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INTRODUCTION
Chronic opioid exposure induces cellular adaptations such as opioid receptor desensitization, downregulation, internalization, and impaired signal transduction, thus attenuating analgesic efficacy over time [1]. As morphine tolerance develops, ineffective pain relief leads to suffering and reduced function, if alternative strategies are unavailable. It can also contribute to opioid-related harms by increasing total opioid exposure. Dose escalation in the setting of tolerance elevates the risk of adverse effects, including respiratory depression, opioid-induced hyperalgesia, endocrine dysfunction, immunosuppression, and accidental overdose [2]. Moreover, higher cumulative doses are associated with increased likelihood of developing physical dependence and withdrawal symptoms upon dose reduction or interruption, which may further reinforce continued opioid use and contribute to the development of opioid use disorder [3].
Although early studies investigating opioid tolerance emphasized opioid receptor desensitization at the spinal level, clinical and experimental evidence shows that tolerance cannot be fully explained by local receptor changes. Recently, opioid tolerance is increasingly recognized as a process that extends beyond receptor-level adaptations in the spinal cord, involving extensive supraspinal mechanisms within pain- and reward-related brain circuits. Supraspinal mechanisms reported to be involved in the development of opioid tolerance include loss of descending analgesia in the periaqueductal gray and rostral ventromedial medulla [4], increased excitability of neurons in the locus coerleus [5], and alterations in dopaminergic neuronal function in the ventral tegmental area (VTA) and nucleus accumbens (NAc) [6]. Among them, the NAc, which is regulated by dopaminergic projections from the VTA, is not only central to reward but also influences the affective and motivational dimensions of pain [7–9]. These signaling pathways may explain how tolerance emerges from supraspinal circuit dysfunction, links pain relief to reward processes, connects tolerance with addiction risk, and opens avenues for therapeutic innovation [6].
Here, the authors explored the role of dopaminergic neurotransmission in the development of morphine tolerance in the rat neuropathic pain model. The extracellular dopamine level in the NAc was measured using microdialysis study and evaluated the effects of dopaminergic receptor agonists microinjected into the NAc on morphine analgesic tolerance. Dopaminergic fiber expression in the NAc of morphine tolerant animals was assessed using immunohistochemical techniques. Additionally, the effects of the microglial inhibitor minocycline on the extracellular dopamine level in the NAc and the development of morphine tolerance were investigated.
MATERIALS AND METHODS
1. Experimental protocol
The experimental protocol was approved by the Institutional Animal Care and Use Committee of Chonnam National University Medical School (no. CNU IACUC-H-2018-72). In the first series of experiments, the authors aimed to elucidate the role of NAc dopaminergic neurotransmission in the development of morphine tolerance. We measured extracellular dopamine level in the NAc using microdialysis study and evaluated the effects of dopamine receptor agonists administered into the NAc on morphine analgesic tolerance via a von Frey filaments test. To assess the expression level of dopaminergic fiber in the NAc, immunohistochemical staining was performed. The second series of experiments were for clarifying the involvement of microglial activation in the NAc dopaminergic alteration and the development of morphine tolerance. To verify this speculation, minocycline, a microglial inhibitor, was pretreated with morphine during the tolerance induction phase, and microdialysis and behavioral assessment were performed.
2. Animals
Adult male Sprague–Dawley rats weighing 280–320 g were utilized in the current study. The animals were maintained under controlled environmental conditions (22°C–23°C, 12-h light/dark cycle) with ad libitum access to food and water. Neuropathic pain was induced by ligation of the L5 and L6 spinal nerves, following a previously reported technique [10]. Rats showing impaired flexion of the left hind limb, suggestive of L4 injury, were excluded. Animals that developed a 50% paw withdrawal threshold below 4.0 g by postoperative day 5 were regarded as neuropathic. For morphine administration, polyethylene-10 tubing was implanted into the intrathecal space under sevoflurane anesthesia, as previously described [11]. Animals were allowed a 7-day recovery period after catheterization. Thereafter, morphine analgesic tolerance was induced by intrathecal injection of morphine 15 μg twice daily for 7 days (morphine tolerant group). This regimen was determined based on a preliminary study in which the administration schedule was modified from previous reports [12,13] to improve the reproducibility of the model. Control animals received normal saline in the same volume and schedule (morphine-naïve group). Development of morphine tolerance was demonstrated by a progressive decline in analgesic response in a pain behavior test, which was performed 30 minutes after the drug administration, toward the baseline level by day 7 (Fig. 1).
