D1 and D2 neurons in medial nucleus accumbens participate in chronic orofacial pain phenotypes

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Chronic orofacial pain susceptible and resistant phenotypes might be regulated by D1- and D2-receptor–-expressing neurons in medial nucleus accumbens.

Abstract

Introduction:

Trigeminal nerve injuries can lead to different outcomes, such as chronic neuropathic pain. Nevertheless, it is still elusive why different phenotypes of chronic pain development are shown among individuals. Nucleus accumbens (NAc) might be important for chronic pain development. Still, the role of this structure and its neural subpopulations on these phenotypes development in the orofacial region remains unknown.

Objectives:

To evaluate the role of dopamine D1- and D2-receptor–expressing neurons (D1Rn and D2Rn) of medial NAc (mNAc) in developing phenotypes of chronic orofacial pain.

Methods:

C57BL/6 male and female mice underwent a trigeminal nerve compression injury and were assessed with the von Frey test for nociceptive threshold on day 3 and from weeks 1 to 14. To determine the role of mNAc D1Rn and D2Rn, transgenic D1R-Cre and D2R-Cre mice were infected with a genetically engineered caspase, causing specific ablation of such neurons.

Results:

All the injured C57BL/6 mice showed mechanical hypersensitivity during the first weeks. On week 2, mice were classified into high and low threshold (HT and LT). Most HT mice recovered from mechanical hypersensitivity, whereas most LT mice remained hypersensitive during all assessment weeks. Both mNAc D1Rn and D2Rn ablation decreased the percentage of HT mice, and D2Rn ablation increased the time of hypersensitivity recovery, suggesting these populations participate in both nociceptive threshold profiles and recovery capacity.

Conclusion:

mNAc D1Rn and D2Rn might play an important role in determining the phenotypes of chronic orofacial pain development after a nervous injury.

1. Introduction

Chronic neuropathic pain is a debilitating condition that decreases the quality of life of who suffers from it.¹⁴ Trigeminal injuries can have different outcomes, including but not limited to pain.¹³

Accordingly, individuals who develop pain after an event will recover from it, whereas in others, pain becomes chronic.²⁵ In chronic pain development, profiles or phenotypes can occur in both humans and rodents. Recently, it was demonstrated that after a sciatic nerve injury in rats, these would show 2 chronic pain development phenotypes: resistant and susceptible, characterized by their nociceptive threshold to mechanical stimulation.¹¹ After that, the same group found that this behavioral outcome was associated with different proteomic profiles in the nucleus accumbens.¹⁰

Nucleus accumbens (NAc) is one of the striatal complex central nervous system structures. Nucleus accumbens is anatomically divided in Core (NAcC) and, surrounding it, a Shell (NAcSh) portion. Despite this organization seems simple, it is involved in a broad diversity of functions. This can be attributed to the recent findings that have shown that NAc has an important topographical heterogeneity, not only limited to the mentioned portions but also presenting subdivisions, such as medial (mNac) and lateral (latNAc). Consequently, it has been demonstrated that these NAc subdivisions receive afferents and send projections to different brain regions, being implied in the processing of different events.^4,28

Although NAc has been classically associated with processes such as motivated behaviors and learning,²⁶ it has also been related to chronic pain development and determining pain trajectories. For instance, image studies have demonstrated that NAc is part of a circuitry in which changes can predict the transition to chronic pain.³

Despite all the reported studies regarding pain chronification and its phenotypes, it is still unknown whether this phenomenon occurs in the orofacial region. Furthermore, the neural population involved in whether an individual will be susceptible or resistant to chronic orofacial pain remains elusive. We consider this population to be NAc D1- and D2-receptor–expressing neurons (D1Rn, D2Rn). It has been shown that synaptic transmission is decreased for D2Rn in NAc after sciatic nerve injury.²³ Optogenetic modulation of such neural populations participates in the relief of pain induced by a neuropathic injury.²² In addition, it has been considered that NAc and its subdivisions are involved in the processing of hedonic and aversive information, depending on its topographical organization.⁴ In this sense, it has been found that the medial part of nucleus accumbens shell (mNAc) is associated with the induction of aversive behaviors,⁴ and that such region is activated when nociceptive stimuli are applied, independently of their intensity.¹⁸

Here, we aimed to evaluate the role of mNAc D1Rn and D2Rn in the development of chronic neuropathic orofacial pain phenotypes in mice. We investigated whether this phenomenon occurred for the orofacial region after a trigeminal injury, and evaluated the role of the above-mentioned neural populations through their ablation and its effect on mice's nociceptive threshold under the same conditions. We found that not only does the susceptibility/resistance phenomenon occur in the orofacial region but it is also modified by eliminating D1Rn or D2Rn, increasing the manifestation of the susceptible phenotype.

