of Tiam1^fl/fl mice infected with rAAV8-hSyn-GFP (GFP) or rAAV8-hSyn-Cre-GFP (Cre). Actin was used as an internal control. Data are mean ± standard error of the mean (SEM) (n = 4). Two-tailed Student’s t-test. *P < 0.05 (bottom). (B and C) Morphine antinociceptive tolerance. Left: Per cent maximal possible effect (% MPE) for morphine antinociception from the first administration (Day 1: +30 min) compared with the last administration (Day 7: +30 min). von Frey (C) and hotplate (D). Right: The per cent change for each subject. (D and E) Opioid-induced hyperalgesia (OIH). Left: The pre-morphine baseline nociceptive behaviours before the first administration (Day 1: baseline, BL) compared to the last day (Day 7: BL). Right: The per cent change for each subject. (C and E, reflexive antinociception, von Frey; D and F, affective-motivational behaviour, 55°C hotplate). Data are mean ± SEM (n = 8 mice in each group). Repeated measures two-way ANOVA followed by Tukey’s post-test. **P < 0.01, ***P < 0.001. (F–H) Tiam1 deletion from dorsal root ganglion (DRG) neurons reduces morphine antinociceptive tolerance and OIH. (G) Advillin-Cre mice were crossed with Tiam1^fl/fl mice to delete Tiam1 from DRG neurons. (H) Morphine antinociceptive tolerance. Left: Per cent MPE for morphine antinociception to von Frey filament mechanical stimulation from the first administration (Day 1: +30 min) compared with the last administration (Day 7: +30 min). Right: The per cent change for each subject. (I) Morphine hyperalgesia. Left: The pre-morphine baseline nociceptive hypersensitivity before the first administration (Day 1: BL) compared to the last day (Day 7: BL). Right: The per cent change for each subject. Data are mean ± SEM [wild-type (WT), n = 5 mice; Advillin-Tiam1 conditional knockout (cKO), n = 7 mice]. Repeated measures two-way ANOVA followed by Tukey’s post-test. *P < 0.05. Ctrl = control.

As Tiam1 regulates excitatory synapse development and spine morphogenesis in the brain,³² we next investigated the role of brain-expressed Tiam1 in the development of tolerance and OIH by crossing Tiam1^fl/fl mice with CaMKIIα-Cre transgenic mice to delete Tiam1 specifically from postnatal forebrain excitatory neurons.³⁷CaMKIIα-Tiam1 cKO mice exhibited similar antinociceptive tolerance and OIH as littermate controls throughout the 7-day morphine schedule (Supplementary Fig. 5). Given that CaMKII-Cre line only deletes Tiam1 from postnatal forebrain excitatory neurons, we cannot exclude the possible contributions of Tiam1 in other brain regions to morphine tolerance, such as periaqueductal grey (PAG), thalamus and striatum important for morphine tolerance. Together, these data indicated that Tiam1 expressed by the spinal dorsal horn and DRG neurons determines the development of morphine antinociceptive tolerance and OIH.

Inhibiting Tiam1 signalling prevents the development of morphine tolerance and OIH

