Environmental enrichment alleviates neuropathic pain-associated anxiety by enhancing the function of parvalbumin interneurons in the anterior cingulate cortex

Abstract

Chronic neuropathic pain is often accompanied with comorbid anxiety. However, effective interventions for this anxiety are highly limited. This study aims to examine the effect of environmental enrichment (EE) on spared nerve injury (SNI)-induced neuropathic pain-associated anxiety behaviors and explore the mechanisms underlying this effect. EE effectively ameliorated anxiety-like behaviors followed by SNI. EE also significantly reversed the phenotypic loss of parvalbumin (PV) interneurons in the anterior cingulate cortex (ACC) and impaired gamma oscillations under SNI-induced neuropathic pain conditions. In addition, EE reversed the SNI-induced reduction in the number of PV puncta around Ca²⁺/calmodulin-dependent protein kinase II-positive neurons. Furthermore, enhancing the function of PV interneurons could effectively improve the SNI-caused anxiety-like behaviors. In contrast, the inhibition of PV interneurons led to anxiety-like behaviors in naïve mice. Our findings suggest that EE significantly improves anxiety-like behaviors under neuropathic pain conditions, likely by enhancing the function of PV interneurons in ACC.

Keywords: Environmental enrichment; Gamma Oscillation; Neuropathic pain; PV interneurons.

Fig. 1

Environmental enrichment effectively reduced anxiety-like behaviors in mice with neuropathic pain (A) Schematic of the experimental timeline (Created with bioRender.com). (B) Body weight monitoring during the experiment. (C) Paw withdrawal threshold to mechanical stimulation in the plantar test. (D) Paw withdrawal latency to heat stimulation in the plantar test. (E) Trajectories of mice in the OFT. (F) Total distance traveled throughout the area in the OFT. (G) Time spent in the central area of the OFT. (H) Trajectories of mice in the EPMT. (I) Total distance in the EPMT. (J) Time spent in open arms in the EPMT. (K) Schematic of novelty-suppressed feeding test. (L) Latency to initiate feeding in the NSFT. (M) Food consumption in the NSFT. All data represent the mean ± SEM (n = 12 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group; ^#p < 0.05 versus SNI group.

Fig. 2

The phenotypic loss of PV interneurons in the ACC induced by SNI is alleviated by EE (A) Representative images of PV-positive cells in the ACC in different groups of mice. (B) Quantitative results of the number of PV-positive cells contained in the image per square millimeter. (C) Quantitative results of relative fluorescence intensity of PV-positive cells under the same photographic parameters. Scale bars: 50 μm. All data represent the mean ± SEM (n = 9 sections from 3 mice per group). *p < 0.05, ***p < 0.001 vsersus the sham group; ^#p < 0.05 versus SNI group.

Fig. 3

EE improved the gamma oscillations of PV interneurons in ACC of mice with neuropathic pain-associated anxiety (A–B) Representative trace of neural oscillations and power spectral density. (C) Power spectral density analysis of the basal LFP. (D–F) Quantification of fast/medium/low gamma power in different groups. Data are presented as the mean ± SEM (n = 9–12 mice per group). *p < 0.05, versus the sham group, ^#p < 0.01, versus SNI group.

Fig. 4

Effects of EE on CaMKII-positive neurons and pericellular PV points in ACC of mice with neuropathic pain-associated anxiety (A) Representative immunofluorescence images of anti-CaMKIIα and anti-PV co-staining. (B) Quantitative analysis of PV puncta around CaMKII-positive cells. The scale of the overall image is 20 μm, and the scale of the local enlarged image is 10 μm. Data are presented as the mean ± SEM (n = 15 CaMKII cells from 3 mice per group). ***p < 0.001, versus the sham group, ^###p < 0.001 versus the SNI group.

Fig. 5

Chemogenetic activation of PV interneurons alleviated anxiety-like behaviors in mice with neuropathic pain (A) Schematic of the experimental design. (B) Diagram of viral microinjection. (C) Representative image showing the mCherry expression in the ACC (whole figure scale bars: 200 μm, local figure scale bars: 20 μm). (D) Representative images of PV interneurons (green) merged with mCherry (red) in the ACC (scale bars: 20 μm). (E) Quantitative analysis of the proportion of overlapping cells in total PV⁺ cells, and the proportion of overlapping cells in total mCherry⁺ cells. (F–G) Time courses showing paw withdrawal thresholds to von Frey filaments and withdrawal latency to heat stimulation. (H) Trajectories of mice in the OFT. (I) Total distance traveled throughout the arena in the OFT. (J) The time spent in the central area of the OFT. (K) Trajectories of mice in the EPMT. (L) Total distance traveled throughout the arena in the EPMT. (M) Time spent in the open arm in the EPMT. (N) Latency to initiate feeding in NSFT. (O) Food consumption in NSFT. All data represent the mean ± SEM (n = 8–10 mice per group). ^&&&p < 0.001 versus baseline; *p < 0.05, ***p < 0.001 versus mCherry + CNO group; ^#p < 0.05, ^###p < 0.001 versus hM3Dq + VEH group.

Fig. 6

Chemogenetic inhibition of PV interneurons caused anxiety-like behaviors in naïve mice (A) Schematic of the experimental design. (B) Diagram of virus microinjection. (C) Representative image showing the mCherry expression in the ACC (whole figure scale bars: 200 μm, local figure scale bars: 20 μm). (D) Representative images of PV interneurons (green) merged with mCherry (red) in the ACC (scale bars: 20 μm). (E) Quantitative analysis of the proportion of overlapping cells in total PV⁺ cells, and the proportion of overlapping cells in total mCherry⁺ cells. (F–G) Time courses showing paw withdrawal thresholds to von Frey filaments and withdrawal latency to heat stimulation. (H) Trajectories of mice in the OFT. (I) Total distance traveled throughout the arena in the OFT. (J) Time spent in the central area of the OFT. (K) Trajectories of mice in the EPMT. (L) Total distance traveled throughout the arena in EPMT. (M) Time spent in open arms in the EPMT. (N) Latency to initiate feeding in NSFT. (O) Food consumption in NSFT. All data represent the mean ± SEM (n = 8–10 mice per group). *p < 0.05, ***p < 0.001 versus mCherry + CNO group; ^#p < 0.05, ^###p < 0.001 versus hM4Di + VEH group.

Fig. 7

A schematic diagram depicting EE affects neuropathic pain-related anxiety by regulating the function of PV interneurons in ACC. (Image created with Figdraw.com, with permission).