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

Environmental enrichment alleviates neuropathic pain-associated anxiety by enhancing the function of parvalbumin interneurons in the anterior cingulate cortex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Environmental enrichment alleviates neuropathic pain-associated anxiety by enhancing the function of parvalbumin interneurons in the anterior cingulate cortex Zhuo-Yu Ren, Bao-Yu Han, Li-Yuan Zhao, Xue-Jie Lou, Yuan-Xiang Tao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5295650/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Chronic neuropathic pain is often accompanied with comorbid anxiety. However, effective interventions of 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 could effectively ameliorate 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 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 inhibiting function of PV interneurons led to anxiety-like behaviors in native 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. Biological sciences/Neuroscience/Diseases of the nervous system/Anxiety Biological sciences/Neuroscience/Diseases of the nervous system/Chronic pain Environmental enrichment Neuropathic pain PV interneurons Gamma oscillation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Neuropathic pain often is accompanied with many comorbid symptoms, in which anxiety is the most common. Anxiety often further aggravates neuropathic pain and thus forms a vicious circle between pain and anxiety, resulting in the increased burden in the treatment of these disorders. About 50% of neuropathic pain patients reported the anxiety-like symptoms, up to about 30% of whom have been diagnosed with an anxiety disorder [ 1 , 2 ]. Although the mechanisms underlying the anxiety under neuropathic pain conditions have been studied, there are still no effective treatments for neuropathic pain-associated anxiety [ 3 ]. Therefore, new and effective therapies for comorbid anxiety disorders following peripheral nerve injury are urgent to be developed. Environmental enrichment (EE) is an emerging strategy of nondrug treatment for mental diseases [ 4 , 5 ]. It is a low-cost, side-effect-free, and promising mitigation strategy for many mental disorders. More interestingly, EE has been shown to improve anxiety caused by social stress, sleep deprivation, and even Parkinson’s disease [ 6 – 8 ]. However, it is unclear whether EE can prevent neuropathic pain-associated anxiety. The anterior cingulate cortex (ACC) is one of key brain regions that play a critical role in the mechanisms underlying neuropathic pain and associated comorbid anxiety [ 9 ]. The neurons in the ACC could be reliably activated in various neuropathic pain models[ 10 ]. Increased ACC neuronal excitability leads to nociceptive and anxiety-like behavioral responses following peripheral nerve injury [ 11 ], whereas decreased ACC neuronal excitability significantly reduces abdominal hyperalgesia and anxiety in the rats with pancreatitis [ 12 ]. These data suggest that abnormally increased ACC neuronal excitability plays an important role in the occurrence of neuropathic pain and related anxious behaviors. Therefore, this study examined whether EE improved the neuropathic-pain-associated anxiety behaviors likely through regulating the ACC neuronal excitability. Parvalbumin (PV)-positive neurons account for about half of the GABAergic neurons in the cortex and are implicated in regulating neuronal excitability [ 13 ]. Recent studies reported the role of PV interneurons in regulating Alzheimer’s disease-related anxiety and anxiety-like behaviors under chronic stress conditions in rodents [ 14 , 15 ]. However, whether PV-positive neurons participate in neuropathic pain-associated anxiety is still elusive. The present study was designed to explore the therapeutic effects of EE on neuropathic pain and its associated anxiety-like behaviors and to identify the key role of ACC PV interneurons in the alleviated effect of EE on anxiety-like behaviors under neuropathic pain conditions. 2. Methods 2.1 Animals Adult male C57BL/6J mice weighing 22–26 g (8–12 weeks) were obtained from the SPF (Beijing) Biotechnology Co., Lt. The animals were housed in a temperature- and humidity-controlled room (22°C ± 2°C) in a 12-h light/dark cycle with food and water available ad libitum. The experiments adhered to the Committee for Research and Ethical Issues of the International Association for the Study of Pain (IASP) and the National Research Council’s Guide for the Care and Use of Laboratory Animals guidelines. All procedures were conducted in full compliance with the ARRIVE guidelines. In addition, all animals were euthanized with pentobarbital sodium 150mg/kg before obtaining tissue samples. Mice were randomly divided into three groups: (1) in the sham group, mice only received sham surgery and standard environment; (2) in the spare nerve injury (SNI) group, mice received SNI surgery and standard environment; and (3) in the EE group, mice received SNI surgery and EE treatment. 2.2 SNI model establishment To mimic clinical neuropathic pain, a previously established SNI mouse model was used in this study [ 16 ]. In brief, mice were deeply anesthetized by intraperitoneally injecting 60 mg/kg of sodium pentobarbital. Under aseptic surgical conditions, the left sciatic nerve branches were isolated through blunt dissection of the femoral biceps muscle. The peroneal and tibial nerves were both tightly ligated and transected distal to the ligation, with the intact sural nerve. The overlying muscles and skin were then sutured and sterilized postoperatively. Sham surgery was carried out with identical preceding procedures except for nerve ligation and transection. 2.3 Environmental enrichment (EE) Yehezkel et al.’s protocol was used in our EE strategy. Simply put and large polycarbonate cages (60 × 32 × 38 cm) were equipped with running wheels, toys, a maze-like tube system, houses and nesting batting. The configuration was changed every 2 days. Mice were maintained in this EE condition throughout the 5-week light and dark periods. The standard cages (29 × 18 × 16 cm) were used as a control. 2.4 Paw withdrawal threshold (PWT) PWT to mechanical stimulation was examined by using the up-down method as described previously [ 17 ]. Briefly, the mice were placed inside a transparent plexiglass box on metal mesh and adapted for at least 30 min. The Von Frey filaments were taken and punctured vertically into the lateral plantar skin of the left hindlimb. The same position of each mouse was carefully stimulated. The interval of each stimulation was 2–3 min, lasting no more than 6 s. The paw quick withdrawal (lofting) with or without licking and shaking was considered as positive “X”. If no response was seen, a negative “O” was marked. Following the method of Bonin Robert et al. [ 17 ], a sequence with a combination of “O” and “X” was obtained. Finally, the 50% foot-shrinking reaction threshold (g) was calculated according to the open-source program in the literature [ 18 ]. 2.5 Paw withdrawal latency (PWL) A radiant heat test was performed to assess thermal hyperalgesia using a plantar analgesia tester (Infrared Hot Spur Pain Meter, RWD). Briefly, the mice were placed on the glass pane, and the plantar surface was vertically heated by a 5 mm-diameter laser radiant heat source. PWL was averaged with three consecutive tests with at least 5 min interval. A cut-off value of 20 s was applied to avoid possible tissue damage [ 19 ]. 2.6 Open-field test (OFT) The open-field apparatus is a 50 × 50 × 50 cm box with an open top. Mice were placed in the center of the arena and recorded for 5 min by a ceiling-fixed video camera. The SMART 3.0 video-tracking software was used to record the time spent in the central area and the total distance of movement. The chamber was cleaned with 75% ethanol after each test. 2.7 Elevated plus maze test (EPMT) The apparatus consisted of one central area (5 × 5 cm) and four arms (35 × 5 cm) and was elevated 75 cm above the floor. Two opposite arms were opened without a wall, whereas the other two were enclosed by high walls. Testing was carried out in a dimly lit room with a 40 W bulb hung 60 cm above the central part of the maze. Mice were placed in the center square facing an open arm and were allowed to explore the maze for 5 min. The light intensity was about 165 lux. During the testing period, the number of open-arm entries and the time spent in open arms were recorded using SMART 3.0 video-tracking software. If the mice fell off the elevated maze, the mouse was removed from the study. 2.8 Novelty-suppressed feeding test (NFST) The test was performed in a black homemade box (50 × 50 × 50 cm) with 1 cm-thick bedding at the bottom. All mice were deprived of food but not water for 24 h. At the time of testing, the environment needed to be bright, and a food pellet was placed on a piece of paper at the center of the box. Then, the mice were placed in a corner of the box, and the time when they first picked up and ate food was recorded. The latency to feed was recorded with a maximum time of 5 min. After the mice began to eat food, they were immediately transferred to their respective cages alone. Then, a 5-min food consumption test was conducted to rule out the effect of differences in appetite on feed latency. 