Nucleus Accumbens MEF2C Mediates the Maintenance of Peripheral Nerve Injury-induced Physiological and Behavioral Maladaptations

Abstract

Pre-clinical and clinical work has demonstrated altered plasticity and activity in the nucleus accumbens (NAc) under chronic pain states, highlighting critical therapeutic avenues for the management of chronic pain conditions. In this study, we demonstrate that myocyte enhancer factor 2C (MEF2C), a master regulator of neuronal activity and plasticity, is repressed in NAc neurons after prolonged spared nerve injury (SNI). Viral-mediated overexpression of Mef2c in NAc neurons partially ameliorated sensory hypersensitivity and emotional behaviors in mice with SNI, while also altering transcriptional pathways associated with synaptic signaling. Mef2c overexpression also reversed SNI-induced neuronal hyperexcitability and sensitized dopamine release after phasic stimulation. Transcriptional changes induced by Mef2c overexpression were different than those observed after desipramine treatment, suggesting a mechanism of action different from antidepressants. Overall, we show that interventions in MEF2C-regulated mechanisms in the NAc are sufficient to disrupt the maintenance of chronic pain states, providing potential new treatment avenues for neuropathic pain.

INTRODUCTION

Chronic pain affects approximately 20% of the United States population [15]. Globally, approximately 30% of the population suffers from chronic pain [32]. Aside from sensory manifestations, chronic pain conditions are also associated with high rates of co-morbid depression and substance abuse [6,14]. This is likely due to commonalities in regional maladaptations, such as changes in connectivity, activity, and volume of several brain regions associated with the regulation of sensory valence and emotion, including the nucleus accumbens (NAc) [60]. Combined, the complex and variable clinical presentations of chronic pain and affective co-morbidities, as well as the multifaceted regional and molecular maladaptive underpinnings of the disease, make it difficult to comprehensively treat all symptoms.

In this study, we chose the NAc as the region of interest as it has a strong clinical predictive value for pain chronification [4,67,68]. Maladaptive activity in the NAc has also been clinically characterized in chronic pain states [38]. Lastly, the role of the NAc in pain processing has been recapitulated in several murine models, increasing the translational relevance of basic science studies of this region [11,18,52,55]. Our group has previously found that molecular mechanisms in the NAc are also crucial for regulating the efficacy of therapeutics that are clinically effective for treating chronic pain, such as opioids and antidepressants [22,45,64].

Aside from opioids and antidepressants, epigenetic remodelers, such as histone deacetylases (HDACs), have been shown to directly modulate neuropathic pain states and control the efficacy of therapeutic compounds [8,16,49]. Their mechanism of action involves changes to chromatin accessibility and direct interactions with transcriptional regulators, such as transcription factors (TFs). Our group has previously demonstrated roles for NAc HDAC1 [53] and HDAC5 [17] in the maintenance of sensory and affective behavioral abnormalities after prolonged nerve injury in mice. However, because the adverse effects of longitudinal systemic HDAC inhibition are not well understood [26], we wanted to investigate specific downstream targets that are affected by changes in HDAC activity.

In the present study, we analyzed an existing RNA sequencing (RNA-seq) dataset of NAc tissue from mice with peripheral nerve injury or sham surgery and identified myocyte enhancer factor 2C (MEF2C), a Class I and IIa HDAC-regulated member of the MEF2 family of TFs that are broadly relevant for neuronal growth/development and plasticity [29,37], as a critical regulator of the persistence of chronic pain. Using several molecular quantification techniques, we identified a general repression of MEF2C in NAc neurons after SNI. To test if the promotion of MEF2C activity affects sensory hypersensitivity in the SNI model, we performed stereotaxic delivery of neuron promoter driven AAV vectors expressing Mef2c or control Gfp to the mouse NAc. Our findings demonstrate that Mef2c overexpression partially alleviates sensory hypersensitivity and anxiety-like behaviors, while also normalizing maladaptive transcriptomic, electrophysiological, and circuit-level dopamine (DA) neurotransmission signatures. Of note, we utilized mice from two different genetic backgrounds and both sexes throughout this study to determine the robustness of our molecular and behavioral findings across these biological variables, which have been shown to differ markedly across injury models [31,43,62]. This study further underpins the role of master regulators of NAc transcriptomic maladaptations in models of prolonged peripheral nerve injury and highlights new avenues for therapeutic interventions.

