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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Neuropharmacology. 2020 Jan 7;166:107935. doi: 10.1016/j.neuropharm.2020.107935

Attenuated Dopamine Receptor Signaling in Nucleus Accumbens Core in a Rat Model of Chemically-Induced Neuropathy

Dana E Selley 1,*, Matthew F Lazenka 1, Laura J Sim-Selley 1, Julie R Secor McVoy 1, David N Potter 2, Elena H Chartoff 2, William A Carlezon Jr 2, S Stevens Negus 1
PMCID: PMC7654441  NIHMSID: NIHMS1603128  PMID: 31917153

Abstract

Neuropathy is major source of chronic pain that can be caused by mechanically or chemically induced nerve injury. Intraplantar formalin injection produces local necrosis over a two-week period and has been used to model neuropathy in rats. To determine whether neuropathy alters dopamine (DA) receptor responsiveness in mesolimbic brain regions, we examined dopamine D1-like and D2-like receptor (D1/2R) signaling and expression in male rats 14 days after bilateral intraplantar formalin injections into both rear paws. D2R-mediated G-protein activation and expression of the D2R long, but not short, isoform were reduced in nucleus accumbens (NAc) core, but not in NAc shell, caudate-putamen or ventral tegmental area of formalin- compared to saline-treated rats. In addition, D1R-stimulated adenylyl cyclase activity was also reduced in NAc core, but not in NAc shell or prefrontal cortex, of formalin-treated rats, whereas D1R expression was unaffected. Other proteins involved in dopamine neurotransmission, including dopamine uptake transporter and tyrosine hydroxylase, were unaffected by formalin treatment. In behavioral tests, the potency of a D2R agonist to suppress intracranial self-stimulation (ICSS) was decreased in formalin-treated rats, whereas D1R agonist effects were not altered. The combination of reduced D2R expression and signaling in NAc core with reduced suppression of ICSS responding by a D2R agonist suggest a reduction in D2 autoreceptor function. Altogether, these results indicate that intraplantar formalin produces attenuation of highly specific DA receptor signaling processes in NAc core of male rats and suggest the development of a neuropathy-induced allostatic state in both pre- and post-synaptic DA receptor function.

1. INTRODUCTION

The mesolimbic dopamine (DA) system consists of dopaminergic neurons that project from the ventral tegmental area (VTA) in midbrain to forebrain targets including the nucleus accumbens (NAc) core and shell (Ikemoto et al., 2015; Sesack and Grace, 2010). Postsynaptic neurons in NAc consist primarily of two populations defined in part by their expression of either D1-like or D2-like DA receptors (D1R and D2R, respectively) (Beaulieu and Gainetdinov, 2011). Dopaminergic transmission between mesolimbic DA neurons and their postsynaptic NAc targets plays a major role in the neural representation of rewarding stimuli and expression of motivated behavior, and accumulating evidence suggests that pain states impair that signaling (Mitsi and Zachariou, 2016; Taylor et al., 2016; Watanabe and Narita, 2018). For example, we evaluated the effects of intraperitoneal (i.p.) injection of dilute lactic acid in rats as an acute visceral noxious stimulus on both in vivo microdialysis measures of NAc core DA levels and intracranial self-stimulation (ICSS) as a positively reinforced operant behavior dependent on mesolimbic DA signaling (Leitl et al., 2014a). Acid i.p. injection produced concentration-dependent decreases in both NAc DA levels and ICSS, and both effects were blocked by clinically effective analgesics, including morphine and ketoprofen. Manipulations often used in rodent models of chronic pain produce more subtle effects on mesolimbic DA signaling (Mitsi and Zachariou, 2016; Taylor et al., 2016; Watanabe and Narita, 2018) and behavior (Negus, 2019). However, we found previously that intraplantar (Ipl) injection of formalin served as a model of chemically induced neuropathy in rats that produced sustained changes in both a conventional metric of pain-related behavior (hypersensitive paw withdrawal from tactile stimuli delivered via von Frey filaments) together with small but statistically significant depression of ICSS suggestive of sustained depression of mesolimbic DA signaling (Leitl and Negus, 2016; Leitl et al., 2014b). Microdialysis assessment of extracellular DA levels as a measure of DA signaling is best suited to within-subject evaluation of relatively transient changes in DA release over the course of minutes to hours, as in studies with acute noxious or antinociceptive stimuli (Leitl et al., 2014a; Navratilova et al., 2012). More sustained changes in DA signaling, such as that potentially produced by chronic pain states, might be expected to alter more durable components of DA signaling, such as the activity or expression of DA receptors. In the present study, we evaluated the effects of Ipl formalin as a chronic neuropathic-pain stimulus on function and expression of DA receptors and related biomarkers of DA signaling.

Pain states are associated with alterations in striatal dopamine D2R in both preclinical animal models and human imaging studies (Mitsi and Zachariou, 2016; Taylor et al., 2016). Human imaging studies indicate either increased or decreased striatal D2R receptor binding potentials using positron emission tomography in various pain states (Martikainen et al., 2018; Martikainen et al., 2015), although these results could be due to altered DA occupancy, D2R Bmax levels or both. Studies in rodents can avoid these confounds by measuring D2R function and expression levels ex vivo. Although only a limited number of such studies have been reported, they generally indicate decreased D2R levels in chronic pain models. For example, NAc D2R and tyrosine hydroxylase (TH) protein levels were decreased two weeks after spared nerve injury (SNI) in rats (Sagheddu et al., 2015). Similarly, both D2R and D1R mRNA levels were decreased in NAc 28 days after surgery in this same SNI model (Chang et al., 2014). However, there are currently no published reports on DA receptor signaling at the biochemical level along with comprehensive measures of levels of DA receptors and other proteins associated with DA neurotransmission, such as DAT and TH.

Here, we examined the effects of Ipl formalin treatment of male rats on D2- and D1-like receptor-mediated G-protein signaling and protein levels of D2R, D1R, DAT and TH in mesolimbic dopaminergic brain regions, including NAc core and shell, prefrontal cortex (PFC) and VTA. We hypothesized that formalin-induced neuropathy would decrease NAc D2 expression, thereby resulting in decreased D2R-mediated G-protein activation. Our findings indicated attenuation in both D2R and D1R signaling in NAc core, so ICSS studies were then conducted to determine whether these changes were associated with alterations in the ability of D2- and D1-like agonists to affect ICSS responding (Lazenka et al., 2016).

