Dopamine receptor D2,but not D1,mediates the reward circuit from the ventral tegmental area to the central amygdala,which is involved in pain relief

Animals

Male wild-type C57BL/6 mice (8–10 weeks) and DAT-Cre bacterial artificial chromosome transgenic mice (8–10 weeks) were used in this study. Dopamine transporter-Cre mice were purchased from Shanghai NanFang Model Biotechnology Co. The Cre mice were on the C57BL/6 genetic background. The mice were housed under standard conditions on a 12-hour light-dark cycle with ad libitum access to water and food pellets. All behavioral tests were conducted by an investigator blinded to the treatment groups. All experiments were conducted in accordance with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain.

Animal model of plantar incision pain

All mice were anesthetized with 2% halothane delivered via a nose cone. A 0.5-cm longitudinal incision was made in the skin and fascia of the left plantar aspect of the foot starting 0.5 cm from the proximal edge of the heel and toward the toes with a number 11 blade. In most animals, the plantaris muscle was elevated and incised longitudinally. Sham-operated animals were anesthetized, and the left hind paw was cleaned, but no incision was made. Peripheral nerve block was achieved by injection of 100 μL of lidocaine or saline (1%, wt/vol) into the PF under isoflurane anesthesia. The muscle origin and insertion were kept intact. After hemostasis with gentle pressure, the skin was closed with 2 5-0 nylon sutures and an FS-2 needle. The wound site was covered with neomycin, and the animals were allowed to recover in their cages.

Nociception test

Mechanical allodynia was assayed using von Frey filaments as described by Chaplan et al. ¹⁰ The experiment was performed by two authors who were blinded to the experimental treatments. The mice were placed on wire mesh platforms in clear cylindrical plastic enclosures, and filaments were applied to the center of the plantar surface of the unincised hind paw or to the wound edge of the incised hind paw. Withdrawal of the hind paw from the floor was scored as a response. When no response was obtained, the filament of the next highest force in the series was applied to the same paw; if a response was obtained, the with the next lowest force was applied. The paw withdrawal threshold was estimated based on this testing protocol. The “up-down” approach was used to calculate mechanical sensitivity. ¹⁰ The mechanical pain threshold of the mice was measured before light stimulation, and then a blue light (465 nm) with a frequency of 20 Hz and intensity of 10 mW was applied for 2 minutes, and turned off for 2 minutes. Afterwards, the mechanical pain threshold was measured again.

The thermal paw withdrawal latency (PWL) was determined using the plantar test (Ugo Basile, Varese, Italy). Each mouse was placed on a transparent glass plate over a radiant heat generator for 30 min for acclimatization. The intensity of the light source was adjusted so that normal animals exhibited a retraction latency of 12–15 s, and a cut-off time was set in advance to prevent tissue damage. The latency to withdrawal from the heat stimulus was measured three times at an interval of ≥5 min, and the average latency in the three tests was calculated as the PWL.

Conditioned place preference (CPP)

A single-trial conditioning protocol was used to assess CPP as previously described.^7,11 All mice were handled by the experimenter before the preconditioning phase. On the preconditioning day, the mice were placed into the CPP boxes and given access to all chambers; the time spent in each chamber over 15 minutes was determined by an automated system to verify that the mice did not show a chamber preference during preconditioning. Following preconditioning, the mice underwent incision or a sham procedure and were placed in their home cages overnight. On the conditioning day (24 h postincision), the mice received saline (100 μL) injection into the PF and were immediately (within 2 min) placed in the appropriate pairing chamber. Four hours later, the mice received lidocaine injection (1%, wt/vol; 100 μL) into the PF and were placed in the opposite chamber. Chamber pairings were counterbalanced. On the test day, 20 h following the afternoon pairing, the mice were placed in the CPP box and given access to all chambers, and their behavior was recorded for 15 min for analysis of chamber preference.