MATERIALS & METHODS

Animals

C57BL/6J or DBA/2J mice (8-9 weeks old) were obtained from Jackson Laboratories and maintained on a 12-hour light/dark cycle with ad libitum access to food and water. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committees at Mount Sinai and Boston University, and of the Society for Neuroscience. All behavioral testing occurred during the animals’ light cycle. Experimenters were blinded to the experimental group, and the order of testing was counterbalanced during behavioral experiments. Different cohorts of animals were used for various behavioral assays, RNA sequencing studies, and electrophysiology/voltammetry experiments.

Tibial Spared Nerve Injury Surgery

Tibial SNI was performed on the left sciatic nerve under inhaled isoflurane, as described previously [57]. Briefly, using a stereomicroscope, a skin and muscle incision of the left hind limb at mid-thigh level was performed to reach the sciatic nerve. The common peroneal and sural nerves were carefully ligated with 6.0-gauge silk sutures (Patterson Veterinary) and transected, and 1–2 mm sections of these nerves were removed, while the tibial nerve was left intact. Skin and muscle were then closed with 4.0-gauge silk sutures (Patterson Veterinary). In Sham-operated mice, the same procedure was followed, but the nerves were left untouched. Following surgery, mice received two days of topical neomycin.

Stereotaxic Surgeries

Stereotaxic coordinates for bilateral viral vector injections into the NAc were as follows with respect to bregma: AP, 1.60mm; ML: ±1.50mm; DV, −4.40mm at an angle of 10° from the midline. AAV8-hSyn-Mef2c-Gfp and AAV8-hSyn-Gfp were supplied by the Akbarian Lab at the Icahn School of Medicine at Mount Sinai [44]. Approximately 7x10⁹ viral particles were administered into the NAc of each hemisphere. Following surgery, mice received two days of topical neomycin. Viral expression was validated at the end of each experiment by GFP Western blot or Mef2c quantitative real-time polymerase chain reaction (qPCR). We confirmed vector expression in both the core and shell of the NAc by imaging 4% PFA-fixed brain slices four weeks after stereotaxic administration of AAV8-hSyn-Gfp vectors.

von Frey Assay

For the assessment of mechanical allodynia, we used von Frey filaments with ascending forces expressed in grams (Stoetling) as cited [57]. Each filament was applied five times in a row against the ipsilateral hindpaw, with all mice receiving a filament application before returning for the next following to the first mouse. Hindpaw withdrawal or licking was marked as a positive allodynia response. A positive response in three of five repetitive stimuli was defined as the allodynia threshold.

Brush Test

Dynamic mechanical allodynia in SNI and sham mice was assessed with the brush assay, in which a 5/0 caliber paint brush (Royal & Langnickel) was stroked from heel to toe as cited [20]. On week five post-injury, after punctate mechanical allodynia thresholds had partially recovered in Mef2c-SNI mice, a filament was applied to the injured hindlimb once every two minutes, for a total of three applications. Each hindpaw response to a brush application was graded as follows: 0 – no response, 1 – sustained lifting of stimulated paw toward the body, 2 – strong lifting of the stimulated paw above the body level, 3 – flinching or licking the stimulated paw. Scores from the three applications per animal were averaged.

Pin Prick Assay

Noxious signaling in SNI and sham mice was assessed with the pin prick. A 27-gauge needle was affixed to a 2 gram von Frey filament on week 12 post-injury. The needle tip was applied to the injured hindlimb a total of ten times, with at least 2 minutes between each application. Responses were scored as follows: “No response”, “Mild Response” – movement of hindpaw from stimulus without shaking or prolonged leg lifting, “Strong Response” – movement of hindpaw from stimulus with shaking or prolonged leg lifting. Percentages of each response were tallied for statistical analysis.