2. Methods

2.1. Animals

Male and female C57BL/6 wild-type adult mice (20–30 g) and D1-Cre and D2-Cre transgenic adult mice (20–30 g) were used in this study. Mice were housed in polycarbonate cages at 25°C in a 12-hour dark–light cycle (light: 8:00 am–8:00 pm); water and standard food were available ad libitum. Animal care and all procedures were conducted according to the Official Mexican Norm (NOM-062-ZOO-1999) and were approved by the University's Bioethics Committee. All efforts were made to reduce the number of mice used and minimize animal suffering. Sample size was estimated using the previous study by Leite-Almeida group.¹⁰ Animals were grouped through simple randomization using the MATLAB function rand(), and all the surgeries were performed by the same investigator.

2.2. Mental nerve compression injury model

Mental nerve compression (MnC) injury was used as a trigeminal neuropathic pain model. Mice were anesthetized with an intraperitoneal injection of a ketamine/xylazine solution (1.8 mL ketamine + 0.59 mL xylazine; 0.1 mL/10 g). An incision was made on the facial skin, on the edge of the jaw, at the level of the labial commissure. Mental nerve was exposed and compressed with fine forceps 2 times for 30 seconds, with an interval of 15 seconds, and the incision was sutured.¹ This procedure was performed bilaterally, according to previous literature protocols in other mental nerve injury models,⁹ and given to previous observations from our workgroup, where after unilateral injury, the contralateral stimulation evoked similar responses to the observed when ipsilateral stimulation was applied.¹⁷ Control group received no surgical procedures, and there were no sham interventions.

2.3. Orofacial mechanical pain test

To assess evoked responses to mechanical stimuli, the von Frey test was performed. Mice were placed individually in plastic cages and were acclimated for 20 minutes before testing. A series of von Frey filaments selected according to previous literature and compatible with the assessment of the areas innervated by the trigeminal nerve in mice (0.008, 0.02, 0.07, 0.16, 0.4, 1.0, 2.0, and 6.0 gf [grams-force])²¹ was used to measure mechanical nociceptive thresholds. The von Frey filaments were applied on the lower lip either from the right or left side from the lowest force and in the ascending order, and mice response was classified as positive or negative,^6,21 as summarized in Table 1. The nociceptive threshold was the filament force in which a positive response occurred in 3 of 5 stimuli.¹⁵ This classification criteria were established using previous works of orofacial pain assessment,^6,16,21 using both the response scoring and determining whether the responses were positive or negative. This experiment consisted of a baseline evaluation, 3 days and 1 to 14 weeks after MnC injury.

Table 1.

Classification criteria for responses to von Frey filaments.

Response	Behavior
Negative	No response
	Stimulus detection or slow turn away from the filament
	Slow or brief withdrawal that may or may not be followed by a single facial wipe
Positive	Attacking or biting the filament or rapid withdrawal followed by facial wipes
	A rapid withdrawal with multiple facial wipes

2.4. Specific neuronal ablation

To specifically eliminate D1Rn or D2Rn, a caspase 3 genetically modified enzyme was used.²⁷ Activation of such caspase induces apoptosis in a cell. Through stereotactic surgery, the adenoviral vector with the construct AAV-flex-taCasp3-TEVp was injected in the coordinates corresponding to mNAc in Cre-D1R or Cre-D2 mice. This way, apoptosis was induced on a specific neural population and restricted to the injection site, minimizing toxicity damage to adjacent neurons that did not express Cre. Once the injection was made, mice were left for 4 weeks to generate the neural ablation effect.