2. MATERIALS AND METHODS

2.1. Experimental Subjects

Adult male Sprague-Dawley rats (ENVIGO, Frederick, MD) were used for these studies. All rats had ad libitum access to food and water and were housed individually at Virginia Commonwealth University on a 12 hr light-dark cycle (6am – 6pm, lights on) in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. For molecular studies, rats weighed between 300 and 400 g at the time of Ipl injection. For ICSS, rats weighed between 300 and 400 g at the time of surgery to implant stimulating electrodes. Sample sizes were based on prior experience with each of these procedures (Lazenka et al., 2016; Lazenka et al., 2015; Sim-Selley et al., 2011; Yap et al., 2015), which has indicated that samples sizes of at least 5 are sufficient to detect statistically significant effects by various manipulations. All experiments were performed with the approval of the Institutional Animal Care and Use Committee at Virginia Commonwealth University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals 8th edition (National Research Council (U.S.), 2011).

2.2. In vitro functional assays: agonist-stimulated [35S]GTPγS binding and adenylyl cyclase activity.

2.2.1. Dissections.

Fourteen days after Ipl treatment with formalin or saline, rats were euthanized by rapid decapitation, and brains were dissected using anatomical landmarks similar to those described previously in mice (Lazenka et al., 2017b; Wiebelhaus et al., 2015) with necessary modification. The prefrontal cortex was dissected by making a cut at the posterior extent of the anterior olfactory nucleus after which the olfactory nuclei were removed. This sample included frontal association, primary and secondary motor, anterior cingulate, prelimbic and orbital frontal cortices. A cut was then made anterior to the optic chiasm, producing a slice that included both the caudate-putamen and NAc. The nucleus accumbens was isolated by removing the cortex ventrally and laterally, the septum and nucleus of the horizontal limb of the diagonal band medially and separating the remaining tissue inferior to the caudate-putamen. The NAc core was separated from the NAc shell by removing tissue ventral, lateral and medial to the anterior commissure (shell) such that a tear-shaped structure tapering at the lateral ventricle superiorly and containing the anterior commissure was left (core). The remaining caudate-putamen was isolated by removing the corpus callosum and surrounding cortex. To dissect the VTA, a coronal slice was taken by first cutting immediately anterior to the mammillary bodies and a second cut was made anterior to the middle cerebellar peduncle, at the midpoint of the cerebral peduncles. From this slice, the interpeduncular nucleus/mammillary bodies, located ventrally; the substantia nigra, located laterally; and the interfascicular nucleus, located medially were removed. The remaining region ventral to the red nucleus was dissected and comprised primarily the VTA. All dissected tissue was stored at −80°C until use.

2.2.2. Membrane preparation.

Membranes were prepared from dissected brain regions as previously described (Lazenka et al., 2015). Briefly, tissue was thawed in membrane buffer (Tris-HCl, pH 7.4, 3 mM MgCl2, 1 mM EGTA), homogenized and centrifuged at 40,000 x g for 10 min. The pellet was collected and homogenized in assay buffer (Tris-HCl, pH 7.4, 3 mM MgCl2, 0.1 mM EGTA, 100 mM NaCl) and protein was determined by the Bradford method.

2.2.3. [35S]GTPγS binding.

Membranes were pretreated with adenosine deaminase (AD) for 15 min at 30°C prior to assay. [35S]GTPγS (1250 Ci/mmol; Perkin-Elmer, Boston, MA) binding was conducted essentially as previously described (Lazenka et al., 2015). Briefly, membranes (3-6 μg protein) were incubated with varying concentrations of quinelorane (D2-like agonist) or CP55,940 (cannabinoid agonist), 30 μM GDP, and 0.1 nM [35S] GTPγS in assay buffer containing 0.1% BSA, for 2 hr at 30°C in a 0.5 ml total volume. Basal binding was assessed without agonist and nonspecific binding was measured with 20 μM unlabeled GTPγS. The assay was terminated by filtration through GF/B glass fiber filters, followed by 3 washes with ice-cold Tris buffer. Bound radioactivity was determined by liquid scintillation spectrophotometry.

2.2.4. Adenylyl cyclase activity.

Adenylyl cyclase assays were performed as previously described (Sim-Selley et al., 2011) with minor modifications. Membranes (20 μg protein) were pretreated with AD as above and incubated with varying concentrations of SKF82958 (D1-like agonist) or CGS21689 (A2a agonist) in assay buffer containing 0.1% BSA, 50 μM ATP, 50 μM GTP, 0.2 mM DTT, 0.2 mM papaverine, 5 mM phosphocreatine, and 20 U/ml creatine phosphokinase for 15 min at 30°C. The incubation was terminated by addition of ELISA sample diluent, and cAMP was quantified using a cAMP ELISA kit (Arbor Assays, Ann Arbor, MI).

2.2.5. Data analysis for in vitro functional assays.

Data are reported as mean values ± SEM of 5-6 rats per group with samples assayed in duplicate. Concentration-effect curves were subjected to non-linear regression analysis to determine Emax and EC50 values. To control for day-to-day inter-assay variability, Emax values in both groups were also normalized to the saline-treated value on each day of the experiment. Significance of agonist-stimulation and formalin treatment was determined by two-way ANOVA with agonist concentration and formalin-versus-saline treatment as the main factors. Differences in Emax and EC50 values between groups were determined by the two-tailed Student’s t-test. All curve-fitting and statistical analysis was conducted using Prism software (GraphPad, San Diego, CA).

2.3. Protein quantification by immunoblot.

2.3.1. Dissections.

Fourteen days after Ipl treatment with formalin or saline, rats were euthanized by rapid decapitation, and whole brains were frozen in isopentane at −30 °C and shipped frozen on dry ice to McLean Hospital for protein (western) immunoblotting, as described previously (Der-Avakian et al., 2017; Yap et al., 2015). Brains were processed without knowledge of the treatment conditions. Briefly, frozen brains were coronally sectioned on a cryostat (HM 505 E; Microm; Walldorf, Germany) until the following regions were exposed: NAc Core (Bregma 2.52mm), NAc Shell (Bregma 2.52mm), and VTA (Bregma −5.04mm), based on the atlas of Paxinos and Watson. Bilateral tissue punches 1–1.5 mm in length were taken with a 1 mm internal diameter corer (Fine Science Tools; Foster City, CA) and placed in Eppendorf tubes kept on dry ice and then stored at −80° C. Upon obtaining the tissue cores, coronal sections (30 μM) of the exposed face of the brain were taken and Nissl stained with cresyl violet for histological analysis of placements. Only the rats in which the tissue punches were targeted appropriately were included in analyses.