Hot Plate Assay

A 42°C hot plate was used to assess hypersensitivity to a non-noxious thermal stimulus. Briefly, an animal was placed on a hot plate in a plastic cylindrical enclosure. A cutoff time of 120 seconds was used, and the response latency was recorded upon seeing a positive response. This was defined as a hindpaw shake/lick or a jump.

Cold Plate Assay

Cold allodynia was assessed in SNI and sham mice with the cold plate assay as cited. The cold plate was pre-set to 0°C with a cut-off time of 120 seconds. A camera was used to record the mice during this timeframe, and a blinded scorer performed the following measurements. The primary measure was latency to first withdrawal in the form of hindpaw licking/shaking or jumping [2].

Dry Ice Plantar Assay

Cold hyperalgesia was assessed through the dry ice assay on week 16 post-injury. Briefly, a 3 mL syringe was modified to remove the needle attachment point. The syringe was filled with granulated dry ice and compressed by hand. Animals were placed on a glass stand and dry ice was applied to the bottom of the glass plate directly underneath the injured hindpaw [7]. The time to respond was recorded across three trials with a 20-second cutoff.

Hindleg Guarding Assay

Mice were placed on a grid surface and were lifted by their tail such that they were able to maintain forelimb grip, but hindlimbs were elevated off the surface. A camera was used to record the injured hindlimb for 20 seconds. A blinded scorer then calculated the cumulative time spent guarding within that timeframe, with guarding consisting of tucking the leg to the stomach or under the tail.

Marble Burying Assay

After one hour of acclimation to the testing room, mice were placed in a standard hamster cage filled with 15 cm of corn-cob bedding and topped with 20 glass marbles. After 30 minutes, the mice were removed and the number of marbles fully or partially buried (60% buried) was counted by two blinded observers and the percentage of marbles buried was calculated. Marbles that were covered more than 60% were counted as buried.

Voluntary Wheel Running Assay

We used a wireless running wheel activity monitoring system (Low-Profile Wireless Running Wheel for Mouse, Med Associates). Mice were habituated for two days in their home cage with the running wheel apparatus. On testing days, each mouse was monitored for one hour [2]. Mice that ran <100 cycles/hour were excluded from the study. Activity was calculated as the total number of revolutions during the testing period.

Locomotor Assay

Locomotor testing was performed using a beam break apparatus (Med Associates Inc.). Animals were placed in a rat cage and were allowed to explore the environment for three hours. Data were automatically acquired by Med Associates software and all beam breaks were summed per animal in 10-minute intervals.

Open Field Assay

For open field testing, mice were allowed to freely explore the arena (40 × 40 × 30 cm) for 6 minutes. Frequency of entry into the center zone (20 cm × 20 cm) versus the border region of the arena was recorded and analyzed using Noldus EthoVision XT 17.0 software as an index of anxiety-like behavior.

RNA Isolation, cDNA Synthesis, & RT-qPCR

On the day of harvest, brains were removed rapidly, placed into ice-cold PBS, and sliced into 1 mm-thick coronal sections in a slice matrix. Bilateral punches were made from NAc (14 gauge) and flash-frozen in tubes on dry ice. RNA was isolated through a chloroform phase separation protocol as detailed in the TRIzol Reagent User Guide. RNA concentrations were measured by NanoDrop (Thermofisher, MA). 1,000ng of cDNA was synthesized using the qScript cDNA Synthesis kit (QuantaBio, MA) as detailed in the qScript cDNA Synthesis Kit Manual. Exon-exon-spanning primers targeting as many splice variants as possible were designed with Primer-BLAST (National Center for Biotechnology Information, MD). qPCRs were performed in triplicate with 30 ng of cDNA and a master mix of exon-spanning primers (see below) and PerfeCTa SYBR Green FastMix ROX (QuantaBio, MA) on a QuantStudio real-time PCR analyzer (Invitrogen, MA), and results were expressed as fold change (2^−ΔΔCt) relative to the Glyceraldehyde 3-phosphate dehydrogenase housekeeping gene (Gapdh).