As our genetic and viral conditional deletion strategy revealed that Tiam1 loss from spinal dorsal horn neurons or DRG neurons prevented the onset of morphine antinociceptive tolerance and OIH, we reasoned that pharmacological blockade of Tiam1 signalling might similarly alleviate these adverse morphine side effects. In wild-type C57BI/6J mice, we paired injections of morphine (s.c.) and NSC23766 [intraperitoneal (i.p.)], a widely used drug that inhibits Tiam1-Rac1 signalling activation.⁷¹ We assessed the antinociceptive effects of morphine and NSC23766 combination therapy on nociceptive reflexes and affective-motivational behaviours. We found that morphine administration significantly reduced nociceptive reflexes and was effective at alleviating affective-motivational behaviours (Supplementary Fig. 6), and combination treatment with NSC23766 did not alter the acute morphine antinociceptive effects (Supplementary Fig. 6). We next determined whether NSC23766 can effectively reduce morphine antinociceptive tolerance and OIH by treating mice with a combination of morphine (10 mg/kg) and NSC23766 (0.2, 1 or 5 mg/kg) or morphine alone, once daily for 7 days. On the first day of treatment, morphine alone or morphine and NSC23766 combination acutely produced strong antinociception as indicated by significant increases in the threshold of mechanically induced nociceptive paw withdrawal (Fig. 4A, von Frey, reflexive behaviour) and latency of noxious thermal-induced paw attending and guarding (Fig. 4D, 55°C hotplate, affective behaviour). As expected, mice treated with morphine alone developed robust antinociceptive tolerance on both reflexive and affective-motivational measures on Day 7 (Fig. 4B and E). Notably, we found that mice co-treated with morphine plus NSC23766 showed a dose-dependent reduction in the onset of tolerance (Fig. 4B and E). Furthermore, the mice co-administrated with morphine and NSC23766 showed a dose-dependent reduction in the onset of OIH on both reflexive and affective-motivational assays (Fig. 4C and F). We also noticed that direct administration of NSC23766 into the spinal cord via intrathecal injection had similar effects as systematic administration on attenuating morphine tolerance and OIH (Supplementary Fig. 7). Together, these data indicate that inhibiting Tiam1 signalling prevents the development of morphine antinociceptive tolerance and OIH.

Figure 4.

Pharmacological blockade of Tiam1 signalling by NSC23766 prevents the development of morphine antinociceptive tolerance and OIH in a dose-dependent manner. (A and B) Effect of NSC23766 co-administration at multiple doses (0, 0.2, 1, or 5 mg/kg NSC) on morphine reflexive antinociceptive tolerance. (A) Effect of NSC23766 co-administration at multiple doses on acute morphine antinociception. (B) Left: Per cent maximal possible effect (% MPE) for morphine antinociception from the first administration (Day 1: 30 min) compared with the last administration (Day 7: 30 min). Right: The per cent change for each subject. (C) Effect of NSC23766 co-administration at multiple doses of reflexive opioid-induced hyperalgesia (OIH). Left: Changes in the pre-morphine baseline (BL) nociceptive behaviours before the first administration (Day 1: BL) compared to the last administration (Day 7: BL). Right: The per cent change for each subject. (D and E) Effect of NSC23766 co-administration at multiple doses (0, 0.2, 1 or 5 mg/kg NSC) on morphine affective-motivational antinociceptive tolerance. (D) Effect of NSC23766 co-administration at multiple doses on acute morphine antinociception. (E) Left: % MPE for morphine antinociception from the first administration (Day 1: 30 min) compared with the last administration (Day 7: 30 min). Right: The per cent change for each subject. (F) Effect of NSC23766 co-administration at multiple doses of affective-motivational OIH. Left: Changes in the pre-morphine baseline nociceptive behaviours before the first administration (Day 1: BL) compared to the last administration (Day 7: BL). Right: The per cent change for each subject. Data are mean ± standard error of the mean (SEM) (n = 8 mice in each group). Two-way ANOVA followed by Tukey’s post-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Blocking Tiam1 signalling reverses established morphine tolerance and OIH

We next examined whether NSC23766 treatment can reverse established morphine antinociceptive tolerance and OIH. We administrated morphine (10 mg/kg, s.c.) for 9 days and began coadministration of NSC23766 (1 mg/kg, i.p.) on Day 1, Day 3, Day 5 or Day 7 (Fig. 5A). As we previously observed, NSC23766 administered from Day 1 completely prevented morphine tolerance and OIH on both reflexive and affective-motivational behaviours (Fig. 5B–E). Initiation of NSC23766 treatment on Day 3, Day 5 or Day 7 of morphine treatment reversed morphine antinociceptive tolerance within 2 days on both reflexive and affective-motivational behaviours (Fig. 5B and C). Furthermore, we found that initiation of NSC23766 treatment on Day 3, Day 5 or Day 7 also reversed OIH on both reflexive and affective-motivational behaviours (Fig. 5D and E). Together, these data demonstrate that NSC23766 administration can reverse established morphine antinociceptive tolerance and OIH. Importantly, we found that on Day 9 and Day 10, all mice received morphine alone and morphine still elicited high antinociception (Fig. 5), indicating that inhibiting Tiam1 signalling can reverse the process that causes morphine antinociceptive tolerance and OIH.