2.3.2. Immunoblotting.

Tissue was sonicated in 1% sodium dodecyl sulfate (SDS) to dissociate membranes. Total protein concentrations in samples were determined using the Bio-Rad DC Protein Assay kit (Hercules, CA), and the concentration of each sample was adjusted to 2.0 mg/ml protein. Cell lysates were heated to 70°C for 10 min before polyacrylamide gel electrophoresis. Denatured protein (10-20 μg) was loaded per lane on 4-12% Bis-Tris Gels (Invitrogen). Protein was transferred onto PVDF membrane. Membranes were stained with Ponceau S then rapidly imaged for total protein as loading control/normalization before blocking for 2 hours at RT with 5% nonfat dry milk in TBS-T with 0.02% Tween 20, then incubated with mouse monoclonal anti-D2R (1:250; Santa Cruz SC-5303) in TBS-T overnight at 4C to detect long and short forms of D2R at 51 and 48kDa. Other primary antibodies included pTH (cell Signaling S3; 1; 1:1000); TH (Millipore/Sigma AB152; 1:40000); DAT (Millipore AB2231; 1:20000); D1R (Santa Cruz SC 14001; 1:250).

2.3.3. Data analysis.

Data are reported as mean values ± SEM of 5-6 rats per group. Immunoblots were analyzed by normalization of optical densities of immunoreactive protein to Ponceau stain, and then normalization of both groups to the saline condition. Significant differences were determined using Student’s t-test, conducted with Prism software.

2.4. In vivo assessment of ICSS

2.4.1. Drugs for in vivo delivery.

Formalin and (−)-quinpirole HCl (D2-like agonist) were obtained from Fisher Scientific (Waltham, MA) and Sigma-Aldrich (St. Louis, MO), respectively. (±)SKF82958 HBr [(±)-6-Chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide] was provided by the National Institute of Mental Health Chemical Synthesis and Drug Supply Program (Bethesda, MD). Each of the three compounds were dissolved in saline. Reagents used for molecular studies are described above. For all studies, rats received bilateral Ipl injections of 5% formalin (100 ul in each hind paw) or saline, as described previously (Leitl et al., 2014b). For behavioral studies, quinpirole and SKF82958 were administered by i.p. injection, and drug doses are expressed in units of the salt form above.

2.4.2. Surgery.

Rats were anesthetized with isoflurane (3% in oxygen; Webster Veterinary, Phoenix, AZ, USA) until unresponsive to toe-pinch prior to implantation of stainless steel electrodes (Plastics One, Roanoke, VA, USA). The cathode, which was 0.25 mm in diameter and covered with polyamide insulation except at the flattened tip, was implanted utilizing a stereotactic method into the left medial forebrain bundle (MFB) at the level of the lateral hypothalamus using previously published coordinates (2.8 mm posterior to bregma, 1.7 mm lateral to the midsagittal suture, and 8.8 mm ventral to the skull) (Leitl et al., 2014b). Three screws were placed in the skull, and the anode (0.125 mm diameter, un-insulated) was wrapped around one of the screws to act as a ground. Dental acrylic was used to secure the electrode to the screws and skull. Ketoprofen (5 mg/kg) was administered as a postoperative analgesic immediately and 24 hrs following surgery. Animals were allowed to recover for at least one week before ICSS training.

2.4.3. Apparatus.

Operant conditioning chambers consisted of sound-attenuating boxes containing modular acrylic and metal test chambers (29.2 cm X 30.5 cm X 24.1 cm) (Med Associates, St. Albans, VT). Each chamber had a response lever (4.5 cm wide, 2.0 cm deep, 3.0 cm above the floor), a 2-watt house light, three stimulus lights (red, yellow and green) centered 7.6 cm above the lever, and an ICSS stimulator. Bipolar cables routed through a swivel-commutator (Model SL2C, Plastics One) connected the stimulator to the electrode. MED-PC IV computer software controlled all programming parameters and data collection (Med Associates).

2.4.4. Training and Testing.

The behavioral procedure and methods for training were similar to those described previously for studies with formalin (Leitl and Negus, 2016; Leitl et al., 2014b) and with direct dopamine agonists (Lazenka et al., 2017a; Lazenka et al., 2016). Under the terminal schedule, behavioral sessions consisted of three consecutive 10 min components, each of which contained 10 consecutive 60 s trials. The stimulation frequency was 158 Hz for the first trial of each component, and frequency decreased in 0.05 log unit steps during the subsequent nine trials to a final frequency of 56 Hz. Each trial began with a 10 s time-out period, during which responding had no scheduled consequences, and five non-contingent stimulations at the designated frequency were delivered at 1 s intervals during the last 5 s of the time out. During the remaining 50 s of each trial, responding produced both intracranial stimulation at the designated frequency and illumination of the lever lights under a fixed-ratio 1 schedule. ICSS performance was considered to be stable when frequency-rate curves were not statistically different over three consecutive days of training as indicated by lack of a significant effect of ‘day’ in a two-way analysis of variance (ANOVA) with day and frequency as the main effect variables (see Data Analysis below). All training was completed within six weeks of surgery.

Once reliable ICSS baselines were established, testing began using a 16-day protocol. On Day 2 of this protocol, rats were treated bilaterally with Ipl formalin (N=12) or saline (N=12). On Day 1 (the day before formalin/saline treatment), and on Day 16 (14 days after formalin/saline treatment), rats were tested with cumulative doses of either quinpirole (0.0032-0.1 mg/kg) or SKF82958 (0.01-0.32 mg/kg) (N=6 formalin-treated rats and N=6 saline-treated rats for each drug). Cumulative-dosing studies with quinpirole or SKF82958 were accomplished using test sessions that consisted of three baseline components followed by four consecutive 30-min test periods. Each test period consisted of a 10 min time out followed by a pair of 10-min test components. A dose of quinpirole or SKF82958 was administered i.p. in a volume of 1 ml/kg at the start of each time out, and each dose increased the total cumulative dose by a half-log increment. Three-component ICSS baseline sessions were conducted on most other days (excluding weekends).

2.4.5. Data analysis for ICSS.

The first baseline component for each day was considered to be a “warm-up” component, and data were discarded. The primary dependent variable was reinforcement rate in stimulations per min during each frequency trial for all remaining baseline and test components. To normalize these data, raw reinforcement rates from each trial in each rat were converted to percent maximum control rate (%MCR) for that rat. The MCR was defined as the mean of the maximal rates observed during the second and third baseline components for the three consecutive training days preceding the 16-day test protocol (six total baseline components). Subsequently, % MCR values for each trial were calculated as [(reinforcement rate during a frequency trial)/(MCR)]×100. For each rat during each session, data from baseline and test components were averaged to yield baseline and test frequency-rate curves. Baseline and test data were then averaged across rats to yield mean baseline and test frequency-rate curves for each manipulation. Results were compared by repeated measures two-way ANOVA with ICSS frequency as a within-subject factor and either Ipl treatment (between-subjects factor) or dopamine agonist dose (within-subjects factor) as the second factor. A significant ANOVA was followed by the Holm-Sidak post-hoc test, and the criterion for significance was p < .05.