Open field test (OFT)

The open field test (OFT), a classical test for measuring anxiety-like behaviors in rodents, ¹² was conducted in an illuminated chamber (45*45*30 cm) divided into a central zone and an outer zone. In the real-time OFT, the mice were allowed to explore freely for 10 min, and the locomotor activity of each animal in the two zones was video-recorded and analyzed with an automated video-tracking system (EthoVision XT, Noldus Information Technology). The 10 min of the trial was divided into two consecutive 5 min periods: the light was kept off in the first period (control), while light stimulation was applied during the second 5 min period. A reduced time spent in the unprotected central zone (central time) was regarded as an indicator of anxiety-related behavior. The total distance traveled in the entire chamber during the test was recorded and used as a measure of general locomotor activity.

Elevated plus maze (EPM) test

The EPM consisted of a central platform (6 × 6 cm²), two closed arms (30 × 6 × 20 cm³) and two opposing open arms (30 × 6 cm²). The maze was placed 100 cm above the floor. A mouse was placed in the central platform facing a closed arm and was allowed to explore the maze for 5 min. The time spent in the open arms and the number of entries into the open arms were analyzed using Smart3.0 software. The area was cleaned between tests using 75% ethanol. The percentage of time spent in the open arms and the percentage of time spent in the open arms were measured to assess anxiety-like behaviors.

Retrograde tracing

Mice were anesthetized with 2% isoflurane and placed in a stereotaxic frame. Then, 0.2 µl of the retrograde tracer fluorogold (FG) (4%; Thermo Fisher) was injected into the CeA (AP: 1.22 mm; ML: 2.8 mm; DV: 4.8 mm). ¹³ as described previously. ¹⁴ The needle was left in place for an additional 10 minutes before withdrawn from the brain. At 7 days after injection, the mice were deeply anesthetized with isoflurane and perfused with 4% paraformaldehyde for imaging or immunohistochemistry.

Drug administration

A D2 receptor antagonist (1 mg/mL; Sigma) was injected with a 30-gauge needle into the CeA (AP: 1.22 mm; ML: 2.8 mm; DV: 4.8 mm) to regulate the activity of D2 receptor. The injection volume was 1 µL.

Optical stimulation

Four weeks after virus injection, a fiber optic cannula (Doric Lenses) was stereotaxically implanted in the brains of anesthetized mice just above the CeA (AP: 1.22 mm; ML: 2.8 mm; DV: 4.8 mm). After implantation, the animals were singly housed and allowed to recover for 7 d before behavioral tests. For optical stimulation, we used a power supply (Plexon 08-06-A-37) with an output power of 10 mW and a light wavelength of 465 nm. The laser properties were modulated by a waveform generator (Plexon Inc.; 6500 Greenville Avenue, Suite 700, USA) that allowed us to control the frequency, pulse square, and pulse width. Blue light pulses of 20 Hz, 15 ms, and 10 mW were delivered, and the laser intensity was set at 10 mW, with continuous light exposure for optical stimulation. Optical modulation was performed prior to (pre), during (laser-on), and after (post) optical stimulation for 5 minutes each.

Stereotaxic surgeries

All mice were anesthetized with 1%–3% isoflurane and placed in a stereotaxic frame (David Kopf Instruments). Viruses (AAV8-EF1a-DIO-ChR2-mCherry; OBIO Technology; Shanghai) (0.5 µL) were injected into the VTA (AP: 3.1 mm; ML: 0.8 mm; DV: 4.25 mm) with a 1 µL Hamilton syringe at a speed of 0.05 µL/min. The needle was left in place for an additional 10 min and then slowly withdrawn. Immunohistochemistry was used to confirm that the stereotaxically injected viruses adequately transduced the VTA without significant spread to other regions. Optical fibers (200 μm inner diameter, N.A. 0.66; Doric Lenses) were implanted into the CeA in all mice. All injections and implantation procedures were performed in 7- to 8-week-old male DAT-cre animals.