Gapdh Forward: 5’-GGTCGGTGTGAACGGATTTGG-3’

Reverse: 5’-TGATGTTAGTGGGGTCTCGC-3’

Mef2c Forward: 5’-ATCCCGATGCAGACGATTCA-3’

Reverse: 5’-GAGGTGGAACAGCACACAATCT-3’

Hdac5 Forward: 5’-ATTTGCTATCATCCGGCCCC-3’

Reverse: 5’-TGGTGAATATCCCAGTCCACG-3’

Ephb2 Forward: 5’-AAAGCCCCACCAACACAGTC-3’

Reverse: 5’-GTCTGGTACTCGGCTTCTGTC-3’

Sncg Forward: 5’-AATGCCGTGAGTGAAGCTGT-3’

Reverse: 5’-CTTCTCCACTCTTGGCCTCTT-3’

RNAscope

The Fluorescent Multiplex V2 kit (Advanced Cell Diagnostics) was used for RNAscope. Specifically, we used the fresh frozen protocol on 20 micron-thick samples as detailed in the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay User Manual. RNAscope probes were as follows: Mef2c (Mm-Mef2c-C1; Cat. No. 421011), Drd1 for D1R+ cells (Mm-Drd1-C2; Cat. No. 461901-C2), and Drd2 for D2R+ (Mm-Drd2-C3; Cat. No. 406501-C3). Opal dyes (Akoya Biosciences) were used for secondary staining as follows: Opal 690 for C1 and Opal 570 for C3. DAPI was used for nuclear staining. Images were taken on an LSM880 confocal microscope (Zeiss) with identical parameters between Sham and SNI samples. Images were quantified with the HALO FISH Module (version 3.6.4134) and quantified as Area_Mef2c/Area_DAPI.

Western Blot

Western blotting was performed as previously described [23]. Briefly, brains were removed rapidly, placed into ice-cold PBS, and sliced into 1 mm-thick coronal sections in a slice matrix. Bilateral punches were made NAc (14 gauge) and flash-frozen in tubes on dry ice. NAc punches were immersed in 40 uL lysis buffer and sonicated. The BioRad DC Protein Assay kit was then used to measure protein concentrations in each sample. Samples were loaded into a pre-cast 4-20% gradient Mini-PROTEAN TGX gel (BioRad) and proteins were transferred onto a nitrocellulose membrane using a Trans-Blot Turbo transfer apparatus (BioRad). Membranes were briefly washed in 0.1% TBST, followed by one hour of blocking with 5% BSA in 0.1% TBST buffer. Membranes were then cut based on the kDa size of target proteins and exposed to the following primary antibodies overnight at 4°C: Rabbit anti-HDAC5 p-Ser498 (Abcam ab47283), Rabbit anti-HDAC5 p-Ser259 (Abcam ab53693), Rabbit anti-GFP (Abcam ab290), Rabbit anti-GAPDH (Cell Signaling Technology #5174). The next day, membranes were washed 3x for five minutes in 0.1% TBST, followed by one-hour incubation in the following secondary antibody: Peroxidase-conjugated Goat anti-rabbit IgG (Jackson ImmunoResearch #111-035-003). Membranes were then once again washed 3x for five minutes in 0.1% TBST and then incubated in ECL solution for one minute. Blots were imaged on an iBright Imaging System (ThermoFisher) and auto-quantified relative to GAPDH on iBright software using background-corrected density values.

Bisulfite Conversion Sequencing

The existence of CpG islands within the promoter region of Mef2c was confirmed using the UCSD Genome Browser. The predicted CpG islands’ nucleotide sequence was extracted, along with an additional 1,750 nucleotides before and after the CpG target region. This elongated sequence was then entered into MethPrimer for the selection of bisulfite sequencing PCR primers. The software’s CpG island prediction function was used with the following parameters: Window: 100, Shift: 1, Obs/Exp: 0.7, GC%: 50%, resulting in four predicted CpG islands. The following primer pairs were then selected:

CpG Island #1: Forward: 5’-GGTTAAAAGTTTTATATATTTTGGG-3’

Product Size: 244nt Reverse: 5’-ACTAACAAAAAAATCCTTCTAAAATC-3’

CpG Island #2: Forward: 5’-GGATTTTTTTGTTAGTTGGTTTGTAGT-3’

Product Size: 224nt Reverse: 5’-AATAAATCAATCTTTCCATTCCTAACAT-3’