2.9 Microelectrode implantation and local field potential recording and analysis Mice were anesthetized with intraperitoneal injecting sodium pentobarbital (60 mg/kg) and mounted on a stereotaxic apparatus (RWD Life Science) in a flat skull position. To completely expose the skull, the muscle, fascia tissue, and periosteum were removed. After the skull justification, three-dimensional coordinates of the ACC region (AP, + 0.7 mm; ML, ± 0.2 mm; DV, − 1.5 mm) were obtained from the mouse brain atlas [ 20 ]. After a 3 × 3 mm size hole was drilled on the skull, the cortex was fully exposed. A four-channel microelectrode used in our experiment was made of 35 µm nichrome alloy with a spacing of 300 µm. The electrode was slowly moved downward through the hole until the predetermined position was reached. The hole was closed with paraffin, and the electrode was fixed on the skull surface with dental acrylic cement. After waking up, the mice were placed in their original cages. After a 1-week recovery under the original environmental conditions, the basal local field potential (LFP) was recorded in the home cage. A digital preamplifier (Plexon Inc., Dallas, TX) was used to transmit the signals, which were then digitized at a sampling rate of 40 kHz. During the recording session, all other electric appliances were turned off to prevent interference. A specific workstation running professional electrophysiological analysis software was used for data processing. For the LFP analysis, wideband recordings were down-sampled at 1250 Hz. In our study, the bands were filtered as follows: slow gamma, 30–50 Hz; medium gamma, 50–80 Hz; and fast gamma, 80–140 Hz. NeuroExplorer 5 (Plexon Inc., Dallas, TX) was used for all data analyses. 2.10 Viral injection Briefly, mice were intraperitoneally injected with 60 mg/kg of sodium pentobarbital and then restrained in a stereotaxic apparatus (RWD Life Science). The skulls were fully exposed to locate bregma and lambda and drilled using a dental drill (WPI, OmniDrill35) at the target location. For chemogenetic manipulations, rAAV-fPV-CRE-bGH PA (titer, 2.64 × 10 12 µg/ml) and rAAV-Ef1α-DIO-hM3Dq-mCherry-WPRE (titer, 2.72 × 10 12 µg/ml) were premixed at a ratio of 1:1 for the specific activation of PV interneurons, and rAAV-fPV-CRE-bGH PA (titer, 2.64 × 10 12 µg/ml) and rAAV-Ef1α-DIO-hM4Di-mCherry-WPRE (titer, 2.72 × 10 12 µg/ml) were premixed at a 1:1 ratio for the specific inhibition of PV interneurons. A 200 nl volume of the virus was injected into the ACC (AP: +0.7 mm, ML: ±0.2 mm, DV − 1.5 mm) with a Hamilton syringe (needle gauge 31) connected with a micro-syringe pump controller (WPI, USA) at a rate of 20 nl/min. Thereafter, the needle was held for an additional 5 min and then slowly withdrawn to allow diffusion. Finally, the skin was sewn back together. A 4-week expression was allowed for all viruses for maximum results. Clozapine-N-oxide (CNO, 2 mg/kg, Wuhan Brain TVA) was dissolved in 0.5% dimethyl sulfide (Sigma) to activate or inhibit the PV interneuron function (the CNO group). The control group only used the vehicle solvent without adding CNO (the VEH group). All viruses and drugs used in this study were provided by Wuhan BrainTVA Co., Ltd. 2.11 Immunofluorescence examination Mice were anesthetized by intraperitoneally injecting 60 mg/kg of sodium pentobarbital, transcranially perfused by phosphate buffer saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) on postoperative day 35. The brain was collected and post-fixed in the 4% PFA overnight and dehydrated in 20% and 30% sucrose at 4°C overnight. Then 25 µm-thick coronally sections of the ACC were collected. After being blocked with 10% normal goat serum in PBS for 1 h at room temperature, the sections were incubated with primary antibodies including mouse anti-Ca²⁺/calmodulin-dependent protein kinase IIα (CaMKIIα; 1:1000, Abcam) and rabbit anti-PV (1:500, Abcam) in 10% norm goat serum plus PBS at 4°C overnight. After being washed with PBS for 4 × 5 min, the sections were exposed to the secondary antibody goat anti-rabbit (1:500, Proteintech) or goat anti-mouse (1: 500, Proteintech) for 1 h at room temperature. A fluorescence microscope (Olympus, FV1000) was used to capture fluorescent images with fluorescence intensity calculated by Image J Software. When fluorescence intensity was compared, the same exposure parameters are used in all sections. 2.12 Statistical analysis All statistical analyses were performed using GraphPad Prism for Windows version 8.0. All data were presented as mean and standard error (mean ± SEM). The Shapiro–Wilk test was performed to determine the normality for the parametric test. Student’s t-test was conducted to examine differences between two groups, while two-way analysis of variance followed by the Bonferroni post hoc test was used to examine the difference between multiple groups. A statistically significant difference was defined as a two-sided p-value < 0.05. 3 Results 3.1 Environment enrichment effectively reduced anxiety-like behaviors in neuropathic pain mice Figure 1 A showed the flow chart of the experimental design. No significant differences in mouse weights of the mice were seen among three groups during the observation period (Fig. 1 B; p > 0.05). Compared with the sham group, PWT and PWL in the SNI group and EE group were significantly lower on days 7, 14, 21, 28, and 35 post surgery (p < 0.001, Fig. 1 C-D). No significant difference was observed between the SNI group and EE group. These results indicates that EE treatment may not affect the SNI-induced mechanical allodynia and thermal hyperalgesia. In the OFT, the movement track of mice was shown in Fig. 1 E. No significant differences were observed in the total distance of mice among three groups (p > 0.05, Fig. 1 F). as expected, the mice in the SNI group spent less time in the central area compared with that in the sham group (p < 0.01, Fig. 1 G). However, EE significantly blocked the SNI-induced reduction in the time spent in the central area (p 0.05, Fig. 1 I). As predicted, compared with the sham group, mice in the SNI group had a significant reduction in time spent in open arms (p < 0.05, Fig. 1 J). However, EE significantly attenuated the SNI-induced reduction in the duration spent in the open arms (p < 0.05, Fig. 1 J). In the NFST (Fig. 1 K), compared with the sham group, the latency time to feed was significantly increased in the SNI group (p < 0.01, Fig. 1 L). EE significantly reduced the latency time of SNI mice to feed (p 0.05, Fig. 1 M). Taken together, our findings indicate that EE exerts an improving effect on neuropathic pain-associated anxiety. 3.2 Effect of the EE on the SNI-induced reductions of PV-positive interneurons in ACC Figure 2 A shows a representative image of immunofluorescence. The immunofluorescence results depicted that the number of PV-positive interneurons in the ACC region was significantly reduced in the SNI group compared with that in the sham group (p < 0.001, Fig. 2 B). Moreover, the relative fluorescence intensity of PV-positive neurons in ACC was also markedly reduced in the SNI group comparing to the sham group (p < 0.05, Fig. 2 C). Interestingly, The EE treatment blocked the SNI-induced reductions in number and intensity of PV-positive neurons (p < 0.05, Fig. 2 B). 3.3 Effect of the EE on the SNI-induced decrease in fast gamma in the PV neurons of ACC PV interneurons regulate the synchronization of excitatory neurons through producing gamma oscillations [ 13 ]. Gamma oscillation was recorded and analyzed in awake, freely moving mice to further evaluate the role of PV interneurons in the anxiety-like symptoms after SNI. Figure 3 A revealed the power spectral density in the ACC, and Fig. 3 B displayed representative images of the local field potential. Figure 3 C depicted the power spectral density curve. Power spectral analysis demonstrated that fast gamma power was significantly decreased in the SNI group when compared to the sham group (p < 0.05, Fig. 3 D). This decrease was reversed after EE treatment (fast gamma, p 0.05, Fig. 3 E-F). 3.4 Effects of the EE on the SNI-induced reduction in number of PV puncta around CaMKII α -positive neurons in ACC Figure 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 boutons 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 = 3 per group). ***p < 0.001, vs. the sham group, ##p < 0.01, ###p < 0.001 vs. SNI group. 3.5 Chemogenetic activation of PV interneurons alleviated anxiety-like behaviors in neuropathic pain mice Figure 5 A–B revealed the flow chart of the experimental design and viral injection diagram. The specificity of the virus was examined first. The immunofluorescence results identified the cells with mCherry signals in the ACC (Fig. 5 C), which were highly co-localized with PV-positive cells (Fig. 5 D). These double-labeled neurons (PV + and mCherry + -positive cells) accounted for about 76.8% of the total PV positive cells, and for about 80.0% of the total mCherry positive cells (Fig. 5 E). The PWT and PWL in both vehicle and CNO groups were significantly decreased 7, 14, 21, 28, and 35 days after SNI surgery, (Fig. 5 F-G). In the OFT (Fig. 5 H), no differences in the total distances were observed between two groups in SNI mice (p < 0.05, Fig. 5 I). However, compared to the vehicle-treated SNI group, mice in the CNO group spent significantly longer time exploring the center area during OFT in SNI mice (p 0.05, Fig. 5 L). However, compared to the vehicle-treated SNI mice, the CNO-treated SNI mice spent significantly more time in the open arm (p < 0.001, Fig. 5 M). In the NFST, the latency time of feeding was significantly shorter in the CNO-treated SNI group than in the vehicle-treated SNI group (p 0.05, Fig. 5 O). These findings indicate that enhancing the function of PV interneurons in ACC ameliorates the SNI-associated anxiety-like behaviors. 