Western blotting

Mouse brain tissues were fully ground in lysis buffer A (10 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl₂, 5 mM EGTA, 1 mM DTT, 100 mM NP-40, 40 mM leupeptin, and 250 mM sucrose) and stored on ice for 30 min before centrifugation at 4°C and 1000 × g for 15 min. The protein concentration was then measured using a bicinchoninic acid protein assay (Solarbio, Beijing, China). Samples (30 μg total protein) were mixed with protein loading buffer (Solarbio, Beijing, China) and heated at 99°C for 5 min before being loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels (CWBIO, China) for electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes (Immobilon®-P, Merck KGaA, Darmstadt, Germany) at 30 V and 4°C overnight. After being blocked with 3% bovine serum albumin (BSA) (Solarbio, Beijing, China) and incubated overnight at 4°C with primary antibodies against D1R (1:1000; catalog No. MAB5290, Millipore), D2R (1:1000 catalog No. AB5084P, Millipore), and GAPDH (catalog No. RM2002 L, Beijing Ray), the membranes were washed six times for 10 min each in Tris-buffered saline and 1% Tween. The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:1000, Jackson laboratories) for 2 h at room temperature. The signals were visualized after processing with an enhanced chemiluminescence (ECL) prime kit and FluorChem E (AlphaImager ProteinSimple, San Jose, CA, USA). The average band density was determined using ImageJ software.

Immunohistochemistry

Following perfusion, VTA- or CeA-containing brain tissues were cut at a thickness of 25 µm with a cryostat (CM1950, Leica, Chicago, IL). Free-floating slices were blocked in 10% normal goat serum for 2 h followed by incubation with primary antibodies overnight at 4°C. Next, the slices were washed and placed in a corresponding Alexa Fluor 488- or Cy3-conjugated secondary antibody for 2 h at 37°C. Immunofluorescence images were taken and analyzed under a Leica fluorescence microscope (DMi8, Leica). The information for all the antibodies used in this study is listed below.

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Antibody	Dilution	Vendor	Catalog number
Anti-D1 receptor	1:200	Millipore	MAB5290
Anti-D2 receptor	1:200	Millipore	AB5084P
Anti-glutaminase	1:300	Abcam	Ab131554
Anti-GABA	1:250	Sigma	A2052
Anti-tyrosine hydroxylase (TH)	1:400	ABclonal	A12756
Cy3-conjugated mouse anti-rabbit	1:200	Jackson Immuno	211-165-109
Alexa Fluor 488-conjugated donkey anti-mouse	1:200	Jackson Immuno	715-545-150
Anti-GAPDH	1:1000	Beijing Ray	RM2002 L

Statistical analysis

All data were analyzed using Prism software, version 9.0 (GraphPad, La Jolla, CA). The data are presented as the means ± SEMs. One-way analyses of variance (ANOVA) followed by Tukey’s tests for multiple comparisons was used to compare the Western blot results among three or more groups. Two-way ANOVA followed by Bonferroni’s multiple comparison test was used to analyze the behavioral data. Student’s t tests were used for comparisons between two groups. Main and interaction effects were considered significant at p < 0.05.

Experimental plantar incision produced significant mechanical allodynia, thermal hyperalgesia and anxiety–like behaviors in mice

Our study showed that compared with those in the sham group, mice that underwent incision exhibited significant mechanical allodynia and thermal hyperalgesia. Compared with those in the sham group, mice in the incision group displayed a significant reduction in the mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) from 6 h to 3 d after surgery (Figure 1(a), (c)). There was no significant between-group difference in the PWT and PWL on the contralateral side (Figure 1(b), (d)).OPEN IN VIEWER

Pain, as a strong stressor, is known to cause comorbid emotional disorders, including anxiety, stress, and depression in pain patients.^15–17 Our study showed that hind paw incision induced anxiety-like behaviors, as determined by the OFT and EPM test. Compared with the sham group, the incision group showed a significant reduction in the percentage of time spent in the center zone in the OFT (Figure 1(e)) and a significant reduction in the percentage of OA entries and time spent in the OAs in the EPM test (Figure 1(g), (h)). No significant difference in total travel distance in the OFT was observed between the incision group and the sham operation (Figure 1(f)). These results indicated that mice exhibited obvious sensory pain symptoms and negative emotions after surgical incisions.