Figure 5.

Pharmacological blockade of Tiam1 signalling by NSC23766 reverses the established morphine antinociceptive tolerance and OIH. (A) Drug paradigm for morphine and NSC23766 administration. Mice were treated daily with morphine alone (10 mg/kg, s.c.) or morphine plus NSC23766 (1 mg/kg, i.p.) with NSC23766 beginning with Day 1, 3, 5 or 7 of morphine treatment (morphine + NSC-1, morphine + NSC-3, morphine + NSC-5 or morphine + NSC-7, respectively). On Day 9 and Day 10, all mice received morphine alone (indicated by the grey area between Day 9 and Day 10). Mechanical nociceptive thresholds and affective-motivational response to noxious thermal stimuli before and 30 min after each daily injection to evaluate opioid-induced hyperalgesia (OIH) and tolerance, respectively. (B and C) Antinociceptive tolerance (B, reflexive hypersensitivity, von Frey; C, affective-motivational, 55°C hotplate). (D and E) OIH (D, reflexive hypersensitivity, von Frey; E, affective-motivational, 55°C hotplate). Data are mean ± standard error of the mean (SEM) (n = 10 mice in each group). Repeated measures two-way ANOVA followed by Tukey’s post-test. *P < 0.05; **P < 0.01; ***P < 0.001 (morphine alone versus morphine + NSC23766). BL = baseline.

Tiam1 mediates prolonged morphine-induced synaptic structural plasticity

Previous studies have established that morphine can induce morphological changes in the dendritic spines of hippocampal neurons.^72,73 We, therefore, investigated whether prolonged morphine can also induce dendritic spine morphogenesis of spinal dorsal horn neurons and whether Tiam1 mediates this structural remodelling. We conducted intra-spinal dorsal horn microinjections of GFP-expressing AAV virus to sparsely label spinal dorsal horn neurons in wild-type and Tiam1 KO mice. Two weeks after the viral injections, we treated mice with 10 mg/kg morphine or saline. After a 7-day treatment, we used confocal imaging to analyse the dendritic spine morphology of GFP-expressing WDR neurons (Fig. 6A). We found that a 7-day morphine treatment increased the density of WDR neuron dendritic spines (Fig. 6B and C). Dendritic spine remodelling is driven by the polymerization of spine-enriched actin.^74–76 The ratio between filamentous (F)-actin to monomeric globular (G)-actin reflects the balance between actin polymerization and depolymerization.⁷⁷ In wild-type mice, we found that a 7-day morphine treatment increased the F-actin to G-actin ratio in the spinal dorsal horn (Fig. 6D), suggesting that prolonged morphine treatment induces actin polymerization that promotes dendritic spine remodelling. However, in Tiam1 KO mice, 7-day morphine treatment did not change the density of dendritic spines or the F-actin to G-actin ratio (Fig. 6D), indicating that Tiam1 mediates prolonged morphine-induced actin cytoskeletal and dendritic spine remodelling.

Figure 6.

Tiam1 regulates chronic morphine-induced synaptic structural plasticity in the spinal dorsal horn. (A) Experimental paradigm for dendritic spine analysis. (B and C) Representative confocal images and quantification of dendritic spine density show that repeated morphine treatment (7 days) increases the dendritic spine density in dorsal horn spinal neurons of wild-type (WT) mice, but not Tiam1 knockout (KO) mice (scale bar = 10 µm). Data are mean ± standard error of the mean (SEM) (n = 20–23 neurons from three mice in each group). Two-way ANOVA analysis followed by Tukey’s post-test. *P < 0.05, **P < 0.01, ***P < 0.001. NS = no significant difference. (D) Tiam1 deletion attenuates repeated morphine-stimulated actin polymerization in the spinal dorsal horn. Western blots and quantification reveal that compared to saline treatment (S), repeated morphine treatment (M) increased the F-actin (F) to G-actin (G) ratio in the spinal dorsal horn of wild-type mice, but not in Tiam1 KO mice. Data are mean ± SEM (n = 4). Two-tailed Student’s t-test. *P < 0.05.