3.6 Chemogenetic inhibition of PV interneurons increased anxiety-like behaviors in native mice To further examine whether PV interneuron function was sufficient for anxiety-like behaviors in mice, we examined the effects of inhibiting PV neuron function. The experimental design and the location of viral injection were shown in Fig. 6 A–B. The viral specificity was shown in Fig. 6 C–D. The double labeled cells (overlap of PV + and mCherry + ) were accounted for about 74.3% of the total PV-positive cells, and for about 81.4% of the total mCherry-positive cells (Fig. 6 E). The chemogenetic inhibition of PV function had no significant effect on basal PWT and PWL in native mice (p > 0.05, Fig. 6 F–G). In the OFT (Fig. 6 H), no significant differences in the total distance were observed between two vehicle- and CNO-treated groups (p > 0.05, Fig. 6 I). Compared to the vehicle group, mice in the CNO group spent significantly less time exploring the central region during OFT (p 0.05, Fig. 6 L). However, compared to the vehicle mice, the CNO mice spent significantly less time in open arms (p < 0.001, Fig. 6 M). In the NFST, the latency time to food was significantly longer in the CNO group than in the vehicle group (p 0.05, Fig. 6 O). These data indicate that the inhibited function of PV interneurons may lead to anxiety-like behaviors in native mice. 4 Discussion In this study, the 5-week EE can reduce anxiety-like behaviors associated with neuropathic pain. In addition, these anxiety-like behaviors may be related to functional inhibition of PV interneurons in ACC. Therapeutic effect of EE on neuropathic pain-associated anxiety may be mediated through enhancing the function of PV interneurons in ACC. Our results revealed that EE significantly improved anxiety-like behaviors after SNI, as evidenced by increased exploration time in the central region of OFT and extended exploration time in the open arm of the EPMT in SNI mice. However, in the pain behavioral test, our results showed that EE did not affect the SNI-induced mechanical allodynia and thermal hyperalgesia. This result contradicts the previous report, which showed that EE attenuated nerve injury-induced hypersensitivity to mechanical and cold stimuli [ 21 ]. This previous work used a model of neurological damage for up to 3 months followed by 2 months of environmental manipulation. In contrast, the present study simultaneously performed pain behavioral tests and environmental manipulation within 35 days after SNI. These differences in timing and duration of intervention may account for the different therapeutic effects of EE on mechanical allodynia. A previous study reported that different EE programs were effective in reducing anxiety, but they may have the distinct effects on neuropathic pain [ 22 ]. Simple EE (only receiving three different objects) did not improve mechanical and cold stimuli hyperalgesia, whereas enhanced EE (receiving five different objects) completely abolished neuropathic pain. Different EE paradigms may have different potential effects on neuropathic pain. Although the present study did not examine the effects of EE on mice in the sham group, our previous work has demonstrated no marked effect of EE on basal behavioral responses [ 23 , 24 ]. The balance between neuronal excitation and inhibition is required for maintaining normal brain function. The loss of this balance may be implicated in neuropathic pain and its accompanying emotional disorders [ 25 ]. Preclinical studies have reported that inhibitory interneuron disorders led to anxiety or depressive behaviors in chronic pain animals [ 22 , 26 , 27 ]. Our research team focused on the function of PV interneurons, the main subtypes of GABAergic interneurons, in ACC and explored their role in generating neuropathic pain -related anxiety in mice. Our immunofluorescence results showed that SNI decreased the number of PV-positive cells in ACC, consistent with that of previous reports [ 28 ]. Notably, chronic inflammatory pain induced by CFA led to a loss of both bilateral PV- and SOM-positive cells in ACC [ 29 ].Moreover, there were significant reductions in anxiety-like behaviors in chronic inflammatory pain rats after activating PV interneurons [ 29 ]. However, the activation of SOM interneurons did not have any effects on pain and pain-related anxiety. Hence, we focused on the effects of PV interneuron function on anxiety-like behaviors under neuropathic pain conditions. By activating or inhibiting PV interneurons in ACC through pharmacogenetics, the activation of PV interneurons can improve neuropathic pain-associated anxiety-like behaviors. Consistently, inhibition of PV interneurons induced anxiety-like behaviors in native mice. In addition, the PV interneuron as inhibitory neurons exerted its physiological function mainly by regulating peripheral excitatory neurons. Our immunofluorescence results also revealed that the PV puncta regulating CaMKII, a marker of glutaminergic neurons in the forebrain, was significantly reduced in mice after SNI. This reduction may further enhance the glutaminergic neuronal activity in ACC. Interestingly, previous studies revealed that the overactivity of excitatory neurons in ACC might contribute to neuropathic pain genesis [ 30 , 31 ]. Our results further support the idea that glutaminergic neuronal hyperexcitability in ACC is a potential causative factor leading to anxiety-like behaviors in neuropathic pain mice and that this hyperexcitability may be partially attributed to the decreased function of PV interneurons. Gamma oscillations are considered to be an important electrophysiological form that is closely related to the activity of PV neurons, in which inhibitory interneurons regulate the information integration of excitatory neurons [ 32 ]. Here, we demonstrated that SNI significantly reduced the fast gamma oscillation power in ACC neurons. However, how SNI affects gamma oscillations in the brain is still unclear. A previous study found that gamma oscillations in the primary somatosensory cortex (S1) were significantly increased during acute pain and were positively correlated with pain intensity [ 33 ]. However, another previous study reported that gamma oscillations were weakened during chronic pain [ 34 ]. Therefore, more relevant experiments should be designed to explore this controversy. In addition, present study did not observe significant changes in the medium and low gamma oscillations. Given that low gamma may be involved in visual processing and that medium gamma may be associated with memory and cognition [ 35 , 36 ], our analysis suggests that these two types of gamma oscillations may not be primarily involved in processing neuropathic pain and associated mood disorders. Our study indicates that the ameliorating effect of EE on neuropathic pain-associated anxiety-like behaviors may be achieved by improving PV interneuron function in ACC. EE has been reported to improve the function of PV interneurons. A previous study showed that EE improved posttraumatic stress disorder by increasing the number of PV interneurons in the hippocampus [ 37 ]. In addition, EE can also promote the increased PV interneurons in stroke mice and improve the recovery of symptoms poststroke [ 38 ]. These results indicate that EE has great potential to improve the function of PV interneurons. Indeed, the data we provided support the central role of the PV interneuron function in ACC in regulating neuropathic pain-associated anxiety. EE as a potential alternative strategy leads to the resilience of neuropathic pain animals to anxiety through enhancing the PV interneuronal activity in ACC. Recent studies have reported that EE can improve anxiety by inhibiting the reduction of PV-positive interneurons in the medial prefrontal cortex caused by separation from the mother early in life [ 39 ]. These data further indicate that EE plays a therapeutic role in neuropathic pain-associated anxiety likely through enhancing the function of PV interneurons. This may provide a theoretical basis for developing treatment options of neuropathic pain-associated anxiety with low side effects in the future. 5 Conclusion This study demonstrated that the decreased function of PV interneurons in ACC may be involved in anxiety-like behaviors under neuropathic pain conditions and reported that EE improved anxiety-like behaviors associated with neuropathic pain by enhancing the PV interneuron functions in ACC (Fig. 7 ). Present study provides the evidence of a nonpharmacological treatment strategy against anxiety-like behaviors in neuropathic pain mice. EE is a promising approach for restoring network homeostasis between excitability and inhibition of neurons in rodents, protecting the brain from the overactivation of ACC excitatory neurons caused by neuropathic pain, and may have potential applications in treating neurological disorders in humans. Declarations Competing Interests The authors declare no conflict of interest. Ethics approval All experiment received approval from the Animal Care and Welfare Committee of Zhengzhou University. Funding The study was supported by The Programme of Introducing Talents of Discipline to Universities of Henan: Anesthesia and Brain Research (No. CXJD2019008), Shandong Province Natural Science Foundation (ZR2022MH216) and the National Natural Science Foundation of China (82401461). Author Contribution Y.J.J. and Z.G.F. designed and supervised the study. R.Z.Y. and H.B.Y. performed the experiment and wrote the main manuscript text. Z.L.Y. and L.X.J. participated in analyzing the results. T.Y.X. edited and revised the manuscript. All authors reviewed and approved the final manuscript. Data Availability The key data are contained in the figures, tables, and additional files. The datasets used or analyzed during this study can be further obtained from the corresponding author on reasonable request References Finnerup, N. B., Kuner, R. & Jensen, T. S. 