CpG Island #3: Forward: 5’-GTTAGGTAAAGGTAAGAATAAAATGAATTG-3’

Product Size: 235nt Reverse: 5’-CCAAAAAACTATATCCAAAAAAAA-3’

CpG Island #4: Forward: 5’-TTTTTTTGGATATAGTTTTTTGGA-3’

Product Size: 394nt Reverse: 5’-CCACTTAATTCAAAACTACAAACAC-3’

Bilateral NAc was punched and fresh frozen from animals on month three after injury. This timepoint was used to assess the promoter-targeting epigenetic mechanisms regulating Mef2c expression after long-term nerve injury states. DNA was first extracted with the QIAmpt Fast DNA Tissue Kit (Qiagen), followed by bisulfite conversion of samples with the EpiTect Bisulfite Kit (Qiagen). Bisulfite PCR with the aforementioned primers was then performed according to Parrish et al. [51]. Raw amplified samples were sent to GeneWiz for enzymatic cleanup and Sanger sequencing with the company’s “Difficult Template” protocol. The primer direction of choice for each CpG Island’s amplicon was previously selected based on sequencing efficiency with practice samples. The methylation percentage at every target CG was calculated by peak measurement using Chromas software. Based on varying primer efficiencies and difficulty of Sanger sequencing certain amplicons due to high %GC and enzymatic slippage at repeats, 57/62 (91.9%) of predicted methylation-prone CG sites were consistently measured. Data was then ratiometrically compiled for every CG target site.

PROMO software was used to predict transcription factors whose affinity might be affected by differential methylation at specific CG sites. Predicted TFs were further analyzed using the Enrichr DisGeNET gateway.

Bulk Tissue RNA Library Preparation & Sequencing

RNA samples with sufficient material (>50 ng) were passed to whole-transcriptome library preparation using the Stranded mRNA Prep kit (Illumina), following the manufacturer’s instructions. Briefly, total RNA inputs were normalized to 100ng/10uL going into preparation. Total RNA was first enriched for mRNA, prior to cDNA generation. 3’ ends of cDNA were then adenylated prior to ligation with adapters utilizing unique dual indices (96 UDIs) to barcode samples to allow for efficient pooling and high throughput sequencing. Libraries were enriched with PCR, with all samples undergoing 13 cycles of amplification prior to purification and pooling for sequencing. Completed libraries were quantified using Quant-iT reagents and equimolar pools were generated and sequenced on the NextSeq 500/550 Hi Output Kit v2.5 (150 CYS) flow cell with 2x50bp paired-end reads, generating a mean of 25 million paired-end reads per sample.

Bioinformatic Analysis

FASTQ files were aligned to the mouse genome (mm10) using the NGS-Data-Charmer pipeline (https://github.com/shenlab-sinai/NGS-Data-Charmer). In brief, the pipeline first trimmed the reads of adaptors and poor-quality bases. The paired-end trimmed reads were aligned to the genome using HISAT2 (version 2.2.1). Duplicate reads were then removed from the alignment files. To obtain a gene expression matrix, the unique reads were processed using FeatureCounts (flags: -p -O -t gene fraction) and the murine transcriptome (GENCODE vM25). In the QC step, samples were examined for the presence of poor sequencing quality and potential outliers. DESeq2 (version 1.32.0) was used to identify differentially expressed genes. Differential transcripts were classified as having a P-nominal<0.05, with a log₂FC > |0.32| cutoff for comparison against SNI-Saline versus Sham-Saline-specific DEGs in the Mef2c and desipramine meta-analysis. Visualizations of the DESeq2 results (Venn/petal diagrams and heatmaps) were generated in R (version 4.1.1). Ontology analysis was performed using Enrichr and Ingenuity Pathway Analysis (IPA, Qiagen, Germany) with a P-nominal < 0.05 cutoff.

In order to assess the relative proportions of cell types contributing to each bulk RNA-seq sample, cell type deconvolution was performed using CIBERSORTx [48]. In brief, the goal of cell type deconvolution is to estimate the abundances of member cell types in a mixed cell population. For deconvolution, the single-cell dataset obtained from mouse brain (GSE129788), down-sampled to 1000 cells per cell type, was used as a reference. Gene expression values for both the single-cell reference and bulk mixture file were provided as counts per million (CPM) to the deconvolution algorithm.