To provide an additional summary measure of ICSS performance, the total number of stimulations per component was determined across all 10 frequency trials of each component. To normalize these data, raw numbers of stimulations per component in each rat were converted to the percent baseline number of stimulations per component for that rat, with the baseline defined as the mean number of stimulations per component during the second and third components on the three consecutive baseline days preceding testing. Thus, % Baseline Stimulations was calculated as (mean total stimulations during a component/mean total stimulations during baseline components) x 100. These data were then averaged across rats. Baseline data in formalin- and saline-treatment groups on a given day were compared by t-test. Drug effects in formalin- and saline-treatment groups on a given day were compared by two-way mixed ANOVA with dose as a within-subjects factor and Ipl treatment as a between-subjects factor. A significant ANOVA was followed by the Bonferroni Holm-Sidak post-hoc test, and the criterion for significance was p < 0.05. All statistical analyses were conducted with Prism software.

3. RESULTS

3.1. Formalin treatment attenuates D2-like receptor-mediated G-protein activation in NAc core.

The effect of Ipl formalin treatment on G-protein activation by dopamine D2-like receptors was determined with quinelorane-stimulated [35S]GTPγS binding in membranes prepared from NAc core and shell, caudate-putamen (CPu) and VTA. Basal [35S]GTPγS binding varied among regions, but was unaffected by formalin treatment in any region examined (Supplementary Figure 1A). Concentration-effect curves of quinelorane-stimulated [35S]GTPγS binding were examined in NAc core versus shell. Results in NAc core showed decreased D2R-stimulated activity in formalin- relative to saline-treated-rats (Figure 1A). Curve-fitting analysis revealed trends toward a significant decrease in Emax and increase in log EC50 values (Table 1). Normalization of Emax values in formalin-treated rats to each corresponding value in saline-treated rats revealed a significant decrease in formalin- compared to saline-treated rats (Table 1).

Figure 1. Formalin treatment decreases D2-like agonist-stimulated G-protein activity in NAc core but not shell.

Figure 1.

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Concentration-effect curves were conducted for stimulation of [35S]GTPγS binding by quinelorane (A, C) or CP55,940 (B) in membranes from NAc core (A, B) or shell (C). Data are mean % stimulation of [35S]GTPγS binding ± SEM (n = 5). Quinelorane-stimulated [35S]GTPγS binding was reduced in NAc core (A) but not shell (C) of formalin-relative to saline-treated rats. Two-way ANOVA in NAc core showed a main effect of qunelorane concentration [F(7, 59) = 23.05, p < 0.0001] and formalin treatment [F(1,59) = 20.73, p < 0.0001], whereas in NAc shell there was a main effect of qunelorane concentration [F(7,61) = 19.62, p < 0.0001] but not of formalin treatment, with no significant interactions between factors in either region. There was no difference between experimental groups in CP55,940-stimulated [35S]GTPγS binding in NAc core (B). Two-way ANOVA revealed a main effect of CP55,940 concentration [F(6,56) = 61.51, p < 0.0001] but not of formalin treatment nor was there an interaction.

Table 1.

Emax and EC50 values of agonist-stimulated [35S]GTPγS binding in formalin- and vehicle-treated rats

Treatment Agonist Emax (% Stim.) Emax (% Saline) EC50 (log M)
NAc Core:
Saline Quinelorane 50.3 ± 6.1 100.0 ± 12.1 −6.91 ± 0.04
Formalin Quinelorane 32.0 ± 5.5† 61.9 ± 6.2* −6.72 ± 0.07†
Saline CP55,940 103.1 ± 7.7 100.0 ± 7.5 −8.22 ± 0.05
Formalin CP55,940 107.6 ± 7.0 105.0 ± 3.1 −8.08 ± 0.06
NAcShell:
Saline Quinelorane 31.1 ± 2.8 100.0 ± 9.1 −6.95 ± 0.11
Formalin Quinelorane 34.4 ± 2.9 114.0 ± 10.0 −7.00 ± 0.08
Saline CP55,940 113.4 ± 4.3 100.0 ± 3.8 −7.93 ± 0.06
Formalin CP55,940 116.8 ± 3.1 104.4 ± 4.0 −7.99 ± 0.05
VTA
Saline Quinelorane 10.2 ± 0.9 100.0 ± 9.1 −7.03 ± 0.19
Formalin Quinelorane 8.4 ± 1.3 88.4 ± 12.3 −7.35 ± 0.16
CPu
Saline Quinelorane 60.7 ± 4.2 100.0 ± 7.0 −7.00 ± 0.17
Formalin Quinelorane 67.3 ± 6.6 110.8 ± 10.8 −6.95 ± 0.08
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Concentration-effect curves of quinelorane- or CP55,940-stimulated [35S]GTPγS binding in NAc core, NAc shell, VTA and CPu were fit by non-linear regression analysis to obtain Emax and EC50 values. Values are presented as mean ± SEM (n = 5-6). % Stim: % stimulation; % Saline: Emax values normalized to the maximal stimulation obtained in saline-treated rats on each day of assay.

*

, p < 0.05 different from saline-quinelorane: normalized Emax: [t(8) = 2.514, p = 0.036]

†

, p < 0.1 trending different from saline-quinelorane: Emax: [t(8) = 1.995, p = 0.081] and log EC50: [t(8) = 2.004, p = 0.080].

To determine whether the formalin-induced decrease in G-protein activation was generalized to other Gi/o-coupled receptors, activity of the CB1 cannabinoid receptor, which has overlapping localization with D2R in NAc (Pickel et al., 2006), was examined in NAc core. In contrast to results with the D2-like agonist quinelorane, G-protein activation by the cannabinoid agonist CP55,940 was unaffected by formalin treatment (Figure 1B). Emax and log EC50 values of CP55,940 did not differ between formalin- and saline-treated rats (Table 1). Formalin treatment did not alter D2R- (Figure 1C) or cannabinoid receptor-stimulated G-protein activation in NAc shell. Neither quinelorane nor CP55,940 Emax and log EC50 values differed between formalin- and saline-treated rats (Table 1).

