Modulatory role of intra-accumbal dopamine receptors in the restraint stress-induced antinociceptive responses

Stress contributes to pain sensation by affecting several neural pathways, including mesolimbic-cortical dopa- mine neurons. Nucleus accumbens, an essential element of the mesolimbic dopaminergic pathway, plays a fundamental role in modulating pain and is differentially influenced by stressful events. Since we previously demonstrated the marked association of intra-NAc dopamine receptors with forced swim stress-evoked analgesia in acute pain state, this research was conducted to consider the contribution of intra-accumbal D1- and D2-like dopamine receptors to modulating effects of exposure to restraint stress in pain-related behaviors during the tail-flick test. Stereotaxic surgery was executed to implant a guide cannula within the NAc in male Wistar rats. On the test day, different concentrations of SCH23390 and Sulpiride as D1- and D2-like dopamine receptor antagonists, respectively, were unilaterally microinjected within the NAc. The vehicle animals received saline or 12 % DMSO (0.5 µ l) instead of SCH23390 or Sulpiride into the NAc, respectively. Five minutes following receiving drug or vehicle, animals were restrained for 3 h and then their acute nociceptive threshold was measured for a 60-min period by the tail-flick test. Our data revealed that RS considerably enhanced antinociceptive reaction in acute pain states. The analgesia evoked by RS dramatically declined following blocking either D1- or D2-like dopamine receptors in the NAc, an effect was more noticeable by D1-like dopamine receptor antagonist. These findings indicated that intra-NAc dopamine receptors are considerably mediated in the RS-produced analgesia in acute pain states, suggesting their possible role in psychological stress and disease.


Introduction
Stress reveals a complex bidirectional impact on pain. During stressful conditions, pain responsiveness to physical or psychological stimuli can be either exacerbated or reduced depending on type, intensity, and period of the stressful stimulus (Valenti et al., 2012;Ferdousi and Finn, 2018). The reduction of pain in response to a stressor characterized as stress-induced analgesia is an adaptive reaction affected by age, gender, and prior experience to stressful stimuli (Butler and Finn, 2009;Ferdousi and Finn, 2018). The stress-evoked antinociceptive reaction is mediated by activating descending inhibitory pain pathway, and the mechanisms involved in this phenomenon can be both endogenous opioid and non-opioid pain inhibitory systems. The opioid pain inhibitory mechanism is suppressed by naloxone or naltrexone (Lewis et al., 1980;Hough et al., 2014;Kim and Fishman, 2020), whereas the non-opioid system is sensitive to other neurotransmitters, including glutamate, GABA, serotonin and dopamine (Butler and Finn, 2009;Dunn et al., 2019).
Growing evidence suggest that brain and spinal cord dopaminergic pathways contribute to pain modulation. Projection from the ventral tegmental area to the nucleus accumbens encodes stimulus salience in response to not only a rewarding mechanism but a supraspinal painsuppression system (Koyanagi et al., 2008;Benarroch, 2016;Ziolkowska, 2021).
Two types of D1-like and D2-like dopamine receptors are associated with pain modulation in the NAc (Missale et al., 1998;Wang et al., 2021). Both these kinds of receptors are observed in the sub-regions of the NAc, shell and core (Harris and Peng, 2020;Wang et al., 2021;Ziolkowska, 2021). Our previous study indicated the involvement of intra-NAc dopamine receptors in physical stress-evoked antinociceptive response. Findings revealed that the blockade of both D1-and D2-like dopamine receptors in the NAc considerably attenuated the analgesia produced by forced swim stress as a physical stress during the tail-flick test (Noursadeghi et al., 2022).
There is evidence that physical and psychological stress are different in terms of Fos expression, hormonal responses, neurochemical alteration (Li et al., 2019;Liu et al., 2018) as well as the brain regions involved in stress responses (Joels and Baram, 2009). For instance, the brainstem and hypothalamic areas are involved in physical stressors, whereas the amygdala, prefrontal cortex, hippocampus, and prefrontal cortex are engaged in psychological stressors depending on the type of stress mediators (Joels and Baram, 2009).
Accordingly, the involvement of dopamine receptors in psychological stress, such as restraint stress has been reported. There is evidence that RS elevates extracellular dopamine and alters dopaminergic tone in the NAc (Holly and Miczek, 2016). The observations of exposure to RS in chronic inflammatory pain have also indicated the possible role of intra-NAc dopamine receptors in RS-induced analgesia in chronic pain (Faramarzi et al., 2021). Therefore, in this research, we investigated whether or not intra-accumbal dopamine receptors were involved in analgesia produced by restraint stress as a psychological stressor in acute pain states.