3. Behavioral analysis
Behavioral assessment was performed using the von Frey filament test to determine the mechanical withdrawal threshold. A positive response was defined as a rapid paw withdrawal or flinching occurring during or immediately after application of filaments with logarithmically increasing stiffness (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15.0 g). The 50% paw withdrawal threshold was estimated according to the up-down method, with 15 g set as the cutoff [11,14].
On each experimental day, animals were placed in boxes with a wire mesh floor for at least 20 minutes to allow habituation, after which they were randomly assigned to receive either the test drug or vehicle. Following morphine administration, withdrawal thresholds were evaluated at 30, 60, 90, 120, 150, and 180 minutes. All behavioral testing was performed by an investigator blinded to the treatment conditions.
4. Drugs
The following drugs were used in the current study: morphine (BCWORLD Pharm) dissolved in saline, CY 208-243 (1 μg; Tocris Cookson Ltd.), a selective D1-like receptor agonist, and cabergoline (0.1 μg; Tocris Cookson Ltd.), a selective D2-like receptor agonist dissolved in 70% DMSO (dimethyl sulfoxide). These agonists were intracranially injected 5 minutes prior to intrathecal morphine injection. The doses were chosen based on a pilot study that determined maximum doses not affecting pain behavior alone. Minocycline (Sigma-Aldrich) was dissolved in 10% DMSO and a dose of 50 mg/kg was injected intraperitoneally 30 minutes prior to each morphine administration during the tolerance induction phase. The dose of the minocycline was based on the previous report [15]. Intrathecal delivery of the experimental drugs was performed using a hand-driven gear-operated syringe pump via an implanted catheter. A total volume of 10 μL was injected, and the catheter was subsequently flushed with an additional 10 μL of saline. For intracranial injections, a microinjection pump (EPS-64; Eicom Co.) was utilized to deliver the drugs in a 1 μL volume using a 9-mm internal injector cannula (Eicom Co.) via the implanted guide cannula, at a rate of 1 μL/min.
5. Intracranial cannulation
For the intracranial microinjection experiments and microdialysis study, guide cannulas were implanted into the NAc shell according to the coordinates provided in the Paxinos and Watson rat brain atlas [16]. Under sevoflurane anesthesia, rats were placed in a stereotaxic apparatus, and the skull was exposed via a midline incision. After drilling a burr hole, an 8-mm 26-gauge stainless steel guide cannula (AG-8; Eicom Co.) was inserted into the NAc shell using the following coordinates: from the bregma, 1.7 mm rostral, 0.8 mm lateral, and 7.0 mm ventral. The cannula was secured to the skull with screws and dental cement. The incision was closed using 3-0 surgical sutures, and rats were individually housed and allowed to recover for 1 week. At the end of the experiments, to confirm accurate cannula placement, brains were harvested and fixed with 4% paraformaldehyde (PFA) and frozen-sectioned. Finally, cannula tip positions were verified by examining and photographing the sections. Data from animals with misplaced cannulas were removed from the analyses.