D2-like receptor-stimulated G-protein activation was also assessed in VTA and CPu (Supplemental Figure 2). Results in VTA showed low and variable stimulation of [35S]GTPγS binding by quinelorane, which did not differ between saline- and formalin-treated rats. However, it should be noted that modest differences could remain undetected due to the low signal-to-noise ratio in this region. Quinelorane produced more robust stimulation of [35S]GTPγS binding in CPu, but there was no effect of formalin treatment. Quinelorane Emax and log EC50 values in VTA and CPu did not differ between saline- and formalin-treated rats (Table 1). Altogether, these results indicate that Ipl formalin treatment attenuated D2R-mediated G-protein activation in NAc core, but not in NAc shell, CPu or VTA, without affecting CB1 cannabinoid receptor-mediated G-protein activation.

3.2. Formalin treatment attenuates D1-like receptor-mediated AC activation in NAc core.

The effect of Ipl formalin treatment on G-protein signaling by dopamine D1-like receptors was determined with SKF82958-stimulated AC activity in membranes prepared from NAc core and shell. Basal AC activity varied among regions but was unaffected by formalin treatment (Supplemental Figure 1B). Concentration-effect curves of SKF82958-stimulated AC activity were examined in NAc core versus shell. Results in NAc core showed decreased D1R-stimulated activity in formalin- relative to saline-treated rats (Figure 2A). Curve-fitting analysis revealed a significant decrease in both Emax and normalized Emax values of SKF82958 in formalin- compared to saline-treated rats, with no difference in log EC50 values (Table 2). In contrast, AC activation in NAc core by the adenosine A2a agonist CGS21680 was unaffected by formalin treatment (Figure 2B). Emax and log EC50 values of CGS21680 did not differ between formalin- and saline-treated rats (Table 2).

Figure 2. Formalin treatment selectively decreases D1-like agonist-stimulated AC activity in NAc core but not shell.

Figure 2.

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Concentration-effect curves were conducted for stimulation of AC activity by SKF82958 (A, C) or CGS21680 (B) in membranes from NAc core (A, B) or shell (C). Data are mean % stimulation of AC activity ± SEM (n = 5-7). SKF82958-stimulated AC activity was reduced in NAc core (A) but not shell (C) of formalin- relative to saline-treated rats. Two-way ANOVA in NAc core showed a main effect of quinelorane concentration [F(6, 75) = 29.45, p < 0.0001) and formalin treatment [F(1,75) = 11.63, p < 0.0001), whereas in NAc shell there was a main effect of SKF82958 concentration [F(6,52) = 18.47, p < 0.0001] but not of formalin treatment, with no significant interactions between factors in either region. There was no difference between experimental groups in CGS21680-stimulated AC activity in NAc core (B). Two-way ANOVA revealed a main effect of CGS21680 concentration [F(6,70) = 17.25, p < 0.0001] but not of formalin treatment nor was there an interaction.

Table 2.

Emax and EC50 values of agonist-stimulated AC activity in formalin- and vehicle-treated rats

Treatment Agonist Emax (% Stim.) Emax (% Saline) EC50 (log M)
NAc Core:
Saline SKF82958 38.7 ± 2.8 100.0 ± 7.4 −7.92 ± 0.14
Formalin SKF82958 30.0 ± 2.9* 77.1 ± 4.0* −7.98 ± 0.15
Saline CGS21689 22.3 ± 2.5 100.0 ± 11.2 −6.80 ± 0.25
Formalin CGS21689 28.4 ± 2.3 132.4 ± 12.8 −6.72 ± 0.26
NAc Shell:
Saline SKF82958 25.8 ± 3.2 100.0 ± 12.7 −7.64 ± 0.09
Formalin SKF82958 24.2 ± 6.0 93.1 ± 16.8 −7.81 ± 0.05
Saline CGS21689 10.5 ± 1.9 100.0 ± 18.6 −6.13 ± 0.10
Formalin CGS21689 9.1 ± 1.9 86.4 ± 6.2 −6.08 ± 0.11
PFC
Saline SKF82958 28.6 ± 1.8 100.0 ± 5.4 −7.75 ± 0.35
Formalin SKF82958 24.2 ± 1.5 90.1 ± 8.8 −7.66 ± 0.19
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Concentration-effect curves of SKF82958- or CGS21680-stimulated AC activity in NAc core, NAc shell and PFC were fit by non-linear regression analysis to obtain Emax and EC50 values. Values are presented as mean ± SEM (n = 4-7). % Stim: % stimulation; % Saline: Emax values normalized to the maximal stimulation obtained in saline-treated rats on each day of assay.

*

, p < 0.05 different from saline-SKF82958: Emax [t(12) = 2.869, p = 0.014] and normalized Emax: [t(8) = 2.514, p = 0.036].

Formalin treatment did not alter D1R-mediated AC activation in NAc shell (Figure 2C). SKF82958 Emax and log EC50 values did not differ between formalin- and saline-treated rats (Table 2). CGS21680-stimulated AC activity was minimal (Emax ≤ 10%) in NAc shell and showed concentration-dependent stimulation only in 4 of the 5 sample pairs, so 4 samples per group were included in the analysis (data not shown). Emax and log EC50 values of CGS21680 did not differ between formalin- and saline-treated rats in NAc shell (Table 2).

Because D1 and A2a receptors are the major Gs/olf-coupled receptors driving AC activation in each of the two distinct populations of striatal medium spiny neurons, the effect of formalin on the ratio of maximal AC activation by each receptor was determined. The ratio of D1/A2a-stimulated AC activity was decreased by ~41% in NAc core of formalin- relative to saline-treated rats (1.11 ± 0.16 versus 1.87 ± 0.28, respectively [t(12) = 2.430, p = 0.032]). In contrast, formalin treatment did not affect the ratio of D1/A2a-stimulated AC activity in NAc shell (2.79 ± 0.46 versus 2.49 ± 0.20 in formalin- and saline-treated rats, respectively).

Formalin treatment also did not alter D1R-mediated AC activation in the prefrontal cortex (PFC), a dopaminergic terminal field region that is enriched in D1-like receptors (Herve et al., 1992) (Supplemental Figure 3). SKF82958 stimulated AC activity in a concentration-dependent manner, but there was no effect of formalin treatment. Neither Emax nor log EC50 values of SKF82958 differed between saline- and formalin-treated rats (Table 2). Taken together, these results indicate that formalin treatment attenuated D1R-mediated AC activation in NAc core, but not in NAc shell or PFC, without significantly altering adenosine A2a receptor-mediated AC activation. Moreover, the ratio of D1:A2a-stimulated AC activity was significantly decreased in NAc core but not shell.