Tiam1 regulates prolonged morphine-induced synaptic functional plasticity

NMDAR-mediated central sensitization in the spinal dorsal horn has been implicated in morphine tolerance and OIH.^8,24 As Tiam1 loss attenuates prolonged morphine-induced actin polymerization (Fig. 6D) and actin polymerization facilitates synaptic NMDAR activity,⁷⁸ we next investigated whether Tiam1 was required for these NMDAR changes. To determine the levels of synaptic NMDAR subunits, we isolated the synaptic membrane fraction from the spinal dorsal horns of wild-type and Tiam1 KO mice 7 days after saline or morphine treatment (10 mg/kg/day, s.c.). In wild-type controls, 7-day morphine treatment increased the levels of synaptic NMDAR subunits GluN1 and GluN2B relative to saline controls (Fig. 7A and B). In contrast to wild-type mice, synaptic NMDAR subunit levels were unaltered by 7-day morphine treatment in Tiam1 KO mice (Fig. 7A and C). These data suggest that Tiam1 mediates the increase in synaptic NMDAR protein levels in response to prolonged morphine.

Figure 7.

Tiam1 regulates chronic morphine-induced synaptic functional plasticity in spinal neurons. (A–C) Western blots and quantification show that the levels of synaptic NMDAR subunit GluN1 and GluN2B were significantly increased in wild-type (WT) mice treated with morphine (M) (10 mg/kg/day for 7 days, s.c.), but not saline (S). Tiam1 deletion prevented chronic morphine-induced increases in synaptic NMDAR subunits. PSD-95 was used as the loading control. Data are mean ± standard error of the mean (SEM) (n = 4). Two-tailed Student’s t-test. **P < 0.01 (saline versus morphine). (D) Original current traces and mean changes in NMDAR currents elicited by puff application of 100 μM NMDA to spinal dorsal horn neurons in wild-type mice and Tiam1 knockout (KO) mice with 7 days of saline or morphine treatment. Data are mean ± SEM (n = 9–12 neurons from three mice in each group). One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05. (E) Original traces, cumulative plots and mean changes in the baseline frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) and the AP5 effect in spinal dorsal horn neurons recorded from wild-type mice and Tiam1 KO mice with 7-day morphine treatment before (baseline), with (AP5) and after (washout) bath application of 50 μM AP5. Data are mean ± SEM (n = 9–12 neurons from three mice in each group). One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05. (F) Original traces and mean changes in baseline values and the AP5 effect on the amplitude of EPSCs of spinal dorsal horn neurons monosynaptically evoked by dorsal root stimulation in wild-type and Tiam1 KO mice treated with saline (sal) or morphine (mor). These traces are an average of 10 consecutive responses. Data are mean ± SEM (n = 14–19 neurons from 3–5 mice in each group). One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 (versus baseline); ^#P < 0.05 (versus baseline in the wild-type saline group).

Electrophysiological recordings of dorsal horn neurons in acute spinal cord slices further revealed that 7-day morphine treatment significantly increased postsynaptic NMDAR currents elicited by puff application of NMDA in wild-type mice (Fig. 7D), whereas no difference was detected in synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) activity (Supplementary Fig. 8). Seven-day morphine treatment also significantly potentiated presynaptic NMDAR activity, as reflected by the increase in the AP5 sensitive frequency of mEPSCs of dorsal horn neurons in wild-type mice (Fig. 7E). These increases in pre- and postsynaptic NMDAR activity were abrogated in Tiam1 KO mice (Fig. 7D and E). In addition, we recorded EPSCs monosynaptically evoked by stimulation of the dorsal root, reflecting synaptic NMDAR activity. In the dorsal horn neurons from 7-day morphine-treated wild-type mice, bath application of AP5 significantly reduced the amplitude of the evoked EPSCs (Fig. 7F). However, in the dorsal horn neurons of 7-day morphine-treated Tiam1 KO mice, AP5 had no significant effect on the amplitude of evoked EPSCs (Fig. 7F). Collectively, these findings indicate that Tiam1 is essential for the prolonged morphine-induced increase in synaptic NMDAR activity in the spinal cord dorsal horn.