Animals
The present research was conducted on male Wistar rats weighing 180-200 g (Pasteur Institute, Tehran, Iran). All rodents were kept in a standard room with a temperature of 22 ± 2 ℃, a humidity of %50 ± 10, and a 12:12-h light/dark cycle. They accessed to food and water freely in the period of habituation. Experimental procedures were carried out in accordance with the animal care (National Institutes of Health Publication, 8th edition, revised 2011), and approved by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.PHNS.REC. 1400.131), Tehran, Iran.
Various concentrations (0.25, 1 and 4 μg/rat) of both antagonists were unilaterally microinjected into the NAc in a volume of 0.5-µl solution.

Stereotaxic surgery
Rats were placed in the stereotaxic apparatus (Stoelting, USA) after being anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). The stereotaxic surgical procedure was randomly performed on one side of the midline as follows: The 23-gauge, 11-mm stainless steel guide cannula was implanted 1 mm above the right or left NAc in accordance with the atlas of rat brain (AP: 1.6 ± 0.15 mm anterior to bregma, LAT: ± 1.6 mm lateral to midline, and DV: 7.8 mm ventral from the skull surface (Paxinos and Watson, 2006). A dental acrylic cement and steel screws were then applied to secure the cannula to the skull. Following the cement was completely dried, the stereotaxic arm was detached and animals were returned to their home cage to recover for at least 6 days prior to experimentation.

Restraint stress test
Restraint stress as a model of immobility and psychological stress was performed in this study. The narrow Plexiglas tubes were designed with approximately 25 cm length × 6 cm height. They had air holes and stoppers that produced a significant stress response without causing physical injury to the animal. On the day of the experiment, rats were acclimatized for an hour in the experimental room. Five minutes after receiving either drug or vehicle, each rat was restrained in a Plexiglas tube to immobilize the animal for a 3-h period; non-restrained control animals were held in their home cages until the time of testing.

Tail-flick test
The noxious thermal tail-flick reflex was utilized to assess the acute pain threshold (D'Amour and Smith, 1941). Using the tail-flick technique, the tail's dorsal surface was subjected to radiant heat in three different spots of 3, 5, and 7 cm from the caudal tip of the tail. Setting the heat intensity to 45 % of its maximum level resulted in baseline tail-flick latency (TFL) values of 3-4 s. An automatic sensor measured the time of the tail-withdrawal reflex, and the cut-off time was set on 10 s to avoid tail hurt. TFLs were assessed both before the experiment commenced and at 5, 15, 30, 45, 60 min time intervals after receiving drugs/vehicles and exposing rats to RS. Using the following formula, the maximal possible effect (%MPE) was calculated from the measured tail-flick latency at each time during the 60-min tail-flick test period.

Locomotor activity
To determine the potential impact of vehicle or drug administration on the rats' locomotor activity, the whole distance travelled was investigated. After acclimation period, the rats were unilaterally microinjected with either drug (SCH23390 or Sulpiride) or their vehicles, and then placed in the open field (60 *60 cm 2 ) to record their movements. The locomotor activity was assessed throughout a 1-h period, using a 3-CCD camera and Ethovision software.

The effect of restraint stress and vehicle administration on control groups
To consider the possible influences of intra-NAc volume of vehicle administration, and RS on noxious reactions, four distinct groups were considered: No Vehicle + No RS (n = 7), No Vehicle + RS (n = 7), Vehicle + No RS (n = 7), Vehicle + RS (n = 9). In groups of No Vehicle + No RS and No Vehicle + RS, the impact of RS was assessed on nociceptive response. In other groups of Vehicle + No RS and Vehicle + RS, the effect of intra-NAc microinjection of vehicle on the nociceptive threshold was investigated. Of note, in vehicle groups, a 0.5-µl solution of 12 % DMSO was microinjected into the NAc and in the RS ones, animals were exposed to a 3-hour restraining period.