6. In vivo microdialysis and high-performance liquid chromatography analysis of dopamine
A microdialysis probe (FX-I-8-01; Eicom Co.) was inserted into the previously implanted guide cannula and connected to a microinfusion pump. Ringer’s solution (147.0 mmol/L NaCl, 4.0 mmol/L KCl, and 2.3 mmol/L CaCl₂; pH 7.2) was perfused through the inlet tubing at a flow rate of 1 μL/min. The dialysate was collected from the outlet tubing into an auto-injector (EAS-20S; Eicom Co.) in 15 minutes intervals. Each 15 μL fraction was analyzed for dopamine content using high-performance liquid chromatography with electrochemical detection (HTEC-500 system; Eicom Co.). The electrochemical detection conditions were as follows: the mobile phase consisted of 0.1 mol/L ammonium acetate buffer (pH 6.0), 0.1 mol/L acetic acid, methanol (7:3, v/v), 7.10 g/L sodium sulfate, and 50 mg/L EDTA-2Na. Dopamine separation was performed using a EICOMPAC CAX column (2.0 × 200 mm; Eicom Co.) with a glassy carbon working electrode (WE-3G; Eicom Co.) at a flow rate of 0.25 mL/min. The detector potential and column temperature were set at 0.45 V and 35.0°C, respectively. The retention time for dopamine was approximately 7.1 minutes. Once dopamine levels had been stabilized, four consecutive samples (1 hour total) were collected.
7. Immunohistochemical staining
To examine the expression of tyrosine hydroxylase (TH) in the NAc shell or microglia in the VTA, immunohistochemical staining was performed. Rats were anesthetized with pentobarbital sodium and transcardially perfused with 200 mL of phosphate-buffered saline (PBS), followed by 4% PFA on ice. Brains were rapidly removed, post-fixed in 4% PFA for 4 hours, and then immersed in 30% sucrose for 72 hours at 4°C. Brain tissues were embedded in frozen section compound (Leica Biosystems), and coronal sections (30 μm thick) containing the NAc shell or VTA were prepared and mounted on glass slides.
The sections were washed in 0.01 M PBS (pH 7.4; 3 × 5 minutes) and blocked for 2 hours in 3% bovine serum albumin and 0.3% Triton X-100 in PBS. Primary antibodies were applied and incubated overnight at 4°C in a humid chamber, as follows: rabbit anti-TH (1:300; Thermo Fisher Scientific) and rabbit anti-Iba1 (1:200; Wako). The sections were washed with PBS (3 × 5 minutes) and incubated for 2 hours at room temperature with the following secondary antibodies: Alexa Fluor 594-conjugated chicken anti-rabbit IgG (1:500; Thermo Fisher Scientific) or Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500; Thermo Fisher Scientific).
After washing with PBS (3 × 10 minutes), sections were mounted with mounting solution and covered with cover slips. Images were acquired using a fluorescence microscope (LSM800; Zeiss) with tile scan mode, presenting an area of 10 × 10 fields. For quantitative analysis, fluorescence signal intensity was measured as the mean fluorescence intensity (calculated as integrated density/area, IntDen/area). The regions of interest within the NAc shell or VTA were delineated according to the stereotaxic coordinates provided in the Paxinos and Watson rat brain atlas [16]. All data are expressed as the mean ± standard error of the mean (SEM) and analyzed using ImageJ software ver. 1.54g.
8. Statistical analysis
All data are presented as the mean ± SEM. The time course of the behavioral response to repeated morphine administration was analyzed via repeated measure analysis of variance (ANOVA). Quantification of the other behavior tests, microdialysis data, and immunochemical analysis were conducted by calculating the area under the curve (AUC). These data were statistically analyzed using an independent sample t-test for two groups or a one-way ANOVA for three groups comparisons. Statistical analyses were performed using IBM SPSS version 18.0 software (IBM Co.). A P value of less than 0.05 was considered statistically significant.
RESULTS
Daily administration of intrathecal morphine produced a progressive decline in analgesic response in pain behavior by day 7 (Fig. 1). Therefore, all experiments were performed on the 8th day of daily morphine administration.