3.3. Formalin treatment decreases D2L receptor protein levels in NAc core.

The receptor function experiments described above showed decreases in both D2-like receptor-mediated G-protein activation and D1-like receptor-mediated AC activation in NAc core but not shell of formalin-treated rats. Because decreased functional activity could be due to decreased receptor levels, reduced coupling between each receptor and its cognate G-protein or both, D2R and D1R protein levels were determined by immunoblot analysis of NAc core and shell. Results in NAc core (Figure 3A, C and E; Supplemental Figures 3 and 4) showed a ~17% decrease in D2R long isoform (D2L) immunoreactivity in formalin- compared to saline-treated rats, without any difference in D2R short isoform (D2S) levels.

Figure 3. Formalin treatment decreases D2L protein levels in NAc core but not shell.

Figure 3.

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Immunoblots were conducted for D2 receptor protein in punches of NAc core (A, C, E) or shell (B, D, F). (A, B) Immunobot images of D2 receptor immunoreactivity. (C-F) Data are mean ± SEM of fold change in D2L isoform (C, D) or D2S isoform (E, F) in each sample relative to the mean value obtained in saline-treated rats (n = 6). Only the D2L isoform in NAc core was significantly decreased in formalin- relative to saline-treated rats. * p < 0.05 different from saline [t(10) = 2.363, p = 0.020].

In contrast to results in NAc core, no differences in either D2L or D2S receptor levels were seen in NAc shell (Figure 3B, D and F). D1R immunoreactivity also did not differ between saline- and formalin-treated rats in either NAc core or shell (Supplemental Figures 6 and 7). Likewise, D2L, D2S and D1 receptor immunoreactivity did not differ between formalin- and saline-treated rats in VTA or PFC (data not shown). Furthermore, immunoblot analysis of other proteins important in the regulation of dopamine neurotransmission, including DAT, tyrosine hydroxylase (TH), and phospho-TH, also showed no differences between formalin- and saline-treated rats in NAc core or shell, VTA or PFC (data not shown). Altogether, these results indicate that formalin treatment selectively decreased D2LR protein in the NAc core without affecting other proteins directly involved in dopamine neurotransmission.

3.4. Formalin treatment attenuates modulation of ICSS responding by D2 but not D1 receptor activation.

Under baseline conditions, MFB stimulation maintained a frequency-dependent increase in reinforcement rates. The mean ± SEM maximum control rate for all rats in the study was 53 ± 1.9 stimulations per trial, and the mean ± SEM baseline number of stimulations per component was 224 ± 11.5. There were no differences across groups in either baseline ICSS or in drug effects on ICSS on Day 1, before formalin or saline treatment (data not shown). Figure 4 shows that there was also no difference in baseline ICSS performance on Day 16, 14 days after formalin or saline treatment. Likewise, there were no significant differences in baseline ICSS responding over the preceding two weeks (days 3-15) during which baseline ICSS responding was measured every weekday beginning 24 hr after formalin treatment (data not shown). Figure 5 shows effects of quinpirole and SKF82958 on Day 16 in each group. Quinpirole produced dose-dependent rightward/downward shifts in the ICSS frequency-rate curves in both groups; however, quinpirole was less potent to decrease the number of stimulations per component in the formalin-treated group than in the saline-treated group. SKF82958 produced a mixed profile of ICSS facilitation and ICSS depression in both groups. In particular, a dose of 0.1 mg/kg SKF82958 facilitated ICSS for at least one brain-stimulation frequency in both groups, and a higher dose of 0.32 mg/kg SKF82958 decreased ICSS for at least two brain-stimulation frequencies in both groups. There were no significant differences between formalin- and saline-treated groups in SKF82958 effects on the number of stimulations per component.

Figure 4. Baseline ICSS performance was unaffected by Ipl formalin treatment.

Figure 4.

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Panel A: Abscissa: Frequency of electrical brain stimulation in Hz (log scale). Ordinate: Percent maximum control reinforcement rate (% MCR). Two-way ANOVA indicated a significant main effect of frequency [F(9, 198) = 151, p < 0.0001], but no effect of treatment or frequency x treatment interaction. Panel B: Abscissa: Intraplantar treatment group. Ordinate: Percent baseline number of stimulations per component, a summary measure of ICSS performance across all brain stimulation frequencies. Student’s t-test indicated no difference between treatment groups. All data show mean ± SEM of 12 rats.

Figure 5. Formalin treatment decreases the potency of D2-like but not D1-like agonists to modulate ICSS responding.

Figure 5.

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Abscissae: Frequency of electrical brain stimulation in Hz (log scale; A, B, D and E) or dose of drug (C and F). Ordinates: Percent maximum control reinforcement rate (% MCR; A, B, D and E) or percent baseline number of stimulations per component (C and F). Filled symbols (A, B, D and E) show significant differences from baseline as determined by repeated-measures two-way ANOVA followed by the Holm-Sidak post-hoc test, p < 0.05. Filled symbols for panel C show a significant difference between saline and formalin treated rats following a two-way mixed ANOVA and a Holm-Sidak post-hoc test, p < 0.05. All data show mean ± SEM of 6 rats. Statistical results are as follows (only significant interaction results are shown for brevity): (A) significant frequency x dose interaction [F(36, 180) = 10.41, p < 0.0001], (B) significant frequency x dose interaction [F(36, 180) = 3.68, p < 0.0001] (C) significant dose x intraplantar treatment interaction [F(4, 40) = 5.80, p < 0.001], (D) significant frequency x dose interaction [F(36, 180) = 4.78, p < 0.0001], (E) significant frequency x dose interaction [F(36, 180) = 2.32, p < 0.001].

4. DISCUSSION

4.1. Summary of major findings

This study examined the effects of the Ipl formalin model of chronic pain on DA receptor expression and signaling in the mesolimbic DA system and on modulation of ICSS behavior by D1-like and D2-like agonists. Three major novel findings were obtained. First, neuropathy-induced reductions in both D1R and D2R-mediated G-protein signaling were demonstrated within the NAc core. These effects were not observed in NAc shell or other brain regions examined, and they appeared to be receptor-homologous because formalin treatment did not significantly affect activity of the Gs/olf-coupled adenosine A2a receptor or the Gi/o-coupled cannabinoid CB1 receptor. Second, these decreases in NAc core D2R function were paralleled by a decrease in D2LR protein; however, there were no significant changes in levels of D2SR or D1R protein. Third, the decrease in NAc core D2R function and expression was associated with reduced potency of the D2-like agonist quinpirole to decrease positively reinforced operant responding in ICSS, whereas the effects of a D1-like agonist on ICSS were not altered. Taken together, this profile of effects suggests that formalin-induced neuropathy alters dopaminergic responsivity in the NAc core, with predominant downregulation of D2R function.