2.5. Stereotactic surgery

Mice were placed in a stereotactic frame under isoflurane anesthesia (3% induction, 1% maintenance). Scalp hair was removed, and the skull was exposed. Bregma was identified, and the coordinates corresponding to mNAc were placed anteroposterior (AP): +1.7 mm, mesio-lateral (ML): ± 0.6 mm, dorso-ventral (DV): −4.9 mm). The brain was exposed with a dental drill through bilateral clambering. The virus was delivered with a syringe (33 gauge, Hamilton), injecting 0.5 μL in each side. Previous studies in mNAc determined viral vector volume.⁵ To prevent leakage, the injection was made at a rate of 100 nL/min. The needle was left for 10 additional minutes and then withdrawn at 1 mm/min.²⁷ The incision was closed, and mice were allowed to recover for further von Frey test assessment before and after MnC injury.

2.6. Histology

To assess the effectiveness of neural ablation, immunohistochemistry for nuclear neuronal protein (NeuN) was performed. Mice were anesthetized with the previously described ketamine/xylazine solution and perfused with 4% paraformaldehyde. Brains were extracted, dehydrated, and paraffin-embedded. Brain tissues were cut at 4 μm with a microtome and mounted on slides. For immunohistochemical analysis, serial sections of tissue from control and experimental animal groups were stained using a NeuN kit (IHCeasy, Proteintech, Cat. KHC003, Rosemont, IL) following the manufacturer's guidelines. Slides underwent deparaffinization with xylene and graded alcohols, followed by antigen retrieval in a water bath at 97°C for 35 minutes. The primary antibody was applied for one hour at room temperature, followed by the application of the secondary antibody for 30 minutes. The reaction was developed with Diaminobenzidine (DAB) and counterstained using hematoxylin and eosin, ensuring thorough washing between each step. Specificity was confirmed by using the kit's blocking buffer instead of the primary antibody, which showed no signal.

2.6.1. Images acquisition and immunostained neurons analysis

Quantitative analysis was performed based on previous work.⁸ Representative sections from both experimental and control groups were selected for assessment. Nucleus accumbens was defined using the ×10 objective. The areas of interest were analyzed using images taken with a microscope (Olympus CX23Led), with the ×40 objective. Positivity profile was rated by counting the number of immunostained cells in the areas of interest.

After acquiring the microphotographs, right and left areas were evaluated in 4 sections each. The average number of positive cells from both sides was calculated to present the results. In addition, the percentage of positive area was recorded across 20 fields for each group. Images were processed using the Immunohistochemistry (IHC) plugin. After converting the images to binary and applying the Otsu filter, the positive area percentage was analyzed with watershed function.

2.7. Data analysis

Data are shown as the mean ± SEM. Statistical analyses were conducted with GraphPad Prism 9 software. Von Frey test assessment data are displayed normalized as the percentage of baseline. Raw data of the comparison between low threshold (LT), high threshold (HT), and control mice are also displayed (Figure S6, http://links.lww.com/PR9/A379). A 2-way ANOVA analysis followed by a Tukey post hoc test was performed to assess the nociceptive threshold differences between groups, and a square-chi test was conducted to evaluate the differences between susceptible and resistant proportions. For histological analysis, the analysis combined samples from both right and left sides, evaluating 20 fields for each group, whether experimental or control. A two-way ANOVA analysis was conducted, followed by a Tukey test. The level of statistical significance for all the tests was set at P < 0.05.