The role of intra accumbal D1-and D2-like dopamine receptors in analgesia evoked by RS
To consider the possible influences of D1-and D2-like dopamine receptors on modulating RS-produced analgesia within the NAc, three concentrations of SCH23390 and Sulpiride were used, respectively. Five minutes prior to RS exposure, each experimental group was unilaterally microinjected with 0.5 µl SCH23390 or Sulpiride (0.25, 1 and 4 μg/ rat).
Saline and 12 % DMSO in the volume of 0.5 µl were administrated into the NAc as the vehicles of SCH23390 and Sulpiride, respectively. Moreover, to understand whether the sole D1-and D2-like dopamine receptor antagonists have any impact on pain threshold, the two groups of animals received the maximum dose of SCH23390 or Sulpiride (4 μg/ rat) without exposing to RS (Table 1). Briefly, 5 min following microinjection of either drugs or vehicles, rats were restrained for a 3-h period and then, their acute noxious threshold was evaluated by the tail-flick test at intervals of 5, 15, 30, 45 and 60 min.

Histology
The placement of guide cannula in the NAc was approved following these procedures: rats were deeply anesthetized and transcardially perfused with normal saline and formaldehyde solution (10 %). The brains were then detached and sliced coronally in 50-μm sections toward the cannula positions in the NAc using a microtome ( Supplementary  Fig. 1). The observed cannula placement was compared to the position of the NAc illustrated in the rat brain atlas ( Fig. 1A and B) and the data of accurate cannula position was only accepted (Paxinos and Watson, 2006).

Data analysis
In the current study, data were analyzed via GraphPad Prism 6 (GraphPad Software) and exhibited as mean ± SEM. Normality of distribution was assessed by Kolmogorov-Smirnov and the comparison of %MPEs was evaluated by two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test. Ordinary one-way ANOVA followed by Dunnett multiple comparisons test was also utilized to calculate the area under the curve (AUC) of %MPEs values. P < 0.05 was statistically considered significant. To determine the locomotor activity, the obtained results of distance travelled were evaluated via ordinary one-way ANOVA followed by a protected Tukey multiple comparisons test. The 50 % effective dose (ED50) of intra-NAc SCH23390 or Sulpiride as D1-and D2-like dopamine receptor antagonists, respectively, was assessed by a scatter plot designed in excel software according to the trend-line equation option in the excel software (Version 2013). The dose-response curve for the ED50 was evaluated according to three concentrations of SCH23390 and Sulpiride.

The influence of RS exposure on the nociceptive threshold with or without vehicle administration
The effect of RS on the acute pain paradigm was investigated at 5, 15, 30, 45, 60 min intervals throughout the tail-flick test. As illustrated in Fig. 2A, two-way ANOVA with Bonferroni post-hoc test for %MPEs  Additionally, unpaired student's t-test exhibited that mean AUC values of groups exposed to RS with or without DMSO administration were considerably higher than those that were not subjected to RS [t 14 = 6.339, P < 0.0001; Fig. 2B]. Findings also demonstrated that intra-NAc vehicle administration had no significant impact on the nociceptive responses.

The effect of intra-accumbal D2-like dopamine receptor antagonist (Sulpiride) on analgesia produced by RS
Intra-NAc administration of Sulpiride, as a D2-like dopamine receptor antagonist, attenuated antinociceptive responses induced by a 3h exposure to RS. The repeated measures two-way ANOVA followed by Bonferroni post-hoc test demonstrated that intra-accumbal microinjection of maximum concentration of Sulpiride (4 µg/0.5 µl DMSO) considerably diminished the analgesia elicited by RS during measuring tail-flick latency [Treatment effect: F (4, 132) = 9.681, P < 0.0001; Time effect: F (4, 132) = 3.601, P = 0.0081; Treatment and time interaction effect: F (16, 132) = 0.4698, P = 0.9575; Fig. 4A]. Ordinary one-way ANOVA followed by Dunnett multiple comparisons test also indicated that the analgesia evoked by RS was profoundly inhibited by intra-NAc microinjection of maximum dose of Sulpiride (4 µg/0.5 µl) [F (3, 30) = 7.226, P < 0.001; Fig. 4B]. On the contrary, intra-accumbal microinjection of other concentrations of Sulpiride (0.25 and 1 µg/ 0.5 µl DMSO) had no influence on RS-produced antinociceptive responses. Additionally, no remarkable attenuation was detected between the AUC values obtained from the group that received the maximum concentration of Sulpiride (4 µg/0.5 µl) prior subjecting to the RS and animals that only received 4 µg/0.5 µl of Sulpiride without exposing to the RS.

