A Neural Circuit From Paraventricular Nucleus of the Thalamus to the Nucleus Accumbens Mediates Inflammatory Pain in Mice

Abstract

Background: Pain is a prevalent comorbidity in numerous clinical conditions and causes suffering; however, the mechanism of pain is intricate, and the neural circuitry underlying pain in the brain remains incompletely elucidated. More research into the perception and modulation of pain within the central nervous system is essential. The nucleus accumbens (NAc) plays a pivotal role in the regulation of animal behavior, and extensive research has unequivocally demonstrated its significant involvement in the occurrence and development of pain. NAc receives projections from various other neural nuclei within the brain, including the paraventricular nucleus of the thalamus (PVT). In this experiment, we demonstrate that the specific glutamatergic neural circuit projection from PVT to NAc (PVT^Glut→NAc) is implicated in the modulation of inflammatory pain in mice.

Methods: We compared the difference in pain thresholds between complete Freund's adjuvant (CFA)-induced inflammatory pain models and controls. Then in a well-established mouse model of CFA-induced inflammatory pain, immunofluorescence staining was utilized to evaluate changes in c-Fos protein expression within PVT neurons. To investigate the role of PVT^Glut→NAc in the modulation of pain, we used optogenetics to modulate this neural circuit, and nociceptive behavioral tests were employed to investigate the functional role of the PVT^Glut→NAc circuit in the modulation of inflammatory pain.

Results: In the mice with the inflammatory pain group, both the paw withdrawal latencies (PWLs) and paw withdrawal thresholds (PWTs) of the right hind paw were decreased compared to the control group. In addition, compared to the control group, CFA-induced inflammatory pain led to increased c-Fos protein expression in PVT, which means that some of the neurons in this area of the brain region have been activated. Following the injection of retrograde transport fluorescent-labeled virus into NAc, glutamatergic neurons projecting from the PVT to NAc were observed, confirming the projection relationship between PVT and NAc. In the experiments in optogenetic regulation, normal mice exhibited pain behavior when the PVT^Glut→NAc circuit was stimulated by a 473 nm blue laser, resulting in decreased PWLs and PWTs compared to the control group, which means activating this neural circuit can lead to painful behaviors. In the CFA-induced pain group, inhibition of the PVT^Glut→NAc circuit by a 589 nm yellow laser alleviated pain behavior, leading to increased PWLs and PWTs compared to the control group, representing the fact that inhibition of this neural circuit relieves pain behaviors.

Conclusions: The findings unveil a pivotal role of the PVT^Glut→NAc circuit in modulating inflammatory pain induced by CFA in mice.

Keywords: inflammatory pain; nucleus accumbens; optogenetics; paraventricular nucleus of the thalamus.

FIGURE 1

Changes of pain behaviors and c‐Fos at 4 h and 3 days in inflammatory pain mice. (A, B) PWLs and PWTs of saline and CFA mice (mean ± SEM; two‐way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, n = 6, six mice). The PWLs and PWTs in mice injected with CFA were significantly reduced. (C) Coronal sections at different levels showed the expression of c‐Fos protein in PVT brain regions of mice in the saline group, CFA 4 h group, and CFA 3‐day group (scale bar = 100 µm). (D) Quantitative analysis of c‐Fos protein expression in PVT brain regions (mean ± SEM; t‐test *p < 0.05, **p < 0.01).

FIGURE 2

The reverse transport fluorescently labeled virus injecting in the NAc brain region was expressed in PVT glutamatergic cell bodies, and most of them were c‐Fos protein positive expression. (A) Schematic diagram of virus injection and the experimental procedure schedule. (B) Reverse transport of fluorescently labeled viruses injecting in NAc (AP: +1.4 mm; LM: ±0.8 mm; DV: −4.6 mm, 0°) expressed in the NAc axon terminals and PVT (Bregma: −1.3 mm) glutamatergic cell bodies (I and III, scale bar = 200 µm; II and IV, scale bar = 100 µm), demonstrating the glutamatergic neurons in the PVT projecting to the NAc. (C) Schematic diagram of virus injection and the experimental procedure schedule. (D) The c‐Fos protein expression in the PVT brain region with fluorescently labeled virus retrograde projection of NAc to PVT glutamatergic neurons (scale bar = 100 µm), indicating a potential association between the PVT^Glut→NAc circuit and inflammatory pain.

FIGURE 3

Activation of PVT^Glut→NAc projection induces hyperalgesia‐like behaviors. (A) Experiment protocol. (B) Schematic illustration of virus injection and optical fiber implantation (Scale bar = 200 µm). (C) Optogenetic stimulation pattern. (D, E) PWLs and PWTs measured during BL (baseline)‐Laser ON‐laser OFF (mean ± SEM; two‐way ANOVA; *p < 0.05, **p < 0.01, n = 6, eight mice). (F and G) PWLs and PWTs measured during BL‐DAY1‐DAY3‐DAY5‐OFF (mean ± SEM; two‐way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, n = 6, eight mice). These findings suggest that activation of the PVT^Glut→NAc circuit can induce nociceptive behaviors in naive mice.

FIGURE 4

Inhibition of PVT^Glut→NAc projection remits pain in inflammatory pain mice. (A) Experiment protocol. (B) Schematic illustration of virus injection and optical fiber implantation (scale bar = 200 µm). (C) Laser stimulation pattern. (D, E) PWLs and PWTs measured during BL‐Laser ON‐laser OFF (mean ± SEM; two‐way ANOVA; n = 6, eight mice). PWLs and PWTs in CFA mice were not ascended upon optogenetic inhibition of the PVT^Glut→NAc neural circuit with single light stimulation. (F, G) PWLs and PWTs measured during BL‐DAY1‐DAY3‐DAY5‐OFF (mean ± SEM; two‐way ANOVA; *p < 0.05, **p < 0.01, ****p < 0.0001, n = 6, eight mice). These findings suggest that inhibiting the PVT^Glut→NAc circuit with periodic light stimulation can alleviate pain in mice with inflammatory pain.