3. Results

3.1. Injured mice can be classified according to their threshold and recovery pattern

To evaluate the effect of MnC injury on the nociceptive threshold of C57BL/6 mice, the von Frey test was applied before and on day 3 and weeks 1 to 14 after the injury (Fig. 1A). Injured group nociceptive thresholds were compared with a control group, which received no intervention. A decrease in the nociceptive threshold in injured mice was observed as a sign of mechanical hypersensitivity, which was not observed in the control group (Fig. 1B). Nevertheless, through the weeks of evaluation, injured mice showed a tendency to increase their threshold and did not have statistically significant differences with the control group. Such phenomenon was explained because injured mice could be classified according to their nociceptive threshold on week 2. This was observed because they would separate in those whose threshold was lower than 0.07 gf, considered to be LT mice, and those whose threshold was equal or higher than 0.07 gf, considered HT mice (Fig. 1C). High threshold mice showed the tendency to increase their nociceptive threshold along the weeks, whereas LT mice remained hypersensitive. It was also found that the percentage of HT was higher than LT mice (67.5% and 32.5%, respectively) (Fig. 1D). We also assessed whether injured mice would recover their threshold baseline values and in which week of assessment that occurred, if the case, as a sign of recovery. We observed that 15.4% of LT mice recovered, whereas 84.6% of them did not. The opposite occurred for the HT group, where 74.1% recovered, and 25.9% did not (Fig. 1E). It was observed that recovered mice would reach such recovery mostly from weeks 3 to 4 after injury (Fig. 1F). To demonstrate that the differences between pain phenotypes were not due to differences in injury performance among mice, a pressure sensor was used to quantify the force applied by the forceps in multiple compressions, additional to mental nerve histological analysis. None of those evaluations showed significant differences; therefore, differences between pain phenotypes cannot be attributed to such factors (Figs. S1–S2, http://links.lww.com/PR9/A379).

Figure 1.

Injured mice can be classified according to their threshold and recovery pattern. (A). Experimental strategy. (B) Changes in nociceptive threshold in both control and injured mice. Nociceptive threshold changes are displayed as the difference in the baseline. (C) Classification of injured group in low and high threshold mice. The dashed horizontal line represents the mean of the control group's nociceptive threshold. (D) Proportions of low and high threshold mice. In C57BL/6 mice, the high threshold proportion is higher than the low threshold proportion. (E) Proportions of recovered and nonrecovered mice for both low and high threshold groups. Most LT mice did not recover their baseline threshold values, whereas most HT mice did. (F) Recovery week for low and high threshold mice. Control, n = 14; MnC, mental nerve compression, n = 40; LT, low threshold, HT, high threshold; *P < 0.05, MnC vs Control; #P < 0.05, LT vs HT.

The level of participation of mice in the von Frey test (“breakpoint”) was analyzed. Injured mice reach breakpoint at von Frey filaments that exert lower forces compared with their baseline and what was shown by the control group (Figs. 2A and B). For LT mice, it was observed that this occurred at lower forces after injury and remained that way until the last week of assessment (Fig. 2C). By contrast, by the same week, HT group breakpoint was similar to its baseline (Fig. 2D).

Figure 2.

“Breakpoint” according to mechanical stimulus intensity before and after surgery for the control group (A), injured mice before threshold classification (B), low threshold (C), and high threshold mice (D). HT, high threshold; LT, low threshold; MnC, mental nerve compression.

3.2. Medial nucleus accumbens D1Rn ablation prevents chronic orofacial pain-resistant phenotype

To determine the participation of mNAc D1Rn and D2Rn in the development of chronic orofacial pain phenotypes, a viral vector containing an engineered caspase was injected in the mNAc of transgenic mice. Figure 3 shows the injection site for mNAc for either D1-Cre or D2-Cre transgenic mice. Furtherly, MnC was performed. The mNAc D1Rn-ablated mice (D1Rn-) decreased their nociceptive threshold after MnC and remained decreased through all the weeks (Fig. 4A). When injured mice were classified as LT and HT, they did not show significant differences, suggesting that both groups remained hypersensitive. The percentage of LT mice, unlike C57BL/6 mice, was higher than the HT mice percentage (75% and 25%, respectively) (Figs. 4B and C). In addition, for HT mice, the percentage of recovered and nonrecovered mice was the same (Figs. 4D and E). When breakpoint was assessed, mice reached it at lower forces during all the 14 weeks, as a hypersensitivity sign (Fig. 5A), also observed for LT and HT mice (Figs. 5B and C).

Figure 3.

Injection site of viral vector containing a modified caspase to induce apoptosis in mNAc of either D1R neurons of D1-Cre mice or D2R neurons of D2-Cre mice. mNAc, medial nucleus accumbens.

Figure 4.

D1R neurons ablation in mNAc increases susceptibility to chronic pain. (A) Changes in nociceptive threshold in control and injured mNAc D1Rn-ablated mice. (B) Classification of injured group in low and high threshold mNAc D1Rn-ablated mice. The dashed horizontal line represents the mean of the control group's nociceptive threshold. (C) Proportions of low and high threshold mice. (D) Proportions of recovered and nonrecovered mice for both low and high threshold groups. (E) Recovery week for low and high threshold mNAc D1Rn-ablated mice. Control, n = 14; MnC, mental nerve compression, n = 12; LT, low threshold, HT, high threshold; *P < 0.05, MnC vs Control; #P < 0.05, LT vs HT. mNAc, medial nucleus accumbens.