The 50 % effective doses (ED50) of SCH23390 and Sulpiride in alteration of RS-evoked antinociceptive response
The ED50 values of SCH23390 and Sulpiride on analgesia produced by the RS were mathematically calculated by log dose-response curves of different doses of these drugs. As indicated in Fig. 5, the ED50 of SCH23390 (2.61 µg/ 0.5 µl saline) was lesser than that of Sulpiride (3.87 µg/ 0.5 µl DMSO). Thus, it seems that the role of D1-like dopamine receptor within the NAc in reducing analgesia evoked by RS is probably more prominent than that of D2-like dopamine receptor in this region. Moreover, the calculated effect size of the D1-like dopamine receptor antagonist (η2 = 0.67) by GraphPad Prism software was more than that of the D2 antagonist (η2 = 0.44).

The influence of intra-NAc microinjection of dopamine receptor antagonists on locomotor activity
The ordinary one-way ANOVA followed by Tukey multiple comparisons test demonstrated no considerable effect of SCH23390 or Sulpiride on distance travelled as a locomotion index during 60 min in comparison with the controls [F (4, 32) = 0.9057, P = 0.4741; Fig. 6]. As a result, the locomotor activity did not influence the experimental results.

Discussion
In the present research we investigated whether intra-NAc dopamine receptors are associated with restraint stress-produced antinociceptive responses in acute pain states. The current findings demonstrated that: (A) % MPE as a nociceptive item obtained from the tail-flick latencies at 5, 15, 30, 45, and 60 min following drug or vehicle administration. Repeated measures two-way ANOVA followed by the Bonferroni post-hoc test indicated that microinjection of Sulpiride at the maximum dose of 4 µg/0.5 µl DMSO within the NAc decreased the antinociceptive effect of RS during 60-min test. (B) The area under the curves (AUCs) analyzed for %MPEs. Each point indicates the mean ± SEM for 7-9 rats. * P < 0.05, * * P < 0.01, and * ** P < 0.001 compared to Vehicle + RS group. ns, Non-significant. 1) RS remarkably evoked analgesia in acute pain states; 2) Antinociceptive responses produced by RS were considerably diminished by blocking intra-NAc D1-or D2-like dopamine receptors, whereas in the absence of RS, microinjection of those antagonists into the NAc did not attenuate acute pain threshold; 3) The role of intra-NAc D1-like dopamine receptor in alteration of RS-evoked analgesia was more prominent than that of D2-like dopamine receptor.
Stress influences brain activity (Imbe et al., 2004;Suarez-Pereira et al., 2022) and exerts a complex modulatory impact on pain leading to either analgesia or hyperalgesia based on the features of stressors (Valenti et al., 2012;Ferdousi and Finn, 2018). We previously provided evidence that animals' exposure to a 6-min FSS period augmented tail-flick latency and antinociceptive reaction throughout 60 min of the tail-flick test (Noursadeghi et al., 2022).
Similar elevation in nociceptive threshold is observed in the present research. A single exposure to RS as a psychological stressor considerably enhanced pain threshold to the thermal stimuli and produced analgesia in acute pain states. Consistency, the experimental data of acute stress in chronic inflammatory pain demonstrated an enhancement of antinociceptive reaction following a single exposure to RS (Faramarzi et al., 2021). In contrast to different paradigms of acute stress, 6-h exposure to RS for 3 weeks produced hyperalgesia and increased reaction to noxious heat applied to the tail (Imbe et al., 2004).
Accordingly, RS and FSS induce both analgesia and hyperalgesia depending on some parameters such as period of exposure to aversive events. It is evident that mechanisms underpinning analgesia and hyperalgesia are both descending inhibitory and facilitatory pathways. Indeed, alterations in the balance between pain inhibiting and facilitating systems attribute to the gain of stress-induced nociceptive process. Hyperalgesia associated with chronic pain reveals a shift towards descending facilitation, whereas analgesia related to acute pain tends towards descending inhibition. Notably, stimulation of the descending pain pathway moderates nociceptive signal in the dorsal horn of the spinal cord, leading to either inhibition or facilitation (Heinricher et al., 2009).
Growing evidence pointed to considerable attribution of neuropeptides and neurotransmitters including dopamine to descending inhibitory pain system (Butler and Finn, 2009;Bravo et al., 2019;Dunn et al., 2019).
