The subthalamic nucleus-ventral pallidum projection targeting cholinergic circuits modulates chronic pain

Abstract

The cholinergic system shows promise for pain management. Although cholinergic neurons in the ventral pallidum (VP) are involved in neuropathic pain, the anatomical organization and pharmacology of the cholinergic circuit contributing to the processing of acute and chronic pain remain unknown. In the present study, we demonstrate that VP cholinergic neurons in mice received pain signals from and mediated pain modulation by glutamatergic (Glu) neurons in the subthalamic nucleus (STN) in both physiological and neuropathic pain conditions. 

Introduction

Chronic pain is a major public health problem. Approximately 10%−15% of global population has been suffered from debilitating chronic pain with seriously degraded quality of life. In contrast, the currently available therapies with opioid and non-steroid anti-inflammatory drugs fail to meet the demand for controlling symptoms of chronic pain without causing intolerable side effects.

Materials and methods

Ethics statement

The experimental protocols in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Xuzhou Medical University under the Regulations for the Administration of Affairs Concerning Experimental Animals (1988) in China. The approval number is 202208S120

Fiber photometry

Fiber photometry was performed with an instrument (ThinkerTech, Nanjing, China) to monitor GCaMP6 (a green-fluorescent calcium sensor) or eYFP signal in VP ChAT neurons and GACh (a green-fluorescent sensor for acetylcholine) or eYFP signal in the VP . 5–6 mice in each group were examined.

Open field test

Each mouse was placed in the center of a square open field arena (30 × 30, 20 cm in height) and allowed to explore freely for 6 min, while their activities were recorded from a video camera above the arena, which was controlled by Ethovision XT 14.0 software

Concluson 

Single Cell Sequence Specific Amplification Kit was used to reversely transcribe mRNA samples into cDNA according to the manufacturer’s manual (VazymE, China). To improve detection sensitivity, target genes were pre-amplified using a multiplex PCR pre-amplification protocol. The reaction program was as follows: reverse transcription at 50 °C for 60 min; initial denaturation at 95 °C for 3 min

Article information

Citation: Ji Y-W, Xu X-Y, Zhou Q-Q, Cao Y-N, Zhao X-J, Zhang K-R, et al. (2026) The subthalamic nucleus-ventral pallidum projection targeting cholinergic circuits modulates chronic pain. PLoS Biol 24(8): e3003923. https://doi.org/10.1371/journal.pbio.3003923

Academic Editor: Guang Yang, Columbia University Irving Medical Center, UNITED STATES OF AMERICA

Received: September 5, 2025; Accepted: July 13, 2026; Published: August 3, 2026

Copyright: © 2026 Ji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data in the study are included in S1 Data as supplementary information submitted with this manuscript.

Funding: This work was supported by the National Natural Science Foundation of China (https://www.nsfc.gov.cn/) (82071231 to CX; 82271293 to CX; 82471247 to CX; 82171235 to CZ; 82371242 to CZ), the Fund for Jiangsu Province Specially-Appointed Professor (to CX and CZ), Science Fund for Distinguished Young Scholars of Jiangsu Province (https://kxjst.jiangsu.gov.cn/) (BK20240054 to CY), the Natural Science Foundation of Jiangsu Province (https://kxjst.jiangsu.gov.cn/) (BK20211349 to CZ), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (https://info.jse.edu.cn) (23KJA320006 to CZ; 23KJA320007 to CX), Construction Project of High Level Hospital of Jiangsu Province (http://wjw.jiangsu.gov.cn) (LCZX202503 to SL), the Leadership Program in Xuzhou Medical University (https://www.xzhmu.edu.cn/) (JBGS202203 to CX), Basic Research Program of Jiangsu (https://kxjst.jiangsu.gov.cn/) (BK20251114 to YWJ), China Postdoctoral Science Foundation (https://www.chinapostdoctor.org.cn/bshjjh) (2025M782032 to YWJ), Excellent postdoctoral program of Jiangsu Province (https://www.jssrcfwypt.org.cn/web/cdsv/rcbs/bsh) (2025ZB633 to YWJ), the Postgraduate Innovation Program in Jiangsu Province (https://jyt.jiangsu.gov.cn) (KYCX22_2935 to YWJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No authors received a salary from the funders.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: ACh, acetylcholine; ACSF, artificial cerebrospinal fluid; AUC, area under the curve; CNO, clozapine-N-oxide; DB, diagonal band of Broca; EPM, Elevated plus maze; EPSCs, excitatory postsynaptic currents; FST, forced swim test; Glu, glutamatergic; MEC, mecamylamine; nAChRs, nicotinic acetylcholine receptors; NBM, nucleus basalis of Meynert; OFT, open field tests; PBS, phosphate-buffered saline; PPN, pedunculopontine tegmental nucleus; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; qRT-PCR, quantitative real-time PCR; sACSF, sucrose-based artificial cerebral spinal fluid; SD, standard deviation; SEM, standard error of the mean; SNI, Spared nerve injury; STN, subthalamic nucleus; TST, tail suspension test; TTX, tetrodotoxin; VP, ventral pallidum; 4-AP, 4-aminopyridine