Figure 5.

“Breakpoint” according to mechanical stimulus intensity before and after surgery for the mNAc D1Rn ablation group (A), low threshold (B), and high threshold mice (C). HT, high threshold; LT, low threshold; MnC, mental nerve compression; mNAc, medial nucleus accumbens.

3.3. Medial nucleus accumbens D2Rn ablation increases chronic orofacial pain susceptibility

When mNAc D2Rn were ablated, the nociceptive threshold decreased after MnC and remained low during all the weeks (Figs. 6A and B). After classifying D2Rn-ablated mice (D2Rn-) in LT and HT, although there were differences between each other, the percentage of LT mice was higher than HT mice (60.9% and 39.1%, respectively) (Fig. 6C). In addition, for both LT and HT D2Rn- group, the percentage of recovered mice decreased, unlike C57BL/6 mice (Fig. 6D). Recovery week for HT mice did not show a pattern and seemed to be scattered throughout the assessment (Fig. 6E). Breakpoint analysis showed that D2Rn- mice also reached it at lower forces during all the 14 weeks (Fig. 7A).

Figure 6.

D2R neurons ablation in mNAc increases susceptibility to chronic pain. (A) Changes in nociceptive threshold in control and injured mNAc D2Rn-ablated mice. (B) Classification of injured group in low and high threshold mNAc D2Rn-ablated mice. The dashed horizontal line represents the mean of the control group's nociceptive threshold. (C) Proportions of low and high threshold mice. (D) Proportions of recovered and nonrecovered mice for both low and high threshold groups. (E) Recovery week for low and high threshold mNAc D2Rn-ablated mice. Control, n = 14; MnC, mental nerve compression, n = 23; LT, low threshold, HT, high threshold; *P < 0.05, MnC vs Control; #P < 0.05, LT vs HT. mNAc, medial nucleus accumbens.

Figure 7.

“Breakpoint” according to mechanical stimulus intensity before and after surgery for the mNAc D2Rn ablation group (A), low threshold (B), and high threshold mice (C). HT, high threshold; LT, low threshold; MnC, mental nerve compression; mNAc, medial nucleus accumbens.

For LT mice, breakpoint occurred at lower forces after injury and remained that way until the last week of assessment, whereas by the same week, HT group's breakpoint was like its baseline. Low threshold group reached its breakpoint at low forces until week 14 (Fig. 7B), and HT group's breakpoint was like its baseline by the same week (Fig. 7C).

3.4. Effectiveness of D1Rn and D2Rn ablation in medial nucleus accumbens

NeuN-positive density profiles (number of positive cells in areas of interest) were calculated through the area of interest. Control group showed a mean of 115.8 NeuN + neurons in 209.7 μm² against 2 and 0 positive cells for the nonrecovery and recovery groups (Table 2).

Table 2.

Number of positivity neuronal protein cells in each group.

Group	Average num. cells	Positive cells
Control	250.75	115.8
Nonrecovered	424.35	2
Recovered	167.95	0

Area 2.0165 × 104 µm²/field, results presented as the mean of the areas examined, n = 20 fields per group examined.

NeuN expression in antibody-positive cells was localized in the nucleus and, in some occasions, in the cytoplasm. Positive neurons showed a morphology consistent with postmitotic and differentiated cells (Figs. 8A–F).

Figure 8.