Upon the observations, stress influences mesolimbic dopamine concentration, however, the response of this effect varies according to a stressor and its intensity. (Cabib and Puglisi-Allegra, 2012;Holly and Miczek, 2016;Smith et al., 2019).
Further, some studies have demonstrated that rodents exposed to repeated unavoidable stressors experienced declined dopamine output in the NAc shell accompanied by a reduced activity of dopaminergic neurons (Mangiavacchi et al., 2001;Wood, 2004;Koiv et al., 2021).
Altogether, these lines of evidence propose that acute stress enhances dopaminergic tone within the NAc resulting in analgesia. By contrast, chronic stress decreases dopaminergic tone leading to hyperalgesia (Wood, 2004).
Given the importance of dopamine in stress-mediated analgesia, recent findings have shown the correlation of intra-NAc dopamine receptors with a significant reduction in antinoxious evoked by acute RS and FSS (Noursadeghi et al., 2022).
Our present data revealed that blocking either D1-or D2-like dopamine receptors within the NAc remarkably diminished and reversed the antinociceptive responses elicited by a single exposure to restraint stress during the tail-flick test. This outcome aligns with the previous result indicating the pronounced effects of both intra-NAc D1and D2-like dopamine receptors in reducing the noxious threshold produced by FSS exposure (Noursadeghi et al., 2022). Despite the profound influence of intra-NAc dopamine receptors in stress states, little research has been devoted to understanding how neuronal subtypes in this region are regulated following stressful condition. The NAc mainly constitutes two distinct subpopulations of GABAergic medium spiny neurons (MSNs), including D1 dopamine receptor and D2 dopamine receptor (Gerfen et al., 1990;Kupchik et al., 2015;Soares-Cunha et al., 2020). According to some observations, D1-MSNs project both directly to the output nuclei of the basal ganglia, and indirectly through the ventral pallidum (VP), while D2-MSNs convey their information exclusively indirectly via the VP (Kupchik et al., 2015;Soares-Cunha et al., 2020). Importantly, there are contradictory viewpoints about D1 and D2 dopamine receptor functions. A canonical understanding of striatal function is that stimulation of D1and D2-MSNs produces opposing effects. For example, D1-MSNs lead to reward, on the contrary, D2-MSNs result in aversion (Hikida et al., 2010;Lobo et al., 2010;Kravitz et al., 2012;). In this line, an animal study indicated that repeated RS dampened the effect of excitatory synapses on D1-MSNs expression in the NAc, but did not influence on D2-MSNs in the NAc core (Lim et al., 2012). Nonetheless, classical view has demonstrated that both MSN subpopulations may exert a concurrent action and convey positive or negative stimuli via different patterns (Soares-Cunha et al., 2020). For instance, they indicated that optogenetic stimulation of either D1-or D2-MSNs leads to rewards (Natsubori et al., 2017;Soares-Cunha et al., 2016. In support of this hypothesis, our recent findings exhibited the contribution of both D1and D2-like dopamine receptors in the NAc to attenuate analgesia elicited by acute RS in acute pain state, though the log dose-response curve pointed to a prominent role of D1 receptor antagonist. Considered together, little is known about underlying mechanisms of intra-NAc dopamine receptors in stress states, and further electrophysiological, cellular and molecular research according to various stressful conditions are needed to reveal certain contributions of D1-and D2-like dopamine receptors within the two distinct sub-regions, core and shell.
Overall, given the observed role of intra-NAc dopamine receptors in restraint stress-induced analgesia, these data suggest the possible involvement of dopaminergic system in various stress conditions.

CRediT authorship contribution statement
Abbas Haghparast was responsible for the study concept and design. Elham Noursadeghi acquired the behavioral data. Abbas Haghparast assisted with data analysis and interpretation of findings. Elham Noursadeghi drafted the manuscript. Abbas Haghparast provided critical revision of the manuscript for important intellectual content. All authors critically reviewed content and approved final version for publication.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Data will be made available on request. compared to control groups on distance traveled. According to ordinary oneway ANOVA, the locomotor activity was not affected by either the maximum dose of antagonists or vehicles. Each point indicates the mean ± SEM for 7-8 rats.