4.2. Formalin treatment decreased DA receptor signaling and D2LR expression in NAc core.

Preclinical and human functional imaging studies suggest a role for the NAc in both acute and chronic pain (Baliki et al., 2010; Baliki et al., 2013; Becerra and Borsook, 2008; Chang et al., 2014; Magnusson and Martin, 2002; Schwartz et al., 2014). In humans, the NAc core and shell regions respond differently to acute thermal pain (Baliki et al., 2013). The present study showed a decrease in quinelorane-stimulated G-protein activation and a specific decrease in the D2LR isoform in the NAc core but not shell. In general agreement with our results, other studies of neuropathic pain in rodents have shown opposing changes in the excitability of D2R expressing medium spiny neurons in the NAc core and shell (Ren et al., 2016).

In addition to effects on D2R expression and activity, we also found that D1-like receptor-stimulated AC activity was decreased in the NAc core but not shell. To our knowledge, this is the first report of effects of neuropathy on D1R activity in the NAc. D1R activation can reverse acute pain-related behavioral depression induced by i.p. acid administration (Lazenka et al., 2017a), and the present findings of reduced D1R activity in neuropathic rats suggest a potential for reduced effectiveness of D1R agonists. Conversely, A2a receptor activation and D2R inhibition were reported to mediate increased pain sensitivity and behavioral depression associated with sleep deprivation (Sardi et al., 2018), suggesting that increased activity of A2a/D2R-expressing neurons contributes to pain behaviors. The decreased ratio of D1R to A2a receptor-mediated AC activation coupled with reduced D2R expression and activity found in NAc core in the present study suggest that the balance of activity of D1R- versus D2R-expressing medium spiny neurons could be perturbed by neuropathic pain, assuming a significant fraction of the post-synaptic D2R population was affected by formalin treatment. Predominant activity of A2a/D2R- relative to D1R-expressing neurons in NAc core could therefore contribute to pain-induced behavioral depression.

The mechanisms underlying desensitization and/or downregulation of D2R and D1R in NAc core of rats with neuropathy are unclear. A decrease in D2L protein could indicate decreased gene (mRNA) expression, as suggested by prior work on SNI-induced neuropathy (Chang et al., 2014). However, it is also possible that D2R desensitization and downregulation occur post-translationally, for example in response to increased activation by endogenous DA. The most common mechanism of G-protein-coupled receptor (including DA receptor) regulation in response to agonist occupancy is receptor phosphorylation followed by β-arrestin-mediated desensitization and internalization (Gurevich et al., 2016). In addition, a calcium-dependent mechanism mediates DA-induced desensitization of the D2S but not D2L isoform (Gantz et al., 2015). Furthermore, D2 receptors interact with G-protein-coupled receptor-associated sorting protein 1 (GASP1), a post-endocytic trafficking protein that promotes trafficking to lysosomes resulting in receptor degradation (Bartlett et al., 2005; Thompson et al., 2010). This mechanism has been shown to mediate D2R downregulation in response to repeated cocaine treatment. Therefore, the formalin-induced reduction in D2L receptor protein could have been due to enhanced GASP1-mediated lysosomal degradation of the protein. In contrast, D1R protein was unaffected by formalin treatment, consistent with its lack of interaction with GASP1 (Bartlett et al., 2005; Thompson et al., 2010). The reduction in D1-like stimulation of AC activity could have been due to receptor phosphorylation and β-arrestin-mediated desensitization. Indeed, D1R in striatal neurons interact with β-arrestin2 in an agonist-stimulated manner (Macey et al., 2005), and catechol-containing agonists such as endogenous DA have been shown to effectively recruit β-arrestin2 to the D1R (Gray et al., 2018).

Other potential explanations for reduced D1R or D2R signaling in NAc core of formalin-treated rats include increased heteromeric interactions with other G-protein-coupled receptors. For example, D2R signaling to Gi/o can be reduced by switching to Gs/olf or Gq/11 signaling via heteromomerization with CB1 or A2a receptors, respectively (Ferre et al., 2009). However, no significant differences in CB1 or A2a receptor activity were seen between formalin- and saline-treated rats in the present study. Nonetheless, mechanisms including homologous or heterologous receptor regulation and heteromerization are unlikely to be mutually exclusive, and future studies may elucidate the contribution of these molecular processes in DA receptor adaption in neuropathic pain states.

4.3. Formalin treatment decreased D2-like agonist potency in ICSS.

Ipl formalin is well-established in preclinical studies as a chronic-pain stimulus (Fu et al., 2001; Grace et al., 2014), and we reported previously that Ipl formalin produced a small but significant decrease in ICSS that was sustained for up to 14 days (Leitl and Negus, 2016; Leitl et al., 2014b). Although this sustained ICSS depression served as a rationale for the present study to examine DA receptor function and expression 14 days after Ipl formalin, the present study failed to replicate this formalin-induced ICSS depression. Other treatments that produce neuropathy in rats, such as spinal nerve ligation or chemotherapy administration, are also generally ineffective to decrease ICSS and other forms of positively reinforced operant behavior in rats (Ewan and Martin, 2011; Legakis et al., 2018; Okun et al., 2016). These results suggest that Ipl formalin and other neuropathy manipulations are at best weakly and inconsistently effective to depress positively reinforced operant responding in rats. By contrast, clinically relevant neuropathic pain in humans is consistently associated with behavioral depression and functional impairment (Colloca et al., 2017; Dworkin et al., 2005). A better understanding of factors that underlie the resistance of rats to neuropathic pain-related behavioral depression could suggest novel insights into mechanisms that mediate pain behaviors and novel strategies for pain treatment.

Despite the lack of effect by formalin treatment on baseline ICSS responding, it remained possible to examine effects on subsequent sensitivity to the effects of D1-like and D2-like receptor agonists. We reported previously that quinpirole and other D2-like agonists produce dose-dependent decreases in ICSS, whereas D1-like agonists produce a mixed profile with stimulation by low doses and primarily depression by higher doses (Lazenka et al., 2016). In the present study, formalin treatment reduced the potency of quinpirole to decrease ICSS, which agrees with the decrease in D2R-stimulated G-protein activation seen in the NAc core. However, it is not clear if this effect is mediated by the D2R long or short isoforms or dopamine D3 receptors because both quinpirole and quinelorane can activate all of these receptors. The effectiveness of D2-like agonists to depress ICSS is likely due to decreased DA release through activation of D2 (and possibly D3) autoreceptors (Gilbert et al., 1995; Khan et al., 1998; Lindgren et al., 2003; Sokoloff et al., 1990; Usiello et al., 2000). Thus, the formalin-induced decrease in quinpirole potency suggests a decrease in DA autoreceptor activity. This possibility is discussed further below.

