Comprehensive Preclinical Assessment of Sensory, Functional, Motivational-Affective, and Neurochemical Outcomes in Neuropathic Pain: The Case of the Sigma-1 Receptor

Abstract

Chronic pain remains a major health problem and is currently facing slow drug innovation. New drug treatments should address not only the sensory-discriminative but also functional and motivational-affective components of chronic pain. In a mouse model of neuropathic pain induced by partial sciatic nerve ligation (PSNL), we analyzed sensory and functional-like outcomes by hindpaw mechanical stimulation and automated gait analysis (CatWalk). We characterized over time a reward-seeking task based on diminished motivation for natural reinforcers (anhedonic-like behavior). To differentiate the appetitive ("wanting") and consummatory ("liking") aspects of motivational behavior, we quantified the latency and number of approaches to eat white chocolate, as well as the eating duration and amount consumed. We explored a putative chronic pain-induced dysregulation of monoamine function by measuring monoamine levels in the nucleus accumbens (NAc), a well-known brain reward area. Finally, we investigated the role of sigma-1 receptor (σ₁R) modulation, a nonopioid target, in these multiple dimensions by genetic deletion and pharmacological dose-response studies. After 6 weeks, PSNL increased the approach latency and reduced the consumption of white chocolate in 20-25% of the mice, while around 50-60% had one or the other parameter affected independently. After 10 weeks, sham-operated mice also displayed anhedonic-like behavior. PSNL was associated with reduced extracellular baseline dopamine and increased norepinephrine in the NAc and with a suppression of increased dopamine and serotonin efflux in response to the rewarding stimulus. Genetic and pharmacological blockade of σ₁R relieved these multiple alterations in nerve-injured mice. We comprehensively describe sensory, functional, and depression-like impairment of key components of motivated behavior associated with nerve injury. We provide a neurochemical substrate for the depressed mesocorticolimbic reward processing in chronic pain, with a potentially increased translational value. Our results also highlight σ₁R for the therapeutic intervention of neuropathic pain.

Figure 1

Mechanical hypersensitivity and gait alterations induced by nerve injury in WT and σ₁R^–/– mice. Scheme of experimental protocol for assessing paw mechanical sensitivity following partial sciatic nerve ligation (PSNL) (A). Withdrawal hind paw response to mechanical von Frey filament stimulation in nerve-injured (PSNL) wild-type (WT) (N = 7) (B) and σ₁R^–/– (KO) (N = 13) (C) mice compared with sham-operated mice (WT N = 7; KO N = 13). PSNL induced long-lasting mechanical hypersensitivity (i.e., decreased paw withdrawal threshold to von Frey filament stimulation) in WT but not in σ₁R^–/– mice. WT PSNL mice developed mechanical hypersensitivity in the right ipsilateral paw lasting over 7–8 weeks after nerve injury. Sham operation did not modify hind paw sensory withdrawal thresholds. Data are mechanical thresholds in gram force expressed as mean ± SEM. The response of ipsilateral paws of the PSNL group was compared to sham-operated mice for each genotype and time point. *P < 0.05, PSNL vs sham. Scheme of methods and experimental protocol for PSNL-related gait alterations (D). Gait analysis of WT (N = 12) and σ₁R^–/–mice (N = 12) 6 weeks after PSNL compared with sham-operated (N = 12) and naïve mice (N = 11) (E). Nerve injury-induced changes in gait parameters in WT but not in σ₁R^–/– mice. In WT PSNL mice, static parameters measuring contact of the nerve-injured right hind paw on the CatWalk floor, including print width and contact area, were significantly reduced compared with the left unlesioned contralateral paw. Dynamic gait parameters in WT PSNL mice, including the mean stand and duty cycle were also significantly reduced in the ipsilateral right paw compared with the contralateral left one. Sham operation did not induce significant gait changes, but a trend in some parameters. Gait parameters of the PSNL and sham groups were compared with naïve mice for each genotype and time point. *P < 0.05 vs naïve, ^#P < 0.05, PSNL vs sham. Ratio RH/LH: right/left hindlimb ratio. Duty cycle expresses the stance phase as a percentage of the entire step cycle (stand + swing).

Figure 2

Appetitive and consummatory behavior of white chocolate in nerve-injured WT and σ₁R^–/– mice. Scheme of experimental protocol for PSNL-related alterations of hedonic-like behavior (A). A reward-seeking behavior (RSB) task was applied to evaluate the affective consequences of neuropathic pain. The approach and consumption of white chocolate allowed analyzing the appetitive and consummatory aspects of motivational behavior. Latency to eat (B) was increased (mainly at 6 and 10 weeks after nerve injury), the number of approaches to the white chocolate (C) was reduced (mainly at 6 weeks after nerve injury), the amount of white chocolate consumed (D) was reduced (mainly at 6 and 10 weeks after nerve injury), and eating duration (E) was reduced (mainly at 6 weeks after nerve injury) in WT PSNL (N = 14–28) compared with WT naïve mice (N = 8–20). Sham operation (N = 16–27) had little effect on both appetitive and consummatory aspects of the motivational behavior compared with naïve mice, except at week 10 when some behaviors resemble those of nerve-injured mice. The RSB task was not modified in σ₁R knockout mice submitted to PSNL: no differences in appetitive (F, G) and consummatory (H, I) aspects of the motivational behavior were found between PSNL (N = 7), sham (N = 7), and naïve (N = 5) σ₁R^–/– mice 6 weeks post-surgery (time of maximum alteration of motivational behaviors in WT mice). Each experimental group was measured in separate groups of mice at weeks 1, 3, 6, and 10 post-surgery, once a day for 5 consecutive days. *P < 0.05, PSNL vs naïve; ^#P < 0.05, PSNL vs sham.

