Microglia disrupt mesolimbic reward circuitry in chronic pain

Abstract

Chronic pain attenuates midbrain dopamine (DA) transmission, as evidenced by a decrease in opioid-evoked DA release in the ventral striatum, suggesting that the occurrence of chronic pain impairs reward-related behaviors. However, mechanisms by which pain modifies DA transmission remain elusive. Using in vivo microdialysis and microinjection of drugs into the mesolimbic DA system, we demonstrate in mice and rats that microglial activation in the VTA compromises not only opioid-evoked release of DA, but also other DA-stimulating drugs, such as cocaine. Our data show that loss of stimulated extracellular DA is due to impaired chloride homeostasis in midbrain GABAergic interneurons. Treatment with minocycline or interfering with BDNF signaling restored chloride transport within these neurons and recovered DA-dependent reward behavior. Our findings demonstrate that a peripheral nerve injury causes activated microglia within reward circuitry that result in disruption of dopaminergic signaling and reward behavior. These results have broad implications that are not restricted to the problem of pain, but are also relevant to affective disorders associated with disruption of reward circuitry. Because chronic pain causes glial activation in areas of the CNS important for mood and affect, our findings may translate to other disorders, including anxiety and depression, that demonstrate high comorbidity with chronic pain.

Keywords: addiction; affective disorder; chronic pain; dopamine; emotion; opioids.

Figure 1.

Loss of opioid- and cocaine-evoked DA release in chronic pain. Morphine (10 mg/kg) failed to stimulate extracellular DA in the PNI group, but treatment with minocycline (PNI+MC; 30 mg/kg) restored morphine-evoked extracellular DA levels. Similarly, cocaine-evoked (10 mg/kg) DA levels were lower in the PNI group compared with the sham group and cocaine-evoked extracellular DA could be recovered by pretreatment with minocycline (PNI+MC). Amphetamine-evoked (1 mg/kg) extracellular DA was not significantly different between PNI and sham groups and was not altered by MC treatment. ***p < 0.001. Error bars indicate SEM. Insets depict area under the curve (AUC) for the entire measurement period of DA dialysis for each group. *p < 0.05. Error bars indicate SEM.

Figure 2.

Systemic opioid- and cocaine-induced place preference in chronic pain. a, Systemic morphine (10 mg/kg) produced a place preference in control and chronic pain (PNI) groups. When animals received limited conditioning trials (one exposure to 10 mg/kg morphine and one to saline), only the sham group displayed a robust place preference. Systemic low dose of morphine (1 mg/kg) produced a trend toward a place preference, but was not significant in either the sham or the PNI group. *p < 0.05, **p < 0.01. Error bars indicate SEM. b, Systemic cocaine (10 mg/kg) produced a place preference in both sham and PNI groups. **p < 0.01, ***p < 0.001. Error bars indicate SEM. c, Morphine (10 mg/kg) and cocaine (10 mg/kg) antinociception was measured using the tail withdrawal assay. Neither morphine nor cocaine antinociception was significantly different between sham or PNI groups. Further, inhibition of microglia in chronic pain animals (PNI+MC) did not affect morphine antinociception. Error bars indicate SEM. n.s., Not significant; %MPE, percent maximum possible effect.

Figure 3.

Loss of mesolimbic-specific opioid- and cocaine-induced place preference in chronic pain. a, Repeated conditioning to intra-VTA DAMGO (1 ng) failed to produce a robust place preference in the PNI groups, whereas a high dose of DAMGO (25 ng) produced a robust place preference in both the sham and PNI groups. Pretreatment with the microglial inhibitor minocycline (30 mg/kg) recovered intra-VTA DAMGO (1 ng) place preference in the PNI group. Top left pictograph depicts intra-VTA injection sites. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate SEM. b, Cocaine (100 μg) administered directly into the NAc produced a place preference in the sham group, but not the PNI group. Left pictograph depicts intra-NAc injection sites. *p < 0.05. Error bars indicate SEM.

Figure 4.

Chronic pain activates microglia in the VTA. a, Microglia were significantly activated in the VTA of the chronic pain (PNI) group and displayed a robust activated phenotypes. Pretreatment with the microglial inhibitor minocycline (MC; 30 mg/kg) blocked microglial activation in the PNI+MC group. **p < 0.01, ***p < 0.001. Error bars indicate SEM. Scale bar, 75 μm. b, Mechanical withdrawal thresholds of the ipsilateral hindpaw were measured with von Frey filaments. The PNI group showed significantly lowered mechanical thresholds 2 weeks after nerve injury. Systemic treatment with the microglial inhibitor MC did not affect evoked pain thresholds. **p < 0.01, ***p < 0.001. Error bars indicate SEM. n.s., Not significant.

Figure 5.

Chronic pain disrupts Cl⁻ transport in VTA GABAergic through a microglia-BDNF signaling pathway. a, KCC2 labeling in the VTA was primarily localized in processes not labeled for TH. Immunolabeling of KCC2 (green) was identified in non-TH (red)-labeled neurons and the labeling was punctate, which is indicative of terminal arborization. Scale bar, 75 μm. The right micrographs represent a single image of a TH+ neuron (middle, arrowhead) surrounded by KCC2-immunoreactive arbors (bottom). The top image is a merge of the bottom two images. b, The chronic pain (PNI) group exhibited decreased KCC2 expression in the VTA that could be recovered by systemic treatment with minocycline (PNI+MC; 30 mg/kg). Left black and white micrographs are representative images of KCC2 labeling in the VTA of sham and PNI groups. The right histogram shows KCC2 quantification. Error bars indicate SEM. *p < 0.05, **p < 0.01. AU, Arbitrary units. c, Top left, KCC2 function was assayed in VTA slices using MQAE FLIM. The VTA was localized with brain atlas coordinates and confirmed with TH labeling (red) after Cl⁻ imaging assay. Bottom left, Representative micrographs showing MQAE loading in GAD65-GFP cells (white arrows) in the VTA (2-photon optical section ∼1 μm). Top right, Pseudocolor images showing lifetime maps from control VTA slices in the presence of 2.5 or 15 mm KCl. d, Representative lifetime plots from VTA slices. e, Graph plotted by averaging the slope in the lifetime plots immediately after administration of 15 mm KCl. The PNI group had significantly slower fluorescence lifetime change rate indicative of a slower transport of Cl⁻ into the cell. Normal transport rate was restored in animals pretreated with minocycline (PNI+MC) or in slices treated with a TrkB antagonist (PNI+anti-trkB). (*p < 0.05; n = 7 animals with at least 2 slices per animal, 14–20 slices/condition, and 70–100 cells, on average 5/slice). Error bars indicate SEM. f, VTA punches from the PNI group showed significantly elevated BDNF protein, which was blocked with pretreatment with MC. *p < 0.05 compared with sham, #p < 0.05 compared with PNI+saline. Error bars indicate SEM.