1. Decreased extracellular dopamine level in the NAc in morphine tolerant animals
The in vivo microdialysis study revealed that the extracellular level of dopamine in the NAc was significantly decreased in the morphine-tolerant compared to morphine-naive spinal-nerve ligated animals. Fig. 2 shows the extracellular level of dopamine in the NAc in both groups of animals. The AUCs for the 1-hour measurement of dopamine level in the morphine tolerant animals was significantly lower than those in the morphine naïve animals (P = 0.043, Fig. 2). Fig. 3 shows immunohistochemical analysis of dopaminergic fibers in the NAc. A one-way ANOVA of the mean TH fluorescence intensities showed a statistically significant differences among the groups (F [2,12] = 11.984, P = 0.001, Fig. 3). Post-hoc comparison using Tukey HSD (honestly significant difference) test indicated that the TH immunoreactivities were decreased in both the morphine naïve and tolerant neuropathic groups compared to the sham (non-neuropathic) group (P = 0.001 and 0.017, respectively), however, there was no significant difference between the two neuropathic groups (P = 0.317, Fig. 3).
2. The effects of dopamine receptor agonist on established morphine tolerance
To determine whether the decreased extracellular level of dopamine in the NAc is involved in the mechanisms of morphine tolerance, selective D1-like receptor agonist CY 208-243 1 μg or D2-like receptor agonist cabergoline 0.1 μg was microinjected into the NAc 5 minutes prior to the administration of morphine. Analgesic responses to morphine in tolerant animals were improved by both the CY 208-243 and cabergoline pretreatments compared to those receiving the vehicle (P < 0.001 for both, Fig. 4).
3. The effect of minocycline pretreatment on the NAc dopamine level and the development of morphine tolerance
To determine whether the microglial activation in the VTA contributes to the alterations in the dopamine level in the NAc and the development of morphine tolerance, microglial inhibitor minocycline was pretreated with morphine during the tolerance induction phase. The minocycline-treated animals exhibited improved analgesic response to morphine compared to vehicle-treated ones (P = 0.001, Fig. 5). The microdialysis study showed an increased NAc extracellular dopamine level in the minocycline-treated animals compared to that in the vehicle-treated ones (P = 0.049, Fig. 6). Fig. 7 presents decreased IBA1 immunoreactivity in the VTA by minocycline treatment (P = 0.024 for minocycline vs. vehicle group, Fig. 7).
DISCUSSION
In the current study, in vivo microdialysis study demonstrated that the extracellular level of dopamine in the NAc was decreased in the morphine tolerant animals. A dopaminergic D1- or D2-like receptor agonist, when pretreated into the NAc, improved analgesic response to morphine in the tolerant animals. These observations indicate that the decreased dopaminergic neurotransmission in the NAc plays a significant role in the underlying mechanisms of morphine tolerance. When the microglial inhibitor minocycline was administered prior to daily morphine administration, the level of extracellular dopamine in the NAc was partially recovered and the development of morphine tolerance was attenuated. These findings suggest that the microglial activation in the VTA produced by chronic morphine exposure disrupts dopaminergic signaling in the NAc and contributes to the development of morphine tolerance in chronic neuropathic pain.
The NAc is a subcortical structure located in the ventral striatum. Dopaminergic inputs to this region arise from the VTA, while glutamatergic afferents originate from the prefrontal cortex, ventral hippocampus, basolateral amygdala, and thalamus [17]. NAc dopamine is well known as a critical neurochemical signal that underlies the reinforcing, motivational, and learning aspects of addiction [18]. However, accumulating scientific evidence indicates that it also plays a substantial role in pain modulation and analgesic drug responses. Pharmacological and optogenetic manipulations activating dopamine receptors in the NAc produced meaningful pain relief in a rat neuropathic pain model [7]. Stress-induced analgesia in an acute pain model using forced swim stress was significantly attenuated by blocking either D1- or D2-like dopamine receptors directly in the NAc [8]. In spinal nerve-ligated rats, pain relief by intrathecal pregabalin triggered increased dopamine release in the NAc during early but not later phases of neuropathic pain [9].