Representation of the immunohistochemical analysis of mouse tissues stained with the NeuN antibody. (A) A sample from the control group is shown at low magnification in a square, indicating the area examined. Individual cells in this sample exhibit positivity for the NeuN antibody. (B) At a higher magnification, the same sample shows positive cells, with reactivity localized in the cytoplasm and nuclei of the cells. (C) Results from the nonrecovered group at low magnification are shown; this sample displays a higher number of cells, with some cells demonstrating slight positivity. (D) The same sample at higher magnification revealed the presence of slight positivity localized in the nucleus and cytoplasm (white circle). The hematoxylin stain highlighted densely stained cells with enlarged, hyperchromatic nuclei (red circles) and cells with pyramidal projections (green circles). (E) Represents a sample from the recovered mice group. (F) At a higher magnification, the examination reveals numerous densely stained cells with enlarged and hyperchromatic nuclei that also lack antibody expression (red circle). This sample shows a greater number of cells per area, with cells exhibiting large pyramidal projections (blue circle) and features consistent with mitotic figures (purple circle). Finally, image (G) provides a graphical representation of the positive area percentage analysis, highlighting significant differences in NeuN antibody expression between the control and nonrecovered and recovered groups, with P-values less than 0.0001. A total of 20 fields were examined per condition. Statistical analysis using ANOVA revealed a treatment effect across experimental groups, with F (2, 57) = 498.9. Magnifications for images (A), (C), and (E) are at ×20 with a scale bar of 100 µm, while magnifications for (B), (D), and (F) are at ×40 with a scale bar of 50 µm. NeuN, neuronal protein.

The positive area percentages confirmed that the control group shows a higher positivity percentage (85.42% ± 7.3) compared with the nonrecovery group (11.3% + −7.3) and with the recovery group (3.89% ± 6.8). These differences were statistically significant (P = 0.0001) (Fig. 8G). These results suggest that the viral injection reduced the number of NeuN + neurons in the brain tissue.

4. Discussion

Here, we found that (1) susceptibility and resistance phenotypes of chronic pain development occur at the orofacial level after a trigeminal nerve injury, through the existence of low and high nociceptive thresholds, and different recovery patterns; (2) when D1Rn in mNAc were ablated, the occurrence of resistance phenotype decreased and most mice remained hypersensitive; and (3) ablation of D2Rn in mNAc also exacerbates mechanical hypersensitivity after trigeminal injury, increases the percentage of susceptible mice, and modifies their recovery patterns. Altogether, these results suggest that mNAc D1Rn and D2Rn participate in the development of chronic neuropathic orofacial pain phenotypes.

To determine whether chronic pain phenotypes occur in the orofacial region, we first performed MnC injury as a trigeminal neuropathic pain model. Initially, we found that C57BL/6 injured mice seemed to recover over the weeks. This was explained by the fact that mice could be classified according to their threshold, which was high or low, additional to whether they would recover their baseline nociceptive threshold values. Sham groups were not used in this study, given the possibility of inducing hypersensitivity due to the surgical process.¹⁹ High threshold and LT classification, as well as susceptible and resistant phenotypes for neuropathic pain, have been previously described¹⁰ in rodents after sciatic nerve injury, but, to the best of our knowledge, this is the first time these phenotypes are described for the orofacial region. All the injured mice were classified on week 2. This week was chosen because it has been reported that plastic changes in mNAc derived from sciatic nerve injury occur from 9 to 12 days after it,²³ and the study conducted by Guimaraes described before, where they observed that the HT and LT groups began to separate around week 2.

In addition, we consider that the differences found between our groups are not attributable to distinct forces applied when performing the compressions. We corroborated this through a pressure sensor, which showed no differences between compressions (Fig. S1, http://links.lww.com/PR9/A379), as well as the histological analysis of mental nerves, dissected after the 14 weeks (Fig. S2, http://links.lww.com/PR9/A379). We consider that the susceptibility/resistance phenomenon is not (at least not completely) peripheral given that, when mental nerves were dissected, we could still observe tissue damage patterns in all the injured mice, regardless of the group they belonged to.

This phenomenon induced by mental nerve compression occurs unlike reported for other orofacial pain models, where hypersensitivity remains through all the experimentation sessions. This suggests that susceptibility and resistance to chronic orofacial pain phenotypes involve both the mice's nociceptive threshold and their recovery capacity and pattern. Therefore, we consider MnC injury an adequate model for studying chronic orofacial pain phenotypes, not only because it reproduces reliably this phenomenon but also because it is a small and little-invasive procedure, yet effective for observing differences among subjects.