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Electroacupuncture Ameliorates Chronic Inflammatory Pain-Related Anxiety by Activating PV Interneurons in the Anterior Cingulate Cortex. Front. Neurosci. 15 , 691931 (2021). Li, Y. D. et al. Anterior cingulate cortex projections to the dorsal medial striatum underlie insomnia associated with chronic pain. Neuron . 112 (8), 1328–1341e4 (2024). Zhu, D. Y. et al. The increased in vivo firing of pyramidal cells but not interneurons in the anterior cingulate cortex after neuropathic pain. Mol. Brain . 15 (1), 12 (2022). Cardin, J. A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature . 459 (7247), 663–667 (2009). Zhang, Z. G. et al. Gamma-band oscillations in the primary somatosensory cortex–a direct and obligatory correlate of subjective pain intensity. J. Neurosci. 32 (22), 7429–7438 (2012). Li, Z. et al. Gamma-band oscillations of pain and nociception: A systematic review and meta-analysis of human and rodent studies. Neurosci. Biobehav Rev. 146 , 105062 (2023). Han, C. et al. Multiple gamma rhythms carry distinct spatial frequency information in primary visual cortex. PLoS Biol. 19 (12), e3001466 (2021). Mably, A. J. & Colgin, L. L. Gamma oscillations in cognitive disorders. Curr. Opin. Neurobiol. 52 , 182–187 (2018). Sun, X. R. et al. Amelioration of oxidative stress-induced phenotype loss of parvalbumin interneurons might contribute to the beneficial effects of environmental enrichment in a rat model of post-traumatic stress disorder. Behav. Brain Res. 312 , 84–92 (2016). Inácio, A. R., Ruscher, K. & Wieloch, T. Enriched environment downregulates macrophage migration inhibitory factor and increases parvalbumin in the brain following experimental stroke. Neurobiol. Dis. 41 (2), 270–278 (2011). Irie, K. et al. An enriched environment ameliorates the reduction of parvalbumin-positive interneurons in the medial prefrontal cortex caused by maternal separation early in life. Front. Neurosci. 17 , 1308368 (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Dec, 2024 Reviews received at journal 18 Dec, 2024 Reviewers agreed at journal 10 Dec, 2024 Reviews received at journal 24 Nov, 2024 Reviewers agreed at journal 15 Nov, 2024 Reviewers invited by journal 15 Nov, 2024 Editor assigned by journal 15 Nov, 2024 Editor invited by journal 10 Nov, 2024 Submission checks completed at journal 08 Nov, 2024 First submitted to journal 19 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5295650","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":382032846,"identity":"f7ddfb56-d79e-46ed-84fc-366bfa520819","order_by":0,"name":"Zhuo-Yu Ren","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo-Yu","middleName":"","lastName":"Ren","suffix":""},{"id":382032847,"identity":"5bc7b682-b002-46b3-962d-4340c45d07e9","order_by":1,"name":"Bao-Yu Han","email":"","orcid":"","institution":"Jinan Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bao-Yu","middleName":"","lastName":"Han","suffix":""},{"id":382032848,"identity":"a3115603-6564-4c25-91d0-8307b14244cf","order_by":2,"name":"Li-Yuan Zhao","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Li-Yuan","middleName":"","lastName":"Zhao","suffix":""},{"id":382032849,"identity":"a329e82c-903d-4bff-8bc8-2a7cbbb8bc74","order_by":3,"name":"Xue-Jie Lou","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xue-Jie","middleName":"","lastName":"Lou","suffix":""},{"id":382032850,"identity":"5b494c96-0847-45c8-b2dd-be13b68088db","order_by":4,"name":"Yuan-Xiang Tao","email":"","orcid":"","institution":"The State University of New Jersey","correspondingAuthor":false,"prefix":"","firstName":"Yuan-Xiang","middleName":"","lastName":"Tao","suffix":""},{"id":382032851,"identity":"aa783b6d-5c71-44fc-8f3b-857d0c2c281b","order_by":5,"name":"Guang-Fen Zhang","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guang-Fen","middleName":"","lastName":"Zhang","suffix":""},{"id":382032852,"identity":"37f167ea-48d4-496e-b590-5f989e67c603","order_by":6,"name":"Jian-Jun Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYFCCAyCCjZmfmfngA9K0SLazJRuQZpnBeR4zAeJUHjxj+LngFx+78WEGMwaGGptowloOnDGWntnHxmx2mCHtAcOxtNwGIrQYSPP2gLUcN2BsOEyUFuPfIC3GzYxtEsRqMZPm+cHGbMDMzEacFskDx8qseRvYmCUOA7UlEOMXvhuHN9/m+XMsmb///McHH2psCGtRuHHCgIGx7VgymJdASDkIyPe3P2Bg+FNjR4ziUTAKRsEoGKEAALr5QPg3WfeqAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Jian-Jun","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-10-19 17:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5295650/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5295650/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-95220-6","type":"published","date":"2025-03-24T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69877906,"identity":"ecabaadf-d913-4257-b521-938695a0154e","added_by":"auto","created_at":"2024-11-26 08:40:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnvironmental enrichment effectively reduced anxiety-like behaviors in mice with neuropathic pain \u003c/strong\u003e(A) Schematic of the experimental timeline. (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 EPM. (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 NFST. (M) Food consumption in the NFST. All data represent the mean ± SEM (n = 12 per group). *p \u0026lt;0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. the sham group; #p \u0026lt; 0.05 vs. SNI group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/f52735b61494803a83384415.png"},{"id":69877928,"identity":"fe4a4ca7-93d7-4df4-b146-3f6cc791159f","added_by":"auto","created_at":"2024-11-26 08:40:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phenotypic loss of PV interneurons in the ACC induced by SNI is alleviated by EE\u003c/strong\u003e(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 = 3 per group). *p \u0026lt; 0.05, ***p \u0026lt; 0.001 vs. the sham group; #p \u0026lt; 0.05 vs. SNI group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/b43f1aef3ec54430f7bfbc99.png"},{"id":69877911,"identity":"77dbd3d8-b11c-48fd-a51e-54d497de3b22","added_by":"auto","created_at":"2024-11-26 08:40:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":170491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEE improved the gamma oscillations of PV interneurons in ACC of mice with neuropathic pain-associated anxiety\u003c/strong\u003e (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 = 5 per group). *p \u0026lt; 0.05, vs. the sham group, #p \u0026lt; 0.01, vs. SNI group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/bf1e8eaf8252b73099163432.png"},{"id":69877926,"identity":"24777e72-25e1-4f62-a6bb-17b9c67ca274","added_by":"auto","created_at":"2024-11-26 08:40:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of EE on CaMKII-positive neurons and pericellular PV points in ACC of mice with neuropathic pain-associated anxiety\u003c/strong\u003e (A) Representative immunofluorescence images of anti-CaMKIIα and anti-PV co-staining. (B) Quantitative analysis of PV boutons 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 = 3 per group). ***p \u0026lt; 0.001, vs. the sham group, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001 vs. SNI group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/48614e220a67e236b48e1600.png"},{"id":69878535,"identity":"a6b6298c-9a9a-445b-bc82-3fa97e48adbb","added_by":"auto","created_at":"2024-11-26 08:48:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemogenetic activation of PV interneurons alleviated anxiety-like behaviors in mice with neuropathic pain\u003c/strong\u003e (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\u003csup\u003e+\u003c/sup\u003e cells, and the proportion of overlapping cells in total mCherry\u003csup\u003e+\u003c/sup\u003e 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 = 10 per group). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. VEH group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/fe598adb8738116a79d93e51.png"},{"id":69877907,"identity":"c6a33d1a-1f2c-4b63-9a2d-8dfe80693976","added_by":"auto","created_at":"2024-11-26 08:40:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemogenetic inhibition of PV interneurons caused anxiety-like behaviors in native mice\u003c/strong\u003e (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\u003csup\u003e+\u003c/sup\u003e cells, and the proportion of overlapping cells in total mCherry\u003csup\u003e+\u003c/sup\u003e 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 = 10 per group). *p \u0026lt; 0.05, ***p \u0026lt; 0.001 vs. VEH group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/65b69e956cb7271db5926908.png"},{"id":69877908,"identity":"0db141cc-4223-44a3-937b-cb46d4366fcd","added_by":"auto","created_at":"2024-11-26 08:40:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":56574,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic diagram depicting EE affects neuropathic pain-related anxiety by regulating the function of PV interneurons in ACC\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/bd8b909bc51d5f96ff838368.png"},{"id":79604742,"identity":"70e711bc-b300-4ae6-88bf-f99505b6000e","added_by":"auto","created_at":"2025-03-31 16:03:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2031630,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5295650/v1/c28cd362-8a6d-4e45-ab20-ca0c1380d5ae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Environmental enrichment alleviates neuropathic pain-associated anxiety by enhancing the function of parvalbumin interneurons in the anterior cingulate cortex","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNeuropathic pain often is accompanied with many comorbid symptoms, in which anxiety is the most common. Anxiety often further aggravates neuropathic pain and thus forms a vicious circle between pain and anxiety, resulting in the increased burden in the treatment of these disorders. About 50% of neuropathic pain patients reported the anxiety-like symptoms, up to about 30% of whom have been diagnosed with an anxiety disorder [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although the mechanisms underlying the anxiety under neuropathic pain conditions have been studied, there are still no effective treatments for neuropathic pain-associated anxiety [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, new and effective therapies for comorbid anxiety disorders following peripheral nerve injury are urgent to be developed. Environmental enrichment (EE) is an emerging strategy of nondrug treatment for mental diseases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is a low-cost, side-effect-free, and promising mitigation strategy for many mental disorders. More interestingly, EE has been shown to improve anxiety caused by social stress, sleep deprivation, and even Parkinson\u0026rsquo;s disease [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, it is unclear whether EE can prevent neuropathic pain-associated anxiety.\u003c/p\u003e \u003cp\u003eThe anterior cingulate cortex (ACC) is one of key brain regions that play a critical role in the mechanisms underlying neuropathic pain and associated comorbid anxiety [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The neurons in the ACC could be reliably activated in various neuropathic pain models[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Increased ACC neuronal excitability leads to nociceptive and anxiety-like behavioral responses following peripheral nerve injury [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], whereas decreased ACC neuronal excitability significantly reduces abdominal hyperalgesia and anxiety in the rats with pancreatitis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These data suggest that abnormally increased ACC neuronal excitability plays an important role in the occurrence of neuropathic pain and related anxious behaviors. Therefore, this study examined whether EE improved the neuropathic-pain-associated anxiety behaviors likely through regulating the ACC neuronal excitability.\u003c/p\u003e \u003cp\u003eParvalbumin (PV)-positive neurons account for about half of the GABAergic neurons in the cortex and are implicated in regulating neuronal excitability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recent studies reported the role of PV interneurons in regulating Alzheimer\u0026rsquo;s disease-related anxiety and anxiety-like behaviors under chronic stress conditions in rodents [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, whether PV-positive neurons participate in neuropathic pain-associated anxiety is still elusive. The present study was designed to explore the therapeutic effects of EE on neuropathic pain and its associated anxiety-like behaviors and to identify the key role of ACC PV interneurons in the alleviated effect of EE on anxiety-like behaviors under neuropathic pain conditions.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eAdult male C57BL/6J mice weighing 22\u0026ndash;26 g (8\u0026ndash;12 weeks) were obtained from the SPF (Beijing) Biotechnology Co., Lt. The animals were housed in a temperature- and humidity-controlled room (22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) in a 12-h light/dark cycle with food and water available ad libitum. The experiments adhered to the Committee for Research and Ethical Issues of the International Association for the Study of Pain (IASP) and the National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals guidelines. All procedures were conducted in full compliance with the ARRIVE guidelines. In addition, all animals were euthanized with pentobarbital sodium 150mg/kg before obtaining tissue samples.\u003c/p\u003e \u003cp\u003eMice were randomly divided into three groups: (1) in the sham group, mice only received sham surgery and standard environment; (2) in the spare nerve injury (SNI) group, mice received SNI surgery and standard environment; and (3) in the EE group, mice received SNI surgery and EE treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 SNI model establishment\u003c/h2\u003e \u003cp\u003eTo mimic clinical neuropathic pain, a previously established SNI mouse model was used in this study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In brief, mice were deeply anesthetized by intraperitoneally injecting 60 mg/kg of sodium pentobarbital. Under aseptic surgical conditions, the left sciatic nerve branches were isolated through blunt dissection of the femoral biceps muscle. The peroneal and tibial nerves were both tightly ligated and transected distal to the ligation, with the intact sural nerve. The overlying muscles and skin were then sutured and sterilized postoperatively. Sham surgery was carried out with identical preceding procedures except for nerve ligation and transection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Environmental enrichment (EE)\u003c/h2\u003e \u003cp\u003eYehezkel et al.\u0026rsquo;s protocol was used in our EE strategy. Simply put and large polycarbonate cages (60 \u0026times; 32 \u0026times; 38 cm) were equipped with running wheels, toys, a maze-like tube system, houses and nesting batting. The configuration was changed every 2 days. Mice were maintained in this EE condition throughout the 5-week light and dark periods. The standard cages (29 \u0026times; 18 \u0026times; 16 cm) were used as a control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Paw withdrawal threshold (PWT)\u003c/h2\u003e \u003cp\u003ePWT to mechanical stimulation was examined by using the up-down method as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, the mice were placed inside a transparent plexiglass box on metal mesh and adapted for at least 30 min. The Von Frey filaments were taken and punctured vertically into the lateral plantar skin of the left hindlimb. The same position of each mouse was carefully stimulated. The interval of each stimulation was 2\u0026ndash;3 min, lasting no more than 6 s. The paw quick withdrawal (lofting) with or without licking and shaking was considered as positive \u0026ldquo;X\u0026rdquo;. If no response was seen, a negative \u0026ldquo;O\u0026rdquo; was marked. Following the method of Bonin Robert et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], a sequence with a combination of \u0026ldquo;O\u0026rdquo; and \u0026ldquo;X\u0026rdquo; was obtained. Finally, the 50% foot-shrinking reaction threshold (g) was calculated according to the open-source program in the literature [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Paw withdrawal latency (PWL)\u003c/h2\u003e \u003cp\u003eA radiant heat test was performed to assess thermal hyperalgesia using a plantar analgesia tester (Infrared Hot Spur Pain Meter, RWD). Briefly, the mice were placed on the glass pane, and the plantar surface was vertically heated by a 5 mm-diameter laser radiant heat source. PWL was averaged with three consecutive tests with at least 5 min interval. A cut-off value of 20 s was applied to avoid possible tissue damage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Open-field test (OFT)\u003c/h2\u003e \u003cp\u003eThe open-field apparatus is a 50 \u0026times; 50 \u0026times; 50 cm box with an open top. Mice were placed in the center of the arena and recorded for 5 min by a ceiling-fixed video camera. The SMART 3.0 video-tracking software was used to record the time spent in the central area and the total distance of movement. The chamber was cleaned with 75% ethanol after each test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Elevated plus maze test (EPMT)\u003c/h2\u003e \u003cp\u003eThe apparatus consisted of one central area (5 \u0026times; 5 cm) and four arms (35 \u0026times; 5 cm) and was elevated 75 cm above the floor. Two opposite arms were opened without a wall, whereas the other two were enclosed by high walls. Testing was carried out in a dimly lit room with a 40 W bulb hung 60 cm above the central part of the maze. Mice were placed in the center square facing an open arm and were allowed to explore the maze for 5 min. The light intensity was about 165 lux. During the testing period, the number of open-arm entries and the time spent in open arms were recorded using SMART 3.0 video-tracking software. If the mice fell off the elevated maze, the mouse was removed from the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Novelty-suppressed feeding test (NFST)\u003c/h2\u003e \u003cp\u003eThe test was performed in a black homemade box (50 \u0026times; 50 \u0026times; 50 cm) with 1 cm-thick bedding