Experimental studies have consistently reported that chronic opioid-induced reductions or dysregulation of dopamine signaling are closely associated with opioid tolerance. However, fewer studies directly measured the change in release of dopamine in the NAc in response to chronic opioid exposure. Some studies reported blunted responses [19–21], while others presented preserved responses of dopamine release [22]. Such inconsistencies may result from diversities in the doses, routes, or models of chronic opioid administrations. In those studies, observed dopamine releases were phasic responses to subsequent morphine challenge after chronic exposure using a non-chronic pain model. On the other hand, the current study measured the tonic extracellular dopamine level in the NAc in response to repeated morphine administration using a chronic neuropathic pain model, which may be more relevant to clinical situations than previously reported ones. Tonic extracellular dopamine level is thought to be maintained in a too low a concentration to activate postsynaptic receptors, however, in a sufficient range to stimulate autoreceptors regulating dopamine release, thus inhibiting phasic release of dopamine in response to morphine [23]. Although the differential role of change in tonic versus phasic dopamine release in the development of morphine tolerance cannot be clarified in the current study, further research aimed at clarifying the precise role of the NAc in the development of opioid tolerance is likely to yield important advances. Moreover, because the NAc is central to reward and motivation [7–9], elucidating its involvement in opioid tolerance may also shed light on the intersection between tolerance, dependence, and addiction. These insights are expected not only to support the design of safer and more effective opioid-based therapies but also to advance scientific understanding of mesolimbic dopamine circuitry and its role in adaptive and maladaptive motivated behaviors. While further in-depth research is warranted, these academic advances, in conjunction with institutional efforts toward safer opioid prescribing [2], are expected to contribute to reducing opioid-related harms.
Although the involvement of microglia in morphine tolerance is well-supported by multiple animal model and molecular pathway studies, most evidence currently points to spinal microglia as a key driver of tolerance, and microglial participation in the supraspinal regions remains unverified. Nevertheless, microglial activation after chronic opioid exposure has been reported not only in the spinal cord but also in several brain regions including VTA and NAc [6,24]. Pharmacologic inhibition of chronic morphine-activated microglia in the VTA restored reward behavior in opioid-dependent animals, indicating a normalization of NAc dopamine signaling [24]. In a chronic pain model, where VTA microglial activation produced brain-derived neurotrophic factor-dependent suppression of dopamine release in the NAc, inhibition of microglia reinstated accumbal dopamine release [25]. In the current study, using a morphine tolerant rodent model, the authors verified that microglial inhibition restored NAc dopamine release and attenuated morphine tolerance. These findings suggest supraspinal microglia as a potential therapeutic target for the prevention and treatment of opioid tolerance.
The findings from the current study should be interpreted in light of the following considerations. First, both the D1- and D2-like dopamine receptor agonist improved analgesic response to morphine in tolerant animals. Given that each class of receptors produces contradictory neuronal response and is involved in different NAc pathways [26], such results may seem surprising. Although these observations warrant further investigation, it is noteworthy that a subset of NAc neurons simultaneously express both D1 and D2 receptors, forming the dopamine D1–D2 receptor heteromer [27]. Activation of this heteromer by each agonist might have contributed to the current observations. Second, minocycline pretreatment attenuated morphine tolerance much more than the degree of the partial recovery of dopamine level. This result implies that, although the microglial activation and the alterations in dopamine signaling in NAc are closely linked, the development of morphine tolerance is embedded within a wider network of interacting factors and mechanisms.
In conclusion, the authors demonstrated that microglia-related decrease in dopaminergic neurotransmission in the NAc contributes to the development of morphine tolerance. By delineating how alterations in dopamine transmission and related neuroadaptations within the NAc contribute to diminished opioid efficacy, future studies may identify novel molecular and cellular targets for therapeutic intervention. Such knowledge could facilitate the development of strategies that prevent or reverse tolerance while preserving analgesic effectiveness, thereby improving long-term pain management.
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