Despite that NAc is mostly studied in their hedonic and aversive stimuli processing, and learning events,²⁶ it has been reported that it also participates in pain processing and its chronification.¹² Through image studies, there have been reported differences in NAc of patients who experienced chronic pain and found that changes in this structure could predict the transition to chronic pain.^2,3 Regarding experimental studies, Guimaraes found in the aforementioned study that there are changes in proteomic profiles of NAc, associating these changes to the occurrence of chronic pain phenotypes after sciatic nerve injury.¹⁰ Nevertheless, these studies did not address the neural population involved. We hypothesized that D1R and D2R neurons would be involved due to former studies demonstrating their participation in different models of chronic pain. We found that both D1Rn and D2Rn elimination facilitates the occurrence of susceptible phenotype by exacerbating hypersensitivity in injured mice. This result differs from some studies in these 2 populations, which have contrary effects, such as what has recently been reported, where NAc D1Rn optogenetic activation produces the relief of thermal hypersensitivity after sciatic nerve injury. By contrast, the same effect is achieved after silencing D2Rn in the same region.²²

In our study, ablation of both D1Rn and D2Rn produced increased manifestations of susceptible phenotype (Fig. S3, http://links.lww.com/PR9/A379), but they might do it through different means. According to our results, D1Rn might participate in generating of distinct thresholds, and D2Rn might regulate the recovery capacity of the mice. Yet, further investigation is needed to assess these specific mechanisms.

These results are consistent with recent studies that suggest that D1R and D2R neurons do not perform opposite functions. It has been proposed that these populations work similarly in the processing of both hedonic and aversive events,⁷ and that the functional heterogeneity of NAc is not given by its populations but by its topographical organization.⁴

For the specific ablation of mNAc D1Rn and D2Rn, we injected a Cre-dependent viral construct for caspase 3. Our results indicate that such injection significantly reduced the number of neurons in NAc in D1 and D2 Cre + mice. D1Rn ablation was associated with a decrease in the pain-resistant phenotype mice, resulted from a higher LT mice proportion and a lower capacity of recovery from HT mice; meanwhile, D2Rn ablation increased the susceptibility to develop chronic orofacial pain, as indicated by a higher percentage of LT mice and a lower capacity for recovery in both LT and HT. Although previous studies have confirmed the sufficiency of caspase-mediated neural ablation to induce behavioral changes, the similarity in the results obtained from D1Rn or D2Rn ablation might be explained by the idea that the NAc-mediated behavioral response could be dependent on the pattern of neural activity and not on the specific recruited subpopulation.^24,27,29 To further dissect the role of NAc in the pattern of orofacial neuropathic pain recovery, chemogenetic and optogenetic manipulations of both neural populations might be useful, and choosing pivotal moments in the process of recovery.

As in Guimaraes' experiment, where they used 72 rats, we used a large group of mice to ensure the proportions were unbiased. We also showed that if we randomly take 10 from injured mice, the proportion of recovered and nonrecovered mice is maintained (Fig. S4, http://links.lww.com/PR9/A379). We also separated the mice into many subgroups. Although it was not the purpose of this study, we even divided them into females and males to better understand the susceptibility phenomenon and its dependence on sex, as it has been suggested.²⁰ Our results showed no difference in pain response regarding the phase of the estrous cycle (Fig. S5, http://links.lww.com/PR9/A379). Nevertheless, given the number of females included in these experiments, further analysis and experiments should be conducted to obtain conclusive results.

It would also be interesting to test whether the acute inactivation of such neural populations could affect the observed phenotypes with the viral injection. This could be approached with different methods to evaluate different times of inactivation, such as a pharmacological blockade of D1 and D2 receptors, as well as chemogenetic and optogenetic inactivation. Regarding optogenetic modulation, it has been documented that the transitory stimulation of NAc D1Rn and the suppression of D2Rn of the same region would increase the signs of neuropathic pain induced by a sciatic nerve injury.²²

Chronic orofacial pain is an important problem worldwide. Although this work does not have immediate clinical implications, understanding at a deeper level the neural mechanisms that underlie the transition from acute to chronic pain might set the basis for the improvement of its clinical management. Identifying the factors that determine whether an individual develops chronic pain can be a critical step not only to treat but also to predict or even prevent its development, as well as to propose alternatives for chronic pain treatment.

Disclosures

The authors of this work certify that they have no affiliations with or involvement in any organization or entity with any financial interest or nonfinancial interest in the subject matter or materials discussed in this article.

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