Berberine alleviates chronic pain-induced anxiety-like behaviors by inhibiting the activation of VLT-projecting cACC (Cg2) neurons

Abstract

Chronic pain is often accompanied by anxiety, and gradually increasing anxiety makes the pain itself more protracted. Berberine has been found to be able to cross the blood-brain barrier to treat psychiatric disorders, but its neurocirculatory mechanisms remain unclear. Here, we found that neurons in cingulate area 2 (Cg2) of the caudal anterior cingulate cortex (cACC), but not in Cg1 of the cACC, projected to the ventral lateral thalamus (VLT). Next, we induced chronic inflammatory pain by plantar injection of complete Freund's adjuvant (CFA) and observed stable anxiety-like behaviors until two weeks postinjection. We specifically activated VLT-projecting cACC (Cg2) neurons in one-week-old CFA-induced mice without anxiety-like behaviors and in normal control mice to induce anxiety-like behaviors. We inhibited the activation of VLT-projecting cACC (Cg2) neurons in two-week-old CFA-treated mice with anxiety-like behaviors and observed that their anxiety-like behaviors were alleviated. On this basis, we further screened the effective dose of berberine for anxiolysis in two-week-old CFA-treated mice. We observed that the effective dose of berberine obtained above decreased the activity of VLT-projecting cACC (Cg2) neurons. The activation of VLT-projecting cACC (Cg2) neurons abrogated the anxiolytic effect of berberine in two-week-old CFA-treated mice.

Fig. 1. Anxiety-like behaviors were evoked two weeks after CFA injection.

a Schematic of the experimental design. b Gross macroscopic view of left hindpaw after CFA or PBS injection into the left hindpaw of mice. c Statistical results of 50% paw withdrawal thresholds (PWTs). d Statistical results of time spent in the open arms in the EPM test. e Representative images of movement trajectory (top) and activity time (bottom) in the EPM test. f, g Statistical results of time spent in the central area and total distance traveled in the OFT. h Representative images of movement trajectory (top) and activity time (bottom) in the OFT. Data are the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.01. CFA complete Freund’s adjuvant, EPM elevated plus maze, OFT open field test.

Fig. 2. After one week of CFA injection, anxiety-like behaviors could be evoked by activation of VLT-projecting cACC (Cg2) neurons.

a Schematic of the experimental design. b, c Schematic for specific infection of VLT-projecting cACC (Cg2) neurons with mCherry or hM3Dq-mCherry. Scale bar, 200 μm. d Representative images showed that intraperitoneal injection of CNO induced c-Fos expression in VLT-projecting cACC (Cg2) neurons with mCherry or hM3Dq-mCherry. Scale bar, 20 μm. e Percentage of labeled mCherry⁺ neurons expressing c-Fos in mCherry+CNO group and hM3Dq-mCherry+CNO group. f Statistical results of time spent in the open arms in the EPM test. g Representative images of movement trajectory (top) and activity time (bottom) in the EPM test. h, i Statistical results of time spent in the central area and total distance traveled in the OFT. j Representative images of movement trajectory (top) and activity time (bottom) in the OFT. Data are the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant. CFA complete Freund’s adjuvant, VLT ventral lateral thalamus, cACC caudal anterior cingulate cortex, Cg2 cingulate area 2, CNO Clozapine N-oxide, EPM elevated plus maze, OFT open field test.

Fig. 3. Inhibition of VLT-projecting cACC (Cg2) neuron activation alleviated anxiety-like behaviors induced by CFA.

a Schematic of the experimental design. b, c Schematic for specific infection of VLT-projecting cACC (Cg2) neurons with mCherry or hM4Di-mCherry. Scale bar, 200 μm. d Representative images showed that intraperitoneal injection of CNO induced c-Fos expression in VLT-projecting cACC (Cg2) neurons with mCherry or hM4Di-mCherry. Scale bar, 20 μm. e Percentage of labeled mCherry⁺ neurons expressing c-Fos in mCherry+CNO group and hM4Di-mCherry+CNO group. f Statistical results of time spent in the open arms in the EPM test. g Representative images of movement trajectory (top) and activity time (bottom) in the EPM test. h, i Statistical results of time spent in the central area and total distance traveled in the OFT. j Representative images of movement trajectory (top) and activity time (bottom) in the OFT. Data are the mean ± SEM; *P < 0.05, **P < 0.01, NS not significant. CFA complete Freund’s adjuvant, VLT ventral lateral thalamus, cACC caudal anterior cingulate cortex, Cg2 cingulate area 2, CNO Clozapine N-oxide, EPM elevated plus maze, OFT open field test.

Fig. 4. BBR alleviated anxiety-like behaviors induced by CFA.

a Schematic of the experimental design. b Statistical results of 50% paw withdrawal thresholds (PWTs). ***P < 0.001 vs. baseline in the same group. c–e Statistical results of time spent in the open arms in the EPM test and the central area in the OFT after different gradient concentrations of BBR (2.5, 5 and 10 mg/kg/day). Data are the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant. BBR Berberine, CFA complete Freund’s adjuvant, EPM elevated plus maze, OFT open field test.

Fig. 5. Inhibition of VLT-projecting cACC (Cg2) neuron activation was required for the effect of BBR on relieving anxiety-like behaviors induced by CFA.

a Schematic of the experimental design. b, c Schematic for specific infection of VLT-projecting cACC (Cg2) neurons with mCherry. Scale bar, 200 μm. d–h Statistical results of time spent in the open arms in the EPM test and the central area in the OFT after intraperitoneal injection of BBR (10 mg/kg/day). i Representative images showed that intraperitoneal injection of PBS or BBR induced c-Fos expression in VLT-projecting cACC (Cg2) neurons with mCherry. Scale bar, 20 μm. j Percentage of labeled mCherry⁺ neurons expressing c-Fos in CFA + PBS group and CFA + BBR group. k Schematic of the experimental design. l, m Schematic for specific infection of VLT-projecting cACC (Cg2) neurons with mCherry or hM3Dq-mCherry. n Statistical results of time spent in the open arms in the EPM test. o Representative images of movement trajectory (top) and activity time (bottom) in the EPM test. p, q Statistical results of time spent in the central area and total distance traveled in the OFT. r Representative images of movement trajectory (top) and activity time (bottom) in the OFT. Data are the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant. VLT ventral lateral thalamus, cACC caudal anterior cingulate cortex, Cg2 cingulate area 2, BBR Berberine, CFA complete Freund’s adjuvant, EPM elevated plus maze, OFT open field test, CNO Clozapine N-oxide.