at the bottom. All mice were deprived of food but not water for 24 h. At the time of testing, the environment needed to be bright, and a food pellet was placed on a piece of paper at the center of the box. Then, the mice were placed in a corner of the box, and the time when they first picked up and ate food was recorded. The latency to feed was recorded with a maximum time of 5 min. After the mice began to eat food, they were immediately transferred to their respective cages alone. Then, a 5-min food consumption test was conducted to rule out the effect of differences in appetite on feed latency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Microelectrode implantation and local field potential recording and analysis\u003c/h2\u003e \u003cp\u003eMice were anesthetized with intraperitoneal injecting sodium pentobarbital (60 mg/kg) and mounted on a stereotaxic apparatus (RWD Life Science) in a flat skull position. To completely expose the skull, the muscle, fascia tissue, and periosteum were removed. After the skull justification, three-dimensional coordinates of the ACC region (AP, +\u0026thinsp;0.7 mm; ML, \u0026plusmn;\u0026thinsp;0.2 mm; DV, \u0026minus;\u0026thinsp;1.5 mm) were obtained from the mouse brain atlas [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After a 3 \u0026times; 3 mm size hole was drilled on the skull, the cortex was fully exposed. A four-channel microelectrode used in our experiment was made of 35 \u0026micro;m nichrome alloy with a spacing of 300 \u0026micro;m. The electrode was slowly moved downward through the hole until the predetermined position was reached. The hole was closed with paraffin, and the electrode was fixed on the skull surface with dental acrylic cement. After waking up, the mice were placed in their original cages.\u003c/p\u003e \u003cp\u003eAfter a 1-week recovery under the original environmental conditions, the basal local field potential (LFP) was recorded in the home cage. A digital preamplifier (Plexon Inc., Dallas, TX) was used to transmit the signals, which were then digitized at a sampling rate of 40 kHz. During the recording session, all other electric appliances were turned off to prevent interference. A specific workstation running professional electrophysiological analysis software was used for data processing. For the LFP analysis, wideband recordings were down-sampled at 1250 Hz. In our study, the bands were filtered as follows: slow gamma, 30\u0026ndash;50 Hz; medium gamma, 50\u0026ndash;80 Hz; and fast gamma, 80\u0026ndash;140 Hz. NeuroExplorer 5 (Plexon Inc., Dallas, TX) was used for all data analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Viral injection\u003c/h2\u003e \u003cp\u003eBriefly, mice were intraperitoneally injected with 60 mg/kg of sodium pentobarbital and then restrained in a stereotaxic apparatus (RWD Life Science). The skulls were fully exposed to locate bregma and lambda and drilled using a dental drill (WPI, OmniDrill35) at the target location. For chemogenetic manipulations, rAAV-fPV-CRE-bGH PA (titer, 2.64 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e \u0026micro;g/ml) and rAAV-Ef1α-DIO-hM3Dq-mCherry-WPRE (titer, 2.72 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e \u0026micro;g/ml) were premixed at a ratio of 1:1 for the specific activation of PV interneurons, and rAAV-fPV-CRE-bGH PA (titer, 2.64 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e \u0026micro;g/ml) and rAAV-Ef1α-DIO-hM4Di-mCherry-WPRE (titer, 2.72 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e \u0026micro;g/ml) were premixed at a 1:1 ratio for the specific inhibition of PV interneurons. A 200 nl volume of the virus was injected into the ACC (AP: +0.7 mm, ML: \u0026plusmn;0.2 mm, DV \u0026minus;\u0026thinsp;1.5 mm) with a Hamilton syringe (needle gauge 31) connected with a micro-syringe pump controller (WPI, USA) at a rate of 20 nl/min. Thereafter, the needle was held for an additional 5 min and then slowly withdrawn to allow diffusion. Finally, the skin was sewn back together. A 4-week expression was allowed for all viruses for maximum results. Clozapine-N-oxide (CNO, 2 mg/kg, Wuhan Brain TVA) was dissolved in 0.5% dimethyl sulfide (Sigma) to activate or inhibit the PV interneuron function (the CNO group). The control group only used the vehicle solvent without adding CNO (the VEH group). All viruses and drugs used in this study were provided by Wuhan BrainTVA Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Immunofluorescence examination\u003c/h2\u003e \u003cp\u003eMice were anesthetized by intraperitoneally injecting 60 mg/kg of sodium pentobarbital, transcranially perfused by phosphate buffer saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) on postoperative day 35. The brain was collected and post-fixed in the 4% PFA overnight and dehydrated in 20% and 30% sucrose at 4\u0026deg;C overnight. Then 25 \u0026micro;m-thick coronally sections of the ACC were collected. After being blocked with 10% normal goat serum in PBS for 1 h at room temperature, the sections were incubated with primary antibodies including mouse anti-Ca\u0026sup2;⁺/calmodulin-dependent protein kinase IIα (CaMKIIα; 1:1000, Abcam) and rabbit anti-PV (1:500, Abcam) in 10% norm goat serum plus PBS at 4\u0026deg;C overnight. After being washed with PBS for 4 \u0026times; 5 min, the sections were exposed to the secondary antibody goat anti-rabbit (1:500, Proteintech) or goat anti-mouse (1: 500, Proteintech) for 1 h at room temperature. A fluorescence microscope (Olympus, FV1000) was used to capture fluorescent images with fluorescence intensity calculated by Image J Software. When fluorescence intensity was compared, the same exposure parameters are used in all sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism for Windows version 8.0. All data were presented as mean and standard error (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). The Shapiro\u0026ndash;Wilk test was performed to determine the normality for the parametric test. Student\u0026rsquo;s t-test was conducted to examine differences between two groups, while two-way analysis of variance followed by the Bonferroni post hoc test was used to examine the difference between multiple groups. A statistically significant difference was defined as a two-sided p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Environment enrichment effectively reduced anxiety-like behaviors in neuropathic pain mice\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA showed the flow chart of the experimental design. No significant differences in mouse weights of the mice were seen among three groups during the observation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Compared with the sham group, PWT and PWL in the SNI group and EE group were significantly lower on days 7, 14, 21, 28, and 35 post surgery (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). No significant difference was observed between the SNI group and EE group. These results indicates that EE treatment may not affect the SNI-induced mechanical allodynia and thermal hyperalgesia. In the OFT, the movement track of mice was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. No significant differences were observed in the total distance of mice among three groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). as expected, the mice in the SNI group spent less time in the central area compared with that in the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). However, EE significantly blocked the SNI-induced reduction in the time spent in the central area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Similarly, in the EPMT, the movement trajectory of mice was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH. No significant differences were observed in the total distance traveled by mice among three groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). As predicted, compared with the sham group, mice in the SNI group had a significant reduction in time spent in open arms (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). However, EE significantly attenuated the SNI-induced reduction in the duration spent in the open arms (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). In the NFST (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK), compared with the sham group, the latency time to feed was significantly increased in the SNI group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). EE significantly reduced the latency time of SNI mice to feed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). No significant difference was observed in the amount of food intake among three groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Taken together, our findings indicate that EE exerts an improving effect on neuropathic pain-associated anxiety.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of the EE on the SNI-induced reductions of PV-positive interneurons in ACC\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows a representative image of immunofluorescence. The immunofluorescence results depicted that the number of PV-positive interneurons in the ACC region was significantly reduced in the SNI group compared with that in the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, the relative fluorescence intensity of PV-positive neurons in ACC was also markedly reduced in the SNI group comparing to the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Interestingly, The EE treatment blocked the SNI-induced reductions in number and intensity of PV-positive neurons (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Effect of the EE on the SNI-induced decrease in fast gamma in the PV neurons of ACC\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePV interneurons regulate the synchronization of excitatory neurons through producing gamma oscillations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Gamma oscillation was recorded and analyzed in awake, freely moving mice to further evaluate the role of PV interneurons in the anxiety-like symptoms after SNI. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA revealed the power spectral density in the ACC, and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB displayed representative images of the local field potential. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC depicted the power spectral density curve. Power spectral analysis demonstrated that fast gamma power was significantly decreased in the SNI group when compared to the sham group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This decrease was reversed after EE treatment (fast gamma, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Interestingly, there are no significant differences in medium gamma and low gamma among three groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Effects of the EE on the SNI-induced reduction in number of PV puncta around CaMKII\u003c/b\u003eα\u003cb\u003e-positive neurons in ACC\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eEffects of EE on CaMKII-positive neurons and pericellular PV points in ACC of mice with neuropathic pain-associated anxiety\u003c/b\u003e (A) Representative immunofluorescence images of anti-CaMKIIα and anti-PV co-staining. (B) Quantitative analysis of PV boutons around CaMKII-positive cells. The scale of the overall image is 20 \u0026micro;m, and the scale of the local enlarged image is 10 \u0026micro;m. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;3 per group). ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, vs. the sham group, ##p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ###p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. SNI group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Chemogenetic activation of PV interneurons alleviated anxiety-like behaviors in neuropathic pain mice\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B revealed the flow chart of the experimental design and viral injection diagram. The specificity of the virus was examined first. The immunofluorescence results identified the cells with mCherry signals in the ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), which were highly co-localized with PV-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These double-labeled neurons (PV\u003csup\u003e+\u003c/sup\u003e and mCherry\u003csup\u003e+\u003c/sup\u003e-positive cells) accounted for about 76.8% of the total PV positive cells, and for about 80.0% of the total mCherry positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The PWT and PWL in both vehicle and CNO groups were significantly decreased 7, 14, 21, 28, and 35 days after SNI surgery, (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G). In the OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), no differences in the total distances were observed between two groups in SNI mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). However, compared to the vehicle-treated SNI group, mice in the CNO group spent significantly longer time exploring the center area during OFT in SNI mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). In the EPMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK), no significant difference in the total distance was observed between two groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). However, compared to the vehicle-treated SNI mice, the CNO-treated SNI mice spent significantly more time in the open arm (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). In the NFST, the latency time of feeding was significantly shorter in the CNO-treated SNI group than in the vehicle-treated SNI group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). However, food consumption did not show significant differences between two groups of SNI mice (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). These findings indicate that enhancing the function of PV interneurons in ACC ameliorates the SNI-associated anxiety-like behaviors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Chemogenetic inhibition of PV interneurons increased anxiety-like behaviors in native mice\u003c/h2\u003e \u003cp\u003eTo further examine whether PV interneuron function was sufficient for anxiety-like behaviors in mice, we examined the effects of inhibiting PV neuron function. The experimental design and the location of viral injection were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;B. The viral specificity was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;D. The double labeled cells (overlap of PV\u003csup\u003e+\u003c/sup\u003e and mCherry\u003csup\u003e+\u003c/sup\u003e) were accounted for about 74.3% of the total PV-positive cells, and for about 81.4% of the total mCherry-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The chemogenetic inhibition of PV function had no significant effect on basal PWT and PWL in native mice (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u0026ndash;G). In the OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), no significant differences in the total distance were observed between two vehicle- and CNO-treated groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Compared to the vehicle group, mice in the CNO group spent significantly less time exploring the central region during OFT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). In the EPMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK), no significant difference in total distance was observed between two groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). However, compared to the vehicle mice, the CNO mice spent significantly less time in open arms (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). In the NFST, the latency time to food was significantly longer in the CNO group than in the vehicle group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN). However, the food consumption was not significantly different between two groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO). These data indicate that the inhibited function of PV interneurons may lead to anxiety-like behaviors in native mice.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, the 5-week EE can reduce anxiety-like behaviors associated with neuropathic pain. In addition, these anxiety-like behaviors may be related to functional inhibition of PV interneurons in ACC. Therapeutic effect of EE on neuropathic pain-associated anxiety may be mediated through enhancing the function of PV interneurons in ACC.\u003c/p\u003e \u003cp\u003eOur results revealed that EE significantly improved anxiety-like behaviors after SNI, as evidenced by increased exploration time in the central region of OFT and extended exploration time in the open arm of the EPMT in SNI mice. However, in the pain behavioral test, our results showed that EE did not affect the SNI-induced mechanical allodynia and thermal hyperalgesia. This result contradicts the previous report, which showed that EE attenuated nerve injury-induced hypersensitivity to mechanical and cold stimuli [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This previous work used a model of neurological damage for up to 3 months followed by 2 months of environmental manipulation. In contrast, the present study simultaneously performed pain behavioral tests and environmental manipulation within 35 days after SNI. These differences in timing and duration of intervention may account for the different therapeutic effects of EE on mechanical allodynia. A previous study reported that different EE programs were effective in reducing anxiety, but they may have the distinct effects on neuropathic pain [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Simple EE (only receiving three different objects) did not improve mechanical and cold stimuli hyperalgesia, whereas enhanced EE (receiving five different objects) completely abolished neuropathic pain. Different EE paradigms may have different potential effects on neuropathic pain. Although the present study did not examine the effects of EE on mice in the sham group, our previous work has demonstrated no marked effect of EE on basal behavioral responses [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe balance between neuronal excitation and inhibition is required for maintaining normal brain function. The loss of this balance may be implicated in neuropathic pain and its accompanying emotional disorders [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Preclinical studies have reported that inhibitory interneuron disorders led to anxiety or depressive behaviors in chronic pain animals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our research team focused on the function of PV interneurons, the main subtypes of GABAergic interneurons, in ACC and explored their role in generating neuropathic pain -related anxiety in mice. Our immunofluorescence results showed that SNI decreased the number of PV-positive cells in ACC, consistent with that of previous reports [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Notably, chronic inflammatory