Pain relief elicited CPP and activated VTA neurons

The sensation of pain generates an aversive state that demands a behavioral response (for pain, a motivation to seek relief). In contrast, relief of pain and return to homeostatic balance are rewarding.^3,18,19 The specific brain circuits that process pain relief, however, remain elusive. As demonstrated in Figure 1, evoked pain hypersensitivity was still obvious at 6 h. Peripheral nerve block (PNB) with popliteal fossa (PF) lidocaine injection 6 h postincision resulted in a strong preference for the chamber paired with PNB, indicating pain relief. In contrast, in sham-operated animals, CPP for the PNB-paired chamber was not observed (Figure 2(b)). PNB on the side contralateral to the injured hind paw did not result in CPP, confirming that lidocaine at the dose injected into the PF injection did not induce systemic pain relief, as demonstrated previously. ²⁰ These results showed that relief of ongoing incisional pain produced CPP and pain relief-elicited reward.OPEN IN VIEWER

Emerging preclinical data have shown that offset of an acute noxious stimulus activates brain regions, particularly the ventral tegmental area (VTA)^21,22 Activation of brain regions in injured mice following PNB was investigated using immunohistochemistry for FOS, a marker of neuronal activity. The results showed that PNB significantly increased the number of Fos-positive cells in the VTA in injured mice. Incision injury itself or PNB in sham mice did not change FOS expression (Figure 2(c)–(e)). The results indicated that activation of VTA neurons mediated the reward of pain relief. However, the c-Fos + cell count in the NAc or amygdala did not change significantly after pain relief although findings have demonstrated that VTA-NAc circuit participated in pain relief.^23,24

DAergic projections from the VTA to the CeA in mice

To identify VTA projections to the CeA, the retrograde tracer FG was injected into the CeA. This resulted in retrograde labeling of many CeA-projecting neurons in the VTA (Figure 3(a)). These projections were bilateral, with obvious predominance on the ipsilateral side.OPEN IN VIEWER

To determine whether the observed retrogradely labeled neurons in the VTA were catecholaminergic (dopaminergic), we performed immunohistochemistry for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis and a marker of dopaminergic neurons. Our double staining data showed that CeA-projecting neurons in the VTA were dopaminergic (Figure 3(b)), suggesting that the CeA received projections from the VTA and may have been under the control of dopamine.

To characterize the functional connections of the VTA-CeA circuit, AAV8-EF1α-DIO-ChR2-mCherry was injected into the VTA of DAT-Cre mice (Figure 3(c)). Our double staining data showed that ChR2-mCherry-labeled neurons in the VTA were positive for TH, suggesting that the virus successfully transfected local dopaminergic neurons through the DAT promoter (Figure 3(c)). Four weeks later, we examined mCherry expression in the CeA. Abundant mCherry-labeled fibers were observed in the CeA (Figure 3(d)). These results illustrated the existence of strong, target-specific dopaminergic projections from the VTA to the CeA, suggesting that the VTA-CeA circuit might contribute to pain relief.

Activation of the VTA–CeA neural pathway relieved incisional pain

To explore the potential role of dopaminergic projection from the VTA to the CeA in incisional pain, we used an optogenetic approach and DAT-Cre mice injected with AAV8-EF1α-DIO-ChR2-mCherry (AAV-ChR2) or the control AAV8-EF1α-DIO-mCherry (AAV-mCherry) into the VTA. During specific activation of the CeA-projecting fibers and the terminals originating from the VTA, the mechanical thresholds of the bilateral hind paws were tested, and behavior in the open field test and CPP test were assessed (Figure 4(a)).OPEN IN VIEWER

We found that specific excitation of the excitatory opsin ChR2 in dopaminergic projections from the VTA to the CeA with light significantly increased the mechanical threshold of the ipsilateral hind paw in incised mice. Injection of AAV-ChR2 during blue light stimulation exerted obvious analgesic effects on the ipsilateral hind paw in incised mice (Figure 4(b)). In incised mice infected with AAV-mCherry, light stimulation had no effects on the mechanical threshold of the ipsilateral hind paw. Optogenetic stimulation had no effect on the mechanical threshold of the contralateral hind paw in either the AAV-ChR2-injected group or the AAV-mCherry-injected group (Figure 4(b)).