Figure 3

Monoamine levels in the shell of the nucleus accumbens of 6 weeks WT and σ₁R^–/– mice following nerve injury and in response to palatable food presentation. Overview of methods and experimental protocol for PSNL-related alterations of monoamine levels (A). Extracellular baseline norepinephrine (NE) (B), dopamine (DA) (F), and serotonin (5-HT) (J) levels in the shell of the nucleus accumbens (NAc) of WT naïve and PSNL mice 6 weeks after surgery. Neve injury increased NE, reduced DA, and did not modify 5-HT levels in NAc of WT mice. Values are mean ± SEM corresponding to 12 fractions (60 min of baseline period time, per each experimental group). *P < 0.05 PSNL vs naïve. Microdialysis measurements of extracellular monoamine levels in the NAc in the presence of palatable food in naïve and 6 weeks PSNL WT mice (C, G, K). White chocolate presentation increased DA and 5-HT but not NE levels in naïve WT mice, whereas the presentation of the palatable food did not modify levels of any monoamine in nerve-injured (PSNL) WT mice. The plots show the mean ± SEM in perfusates (N = 12 per group). Data from each time point were normalized within each individual animal as percent change from baseline. After the stabilization of basal monoamine levels in perfusates, a Petri dish containing white chocolate was provided to mice for 10 min (shadow gap). Monoamine levels were expressed as a percentage of averaged basal values (−15 to −10 min, B1; −10 to −5 min, B2; and −5 to 0 min; B3). *P < 0.05 PSNL vs naïve. Extracellular baseline NE (D), DA (H), and 5-HT (L) levels in the NAc of σ₁R^–/– naïve and PSNL mice 6 weeks after surgery. PSNL increased NE and 5-HT but not DA levels in NAc of σ₁R^–/– mice. *P < 0.05, PSNL vs naïve. Microdialysis measurements of extracellular monoamine levels in the NAc in the presence of palatable food in naïve and 6 weeks PSNL σ₁R^–/– mice (E, I, M). White chocolate presentation increased 5-HT in PSNL but not in naïve σ₁R^–/– mice. The experimental protocols were the same as described for WT mice (N = 12 per each experimental group). *P < 0.05 PSNL vs naïve.

Figure 4

Pharmacological blockade of σ₁R relieves sensory and affective alterations and restores the reward pathway in nerve-injured mice. Threshold mechanical sensitivity (A) was assessed with von Frey filaments in the ipsilateral and contralateral hind paws of PSNL (N = 9) (B) and sham-operated (N = 9) (C) WT mice administered with the σ₁R antagonist E-52862 (20, 40, and 80 mg/kg) daily as of days 15–17 post-surgery according to a Latin Square design. Treatment dose-dependently inhibited mechanical allodynia in nerve-injured mice and had no effect in sham-operated mice. Paw withdrawal threshold measurements were performed 30 min after drug administration. After a 24-h drug washout (WO) period, the hindpaw withdrawal threshold was also measured (day 18 post-injury). Each point and vertical line represent the mean ± standard error of the mean (SEM). ^$P < 0.05. Effect of the σ₁R antagonist E-52862 on neuropathic pain-induced depressive-like behavior 6 weeks after surgery (D–F). We show the results of the animals that presented deficits in the two parameters, latency and consumption (N = 12). Treatment attenuated the approach (latency to eat) and consummatory (amount consumed) parameters in PSNL mice while having no effect on sham-operated mice (N = 58). E-52862 was administered 30 min prior to the RSB test in a Latin Square dose design. Values are mean ± SEM. *P < 0.05, vs vehicle; ^#P < 0.05, vs sham. See the Methods and Materials section and Figure S3 for further details. Microdialysis measurements of extracellular monoamine levels in the NAc in the presence of palatable food in 6 weeks PSNL WT mice (G–J). Treatment increased DA levels when nerve-injured mice were exposed to palatable food but had no effect on NE and 5-HT. The animals were administered intraperitoneally σ₁R antagonist E-52862 (20 mg/kg) 30 min prior to white chocolate presentation. Monoamine levels in perfusates were automatically analyzed at 5-min intervals. The plot shows the mean ± SEM of monoamine levels in perfusates (N = 7–8 per group). After the stabilization of basal monoamine levels in perfusates, a Petri dish containing white chocolate was provided to mice for 10 min. Monoamine levels were expressed as a percentage of averaged basal values (three points, −15 to −10 min to −5 to 0 min). *P < 0.05 vs vehicle.