pain induced by CFA led to a loss of both bilateral PV- and SOM-positive cells in ACC [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].Moreover, there were significant reductions in anxiety-like behaviors in chronic inflammatory pain rats after activating PV interneurons [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the activation of SOM interneurons did not have any effects on pain and pain-related anxiety. Hence, we focused on the effects of PV interneuron function on anxiety-like behaviors under neuropathic pain conditions. By activating or inhibiting PV interneurons in ACC through pharmacogenetics, the activation of PV interneurons can improve neuropathic pain-associated anxiety-like behaviors. Consistently, inhibition of PV interneurons induced anxiety-like behaviors in native mice. In addition, the PV interneuron as inhibitory neurons exerted its physiological function mainly by regulating peripheral excitatory neurons. Our immunofluorescence results also revealed that the PV puncta regulating CaMKII, a marker of glutaminergic neurons in the forebrain, was significantly reduced in mice after SNI. This reduction may further enhance the glutaminergic neuronal activity in ACC. Interestingly, previous studies revealed that the overactivity of excitatory neurons in ACC might contribute to neuropathic pain genesis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our results further support the idea that glutaminergic neuronal hyperexcitability in ACC is a potential causative factor leading to anxiety-like behaviors in neuropathic pain mice and that this hyperexcitability may be partially attributed to the decreased function of PV interneurons.\u003c/p\u003e \u003cp\u003eGamma oscillations are considered to be an important electrophysiological form that is closely related to the activity of PV neurons, in which inhibitory interneurons regulate the information integration of excitatory neurons [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Here, we demonstrated that SNI significantly reduced the fast gamma oscillation power in ACC neurons. However, how SNI affects gamma oscillations in the brain is still unclear. A previous study found that gamma oscillations in the primary somatosensory cortex (S1) were significantly increased during acute pain and were positively correlated with pain intensity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, another previous study reported that gamma oscillations were weakened during chronic pain [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, more relevant experiments should be designed to explore this controversy. In addition, present study did not observe significant changes in the medium and low gamma oscillations. Given that low gamma may be involved in visual processing and that medium gamma may be associated with memory and cognition [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], our analysis suggests that these two types of gamma oscillations may not be primarily involved in processing neuropathic pain and associated mood disorders.\u003c/p\u003e \u003cp\u003eOur study indicates that the ameliorating effect of EE on neuropathic pain-associated anxiety-like behaviors may be achieved by improving PV interneuron function in ACC. EE has been reported to improve the function of PV interneurons. A previous study showed that EE improved posttraumatic stress disorder by increasing the number of PV interneurons in the hippocampus [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, EE can also promote the increased PV interneurons in stroke mice and improve the recovery of symptoms poststroke [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These results indicate that EE has great potential to improve the function of PV interneurons. Indeed, the data we provided support the central role of the PV interneuron function in ACC in regulating neuropathic pain-associated anxiety. EE as a potential alternative strategy leads to the resilience of neuropathic pain animals to anxiety through enhancing the PV interneuronal activity in ACC. Recent studies have reported that EE can improve anxiety by inhibiting the reduction of PV-positive interneurons in the medial prefrontal cortex caused by separation from the mother early in life [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These data further indicate that EE plays a therapeutic role in neuropathic pain-associated anxiety likely through enhancing the function of PV interneurons. This may provide a theoretical basis for developing treatment options of neuropathic pain-associated anxiety with low side effects in the future.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study demonstrated that the decreased function of PV interneurons in ACC may be involved in anxiety-like behaviors under neuropathic pain conditions and reported that EE improved anxiety-like behaviors associated with neuropathic pain by enhancing the PV interneuron functions in ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Present study provides the evidence of a nonpharmacological treatment strategy against anxiety-like behaviors in neuropathic pain mice. EE is a promising approach for restoring network homeostasis between excitability and inhibition of neurons in rodents, protecting the brain from the overactivation of ACC excitatory neurons caused by neuropathic pain, and may have potential applications in treating neurological disorders in humans.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e All experiment received approval from the Animal Care and Welfare Committee of Zhengzhou University.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was supported by The Programme of Introducing Talents of Discipline to Universities of Henan: Anesthesia and Brain Research (No. CXJD2019008), Shandong Province Natural Science Foundation (ZR2022MH216) and the National Natural Science Foundation of China (82401461).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.J.J. and Z.G.F. designed and supervised the study. R.Z.Y. and H.B.Y. performed the experiment and wrote the main manuscript text. Z.L.Y. and L.X.J. participated in analyzing the results. T.Y.X. edited and revised the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe key data are contained in the figures, tables, and additional files. The datasets used or analyzed during this study can be further obtained from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFinnerup, N. B., Kuner, R. \u0026amp; Jensen, T. S. Neuropathic Pain: From Mechanisms to Treatment. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cb\u003e101\u003c/b\u003e (1), 259\u0026ndash;301 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeingold, D. et al. 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Neurosci.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 1308368 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Environmental enrichment, Neuropathic pain, PV interneurons, Gamma oscillation","lastPublishedDoi":"10.21203/rs.3.rs-5295650/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5295650/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic neuropathic pain is often accompanied with comorbid anxiety. However, effective interventions of 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 could effectively ameliorate 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 number of PV puncta around Ca\u0026sup2;⁺/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 inhibiting function of PV interneurons led to anxiety-like behaviors in native 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.\u003c/p\u003e","manuscriptTitle":"Environmental enrichment alleviates neuropathic pain-associated anxiety by enhancing the function of parvalbumin interneurons in the anterior cingulate cortex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 08:39:30","doi":"10.21203/rs.3.rs-5295650/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-19T06:59:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-18T05:34:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318206334484474976368930654241409606603","date":"2024-12-11T03:28:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-25T01:34:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82316334186021654341134831735080087693","date":"2024-11-16T00:30:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-15T14:02:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-15T13:59:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-10T14:10:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-08T06:56:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-19T17:14:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"583718ff-98a0-486c-91b3-ad39d32a8b3c","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40683206,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Anxiety"},{"id":40683207,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Chronic pain"}],"tags":[],"updatedAt":"2025-03-31T15:58:25+00:00","versionOfRecord":{"articleIdentity":"rs-5295650","link":"https://doi.org/10.1038/s41598-025-95220-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-24 15:56:57","publishedOnDateReadable":"March 24th, 2025"},"versionCreatedAt":"2024-11-26 08:39:30","video":"","vorDoi":"10.1038/s41598-025-95220-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-95220-6","workflowStages":[]},"version":"v1","identity":"rs-5295650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5295650","identity":"rs-5295650","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}