The percentage of time that AAV-ChR2-injected incised mice spent in the center zone in the OFT was significantly increased during light stimulation. However, optogenetic stimulation had no obvious effect on the total distance traveled in either the AAV-ChR2-injected group or the AAV-mCherry-injected group (Figure 4(c)).

According to the CPP test, the time spent in the chamber paired with light stimulation was significantly increased in the AAV-ChR2-injected group, whereas the time spent in the chamber paired with light stimulation was not significantly changed in the AAV-mCherry-injected group (Figure 4(d)). This directly demonstrated that activation of the VTA–CeA pathway contributed to reward seeking resulting from pain relief.

These results of the optogenetic experiments suggested that specific activation of the VTA–CeA neural pathway produced analgesic (pain relief) and anxiolytic effects in incised mice. The VTA–CeA neural pathway was involved in the modulation of nociception and comorbid emotional behavior deficits.

Pain relief decreased dopaminergic D2 receptor expression in injured mice

The above results provide functional evidence that specific activation of VTA-projecting dopaminergic neurons in the CeA via stimulation of fibers from the VTA produce analgesic and anxiolytic effects in incised mice. However, the potential underlying mechanism remains unknown.

The expression of the D1 receptor and D2 receptor in the CeA was assessed by using Western blotting. The expression of D2 receptor was significantly decreased in the CeA in incised mice treated with PNB compared with incised mice treated with saline. However, there was no significant difference in dopamine D1 receptor expression between groups (Figure 5(a)).OPEN IN VIEWER

Furthermore, we examined the neuronal subtypes expressing the two dopamine receptors using double immunofluorescence. Glutaminase and GABA (γ-aminobutyric acid) are markers of glutamatergic excitatory neurons and GABAergic inhibitory neurons, respectively. We observed that in the CeA, D1R-positive neurons colabeled with glutaminase were more abundant than those colabeled with GABA, that D2R-positive neurons were primarily colabeled with GABA, and that only a few D2R-positive neurons were colabeled with glutaminase. Our data indicated that most D1 receptors were expressed in excitatory neurons in the CeA but that D2 receptor was predominantly expressed in inhibitory neurons in the CeA (Figure 5(b)).

D2 receptor in the CeA is involved in pain relief

According to the above results, we found that specific activation of CeA-projecting dopaminergic neurons in the VTA produced obvious analgesic effects. The expression of D2 receptor in the CeA was decreased after pain relief, and the expression of D1 receptor did not change significantly. To assess the role of D2 receptor in the VTA-CeA pathway, we combined optogenetic approaches with behavioral pharmacologic manipulations. Before specifically activating the VTA-CeA pathway, a D2 receptor agonist (quinpirole, 5 µg/µl) was injected intraperitoneally, and then mechanical threshold measurements, the OFT, and the CPP test were performed.

The results showed that compared with DMSO injection, quinpirole injection significantly reversed the pain relief caused by activation of the VTA–CeA neural pathway in incised mice. The mechanical threshold of the ipsilateral hind paw during light stimulation was significantly decreased in the quinpirole injection group compared with the DMSO injection group (Figure 6(a)). The percentage of time spent in the central area in the OFT during light stimulation was significantly decreased by quinpirole injection (Figure 6(b)). However, quinpirole injection had no obvious influence on the mechanical threshold of the ipsilateral hind paw or the percentage of time spent in the central area in the OFT in the absence of no light stimulation.OPEN IN VIEWER

In the CPP test, the time spent in the chamber paired with quinpirole injection was significantly reduced compared with that spent in the chamber paired with DMSO injection following light stimulation (Figure 6(c)). There was no significant difference between the quinpirole injection group and the DMSO injection group in the absence of light stimulation. These results demonstrated that administration of a D2 receptor agonist reversed the pain relief caused by light-induced activation of the VTA-CeA pathway. Dopamine receptor D2, but not D1, mediates the circuit from the ventral tegmental area to the central amygdala, which is involved in pain relief.