In addition to D2R downregulation, formalin treatment also decreased D1R-stimulated AC activity without a reduction in D1R protein in the NAc core. Despite this reduction in D1R signaling, there was no significant change in the effects of a D1-like agonist on ICSS responding, although the intermediate dose of 0.1 mg/kg SKF82958 generally produced weaker ICSS facilitation in formalin- than saline-treated rats. One explanation for this lack of effect may be the biphasic nature of D1-like agonist effects on ICSS as noted above (Lazenka et al., 2016), making subtle changes difficult to detect. Furthermore, the reinforcing effects of ICSS may be more reliant on D1R expression and function in the NAc shell than core (Cheer et al., 2007), which would be consistent with our observation that formalin did not alter D1R-stimulated AC activity in the shell.

4.4. D2 autoreceptor adaptation as a hypothesis to explain both molecular and behavioral changes in the D2R system in formalin-induced neuropathy

Attenuation of D2 autoreceptor signaling could explain several molecular and behavioral findings of this study. Under ordinary conditions, D2 autoreceptors mediate one form of negative feedback that constrains DA neuronal activity and DA release (Ford, 2014). Here, we suggest that downregulation and/or desensitization of D2 autoreceptors may serve as a compensatory response to oppose pain-related decreases in mesolimbic DA release and behavior (Leitl et al., 2014a), maintain baseline DA release and behavior, and reduce sensitivity to exogenous D2R agonists like quinpirole. In this scenario, at least some of the decrease in D2-like agonist-stimulated G-protein activation in NAc core of formalin-treated rats would be due to decreased signaling of D2 autoreceptors. In agreement with this interpretation, agonist-stimulated G-protein activation was reduced by 38% in formalin- relative to saline-treated rats, whereas D2LR protein was only reduced by 17%. While this difference in effect size could be the result of desensitization of a portion of the remaining D2LR, it could also result from D2SR being desensitized without downregulation of D2SR levels. Moreover, although D2SR comprises the majority of D2 autoreceptors and D2LR is largely post-synaptic (Ford, 2014), the D2LR is also expressed in DA neurons and can function as an autoreceptor (Ford, 2014; Gantz et al., 2015; Neve et al., 2013). It is therefore possible that decreased D2LR expression in formalin-treated rats was due to downregulation of D2LR autoreceptors.

D2 autoreceptors are synthesized in dopaminergic cell bodies and exhibit both axonal and somatodendritic expression (Ford, 2014). Therefore, if D2LR mRNA was downregulated in the current study, then decreases in D2LR protein would have been expected to occur in both the VTA and NAc core. However, our results indicated reduced D2LR expression only in NAc core, although it is possible that small decreases in VTA would require larger sample sizes to detect. Nonetheless, it is conceivable that D2LR autoreceptors were selectively downregulated by a non-transcriptional mechanism, such as differential degradation of the D2LR in presynaptic terminals or reduced axonal transport of D2LRs. Regardless of the mechanism, one intriguing and novel implication of this hypothesis is that D2LR may be the autoreceptor downregulated as a compensatory response to maintain DA signaling under conditions of low DA neuronal activity, whereas D2SR desensitization may contribute to other forms of plasticity (e.g., by amplifying DA release under conditions of high DA neuronal activity; (Gantz et al., 2015)).

The hypothesis that formalin-induced neuropathy reduces D2 autoreceptor expression and signaling is consistent with reports that other chronic stress-related manipulations decrease both NAc D2 density and quinpirole potency to produce D2 autoreceptor-mediated effects (Acri et al., 2001; Izenwasser et al., 1998; Qiao et al., 2019). For example, acute treatment with kappa-opioid receptor agonists decreases mesolimbic DA neuronal activity and NAc DA release (similar to our findings with an acute pain stimulus; (Leitl et al., 2014a; Navratilova et al., 2012); however, after repeated kappa agonist treatment, basal DA levels recover to baseline levels in association with both reduced NAc D2 receptor density and tolerance to quinpirole-induced decreases in NAc DA levels (Acri et al., 2001; Izenwasser et al., 1998). These findings were interpreted as evidence of compensatory downregulation of D2 autoreceptors to oppose acute kappa agonist effects. In another example, a recent study found that chronic unpredictable stress in rats had no effect on baseline locomotor activity but decreased both NAc D2 receptor density and quinpirole potency to decrease locomotion (Qiao et al., 2019).

This “D2 autoreceptor adaptation” hypothesis describes a pain-related allostatic state of mesolimbic DA signaling that may be sufficient to maintain or restore DA release and normal behavior while rendering the individual vulnerable to further challenges. More studies will be required to explore this hypothesis and consider its implications for treatment. For example, one goal of treatment might be to block or reverse mechanisms by which pain states reduce DA neuronal activity in the first place; such a treatment would render D2 autoreceptor adaptation unnecessary as a compensatory response. Alternatively, if pain-related decreases in DA neuronal activity cannot be blocked, then other treatments might be considered to complement D2 autoreceptor adaptation in maintaining normal levels of DA signaling. For example, the actions of dopamine transporter inhibitor antidepressants (bupropion, amitifadine) might synergize with D2 autoreceptor downregulation to maintain extracellular DA levels; consistent with this possibility, bupropion and amitifadine alleviated pain-related ICSS depression in rats (Legakis et al., 2019; Leitl and Negus, 2016; Miller et al., 2015; Rosenberg et al., 2013), and bupropion also alleviated neuropathic pain in humans (Semenchuk et al., 2001). Altogether, our findings further support the concept that chronic neuropathy can produce lasting perturbations in mesolimbic dopaminergic system function and point to NAc core as a key region in these allostatic adaptations. Elucidation of the neurobiological mechanisms of this allostasis could provide important insights into the development of novel treatments for chronic pain.

Supplementary Material

Selley et al. Supplementary Material_PMC

5. ACKNOWLEDEMENTS

Mr. Rolando Mendez provided technical assistance with the [35S]GTPγS binding assays. This work was supported by National Institutes of Health grant R01-NS070715.

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