NSC23766 and morphine combination therapy provide long-lasting relief from chronic pain

We next assessed the antinociceptive efficacy of morphine and NSC23766 combination therapy in chronic pain management. We used the CFA mouse model of inflammatory pain wherein CFA injection induced pain hypersensitivity in both reflexive and affective-motivational assays, as indicated by the decreased mechanical and thermal thresholds (Fig. 8B, D and F). To test whether inhibiting Tiam1 signalling can reduce opioid tolerance in chronic pain management, we treated CFA mice with morphine or morphine (10 mg/kg) plus NSC23766 (1 mg/kg) once daily for 7 days. As a positive control, we treated CFA mice with morphine only on Day 1 and Day 7. From Day 2 to Day 6, we treated mice with saline to avoid morphine analgesic tolerance on Day 7. As a native control, we treated CFA mice only with NSC23766 (1 mg/kg). On Day 1 and Day 7, we measured mechanical and thermal thresholds 30 min after drug administration to evaluate the morphine analgesic effect (Fig. 8A).

Figure 8.

Combination therapy with Tiam1 signalling inhibitor NSC23766 reduces morphine tolerance in chronic pain management. (A) Drug paradigm for morphine and NSC23766 administration. (B, D and F) Time course for nociceptive hypersensitivity and affective-motivational behaviour before Complete Freund’s adjuvant (CFA) injection (pre-injury), following CFA injection [post-injury day (PID) 1], and the effect of first treatment (PID 1: +30 min) compared to the last treatment (PID 7: +30 min). Treatments of 10 mg/kg morphine or 10 mg/kg morphine + 1 mg/kg NSC23766 (M+NSC) were administered once daily for 7 days. As a positive control, the saline group was only administered acute morphine on Day 1 and Day 7. (B, von Frey; D, 55°C hotplate; F, affective-motivational, 55°C hotplate) (C, E and G). Antinociceptive tolerance. Left: Maximum possible effect (MPE) for morphine antinociception from the first administration (PID 1: +30 min) compared with the last administration (PID 7: +30 min) (C, von Frey; E, 55°C hotplate; G, affective-motivational, 55°C hotplate). Right: Per cent change for each subject. Two separate one-way ANOVAs were run for pre-injury versus CFA injection (PID 1), and first treatment (PID 1: +30 min) versus last treatment (PID 7: +30 min). Two-way ANOVA analysis followed by Tukey’s post-test. Data are mean ± standard error of the mean (SEM) (n = 8 mice in each group). *P < 0.05; **P < 0.01; ***P < 0.001. The NSC23766 treatment data are available in the Supplementary material.

On the first day of treatment, morphine alone and morphine with NSC23766 coadministration produced strong analgesia in nociceptive sensory-reflexive behaviours (Fig. 8B–E) and affective-motivational behaviours (Fig. 8F and G). However, after 7-day treatment, morphine alone was no longer effective at reducing CFA inflammatory pain. Morphine and NSC combination therapy still produced strong analgesia against mechanical and thermal reflexive hypersensitivity (Fig. 8B–E) as well as against affective-motivational pain response (Fig. 8F and G), with no indication of tolerance. NSC23766 (1 mg/kg) alone did not significantly reduce CFA-induced pain hypersensitivity either 1 day or 7 days after CFA injection (Supplementary Fig. 9). Taken together, our pharmacological results highlight the potential benefit of inhibiting Tiam1 signalling to reduce tolerance and prolong morphine use in chronic pain management.

Discussion

Our findings identify Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network as the downstream maladaptive mechanisms of morphine action on primary afferent nociceptors, which underlies the development and maintenance of morphine tolerance and OIH. Moreover, we show that inhibiting Tiam1 signalling is sufficient to abrogate morphine tolerance and prolong morphine use in chronic pain management. Previous studies have established that morphine action triggers glutamate release from primary afferents and brain-derived neurotrophic factor (BDNF) release from spinal microglia, resulting in the activation of NMDARs and TrkB receptors in spinal neurons, respectively.^9,13,19 As Tiam1 interacts with activated NMDAR and TrkB receptors and mediates Rac1-dependent synaptic remodelling in brain development and neuropathic pain,^{28,33,34,37,38,79} we propose that Tiam1 links prolonged morphine-activated NMDARs and TrkB receptors to Rac1 signalling that orchestrates synaptic structural plasticity via dendritic spine remodelling and functional plasticity via synaptic NMDAR stabilization in spinal neurons, which together underlies morphine tolerance and OIH (Supplementary Fig. 10). Thus, Tiam1-dependent synaptic plasticity in spinal neurons serves as the maladaptive mechanism underlying morphine tolerance and OIH, and inhibiting Tiam1 signalling provides a promising therapeutic strategy to reduce tolerance and prolong morphine use in chronic pain management.

Tiam1 is well placed to propagate the signalling from maladaptive plasticity in primary afferent nociceptors to morphine tolerance and OIH via modulation of spinal dorsal horn neuron synapses. As a Rac1-GEF that couples synaptic receptors to Rac1 signalling, Tiam1 is highly expressed in the spinal cord as well as the developing and adult brain.³² We and others have previously shown that Tiam1 is recruited to activated synaptic NMDAR and TrkB receptors during brain development, resulting in its phosphorylation and activation, enabling Tiam1 to induce localized Rac1 signalling and actin remodelling that drives dendritic spine and synapse development.^28,33,34,79 During the development of tolerance and OIH, morphine not only depresses neurotransmission between nociceptors and spinal dorsal horn neurons but can also trigger glutamate release from primary afferents and BDNF release from spinal microglia, resulting in the activation of synaptic NMDAR and TrkB receptors.^13,21,22,24 By functioning downstream of these activated receptors in the spinal dorsal horn, Tiam1 likely acts as a convergence point to integrate multiple signalling inputs that promote robust Rac1-dependent maladaptive synaptic structural and functional plasticity in spinal dorsal horn neurons, which increases excitatory plasticity and leads to OIH and tolerance.

Accumulating evidence supports the idea of similar molecular mechanisms underlying morphine tolerance/OIH and neuropathic pain at the level of DRG nociceptors, spinal dorsal horn and brain.^7,13,67–69 In particular, enhanced NMDAR function and attenuated GABAergic inhibition in the spinal nociceptive network have been implicated in the development of neuropathic pain and morphine tolerance/OIH. Other studies have shown that spinal disinhibition results from BDNF-TrkB signalling upon nerve injury or prolonged morphine treatment.¹³ The current study and our recent findings suggested that Tiam1 links both prolonged morphine- and nerve injury-induced NMDARs and/or TrkB receptor activation to Rac1 signalling that stabilizes synaptic NMDAR in spinal neurons, which drives morphine tolerance/OIH and neuropathic pain hypersensitivity.³⁸ Furthermore, a key concept emerging from our findings is that prolonged morphine induces dendritic spine remodelling in nociceptive dorsal horn neurons. Because of the strong correlation between spine structure and synaptic function, maladaptive dendritic spine remodelling could directly contribute to long-lasting, maladaptive synaptic activity accompanying OIH states after morphine withdrawal, which contributes to the long-term maintenance of tolerance and OIH. In both morphine tolerance/OIH and neuropathic pain, Tiam1 regulates dorsal horn spinal neuron dendritic spine structure via actin cytoskeleton remodelling. However, more work needs to be done to determine whether Tiam1-mediated synaptic structural and functional plasticity occurs in the same group of spinal neurons during the development of morphine tolerance/OIH and neuropathic pain. Taken together, our findings identify Tiam1-mediated pronociceptive plasticity in spinal neurons as the neural substrate for morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Funding

This work was supported by grants from the National Institute on Drug Abuse (DA056673), National Institute of Neurlogical Disorders and Stroke (NS124141), and the Mission Connect/TIRR Foundation 020-102.

Competing interests

The authors report no competing interests.

Supplementary material

Supplementary material is available at Brain online.