Brain Slice Electrophysiology

Mice were deeply anesthetized with isoflurane and decapitated. The brain was rapidly removed and chilled in cutting artificial CSF (ACSF) containing (in mM): N-methyl-D-glucamine 93, HCl 93, KCl 2.5, NaH2PO4 1.2, NaHCO3 30, HEPES 20, glucose 25, sodium ascorbate 5, thiourea 2, sodium pyruvate 3, MgSO4 10, and CaCl2 0.5, pH 7.4. The brain was embedded in 2% agarose and coronal slices (300 μm thick) were made using a Compresstome (Precisionary Instruments). Brain slices were allowed to recover at 33±1°C in ACSF for 30 minutes and thereafter at room temperature in holding ACSF, containing (in mM): NaCl 92, KCl 2.5, NaH2PO4 1.2, NaHCO3 30, HEPES 20, glucose 25, sodium ascorbate 5, thiourea 2, sodium pyruvate 3, MgSO4, and CaCl2 2, pH 7.4. After at least one hour of recovery, the slices were transferred to a submersion recording chamber and continuously perfused (2-4 mL/min) with recording ACSF containing (in mM, unless indicated otherwise): NaCl 124, KCl 2.5, NaH2PO4 1.2, NaHCO3 24, HEPES 5, glucose 12.5, MgSO4 2, and CaCl2 2, and 100 μM gabazine; pH 7.4. For excitability experiments, 10 μM CNQX was added to the ACSF, and for mEPSC studies the ASCF included 1 μM tetrodotoxin. All solutions were continuously bubbled with 95% O2 / 5% CO2. GFP-expressing neurons were visually identified with infrared differential interference contrast optics (BX51; Olympus). For excitability studies, the recording pipettes (3-5 MΩ) were filled with (in mM): K-gluconate 115, HEPES 10, KCl 20, MgATP 5, MgCl2 1.5, Na2GTP 0.5, Na-phosphocreatine 10, and EGTA 2; pH 7.25. For mEPSC studies, the pipettes contained (in mM) Cs-gluconate 122, HEPES 10, KCl 5, MgATP 5, Na2GTP 0.5, QX314 1, and EGTA 1; pH 7.2. Whole-cell patch-clamp recordings were performed at room temperature using a Multiclamp 700 A amplifier (Molecular Devices). Following breakthrough, cells were allowed to stabilize for three minutes after breakthrough in I=0 mode. For excitability studies, action potentials were evoked in current-clamp mode with a series of 1-s depolarizing pulses (20 steps in 10 pA intervals). For mEPSC recordings, membrane potential was clamped at −90 mV. All data were digitized at 10 kHz and filtered at 3 kHz, and acquisition and analysis were performed using pClamp 11 software (Molecular Devices). Only cells with stable input resistances were included in the analysis. Recording and analysis were conducted in a blinded manner.

Ex vivo Fast Scan Cyclic Voltammetry

Ex vivo fast-scan cyclic voltammetry (FSCV) was used to characterize the effects of SNI and Mef2c overexpression in the NAc core and shell. Briefly, mice were sacrificed three hours into the light cycle and their brains were rapidly removed and immersed in ice-cold oxygenated high sucrose cutting solution (2.5 mM KCl, 1.2 mM NaH2PO4, 0.5 mM CaCl2, 7.0 mM MgCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, 5.0 mM glucose, 206 mM sucrose, 5 mM HEPES, and the pH was adjusted to 7.4). A vibrating tissue slicer was used to prepare 300 μm thick coronal brain sections containing NAc core and shell subregions. These slices were then moved to a recording chamber with oxygenated artificial cerebrospinal fluid (aCSF; 32°C; 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 2.4 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 11 mM glucose, 0.4 mM L-ascorbic acid and the pH was adjusted to 7.4) flowing at 1 mL/min. Endogenous DA release was evoked by single electrical pulse stimulation (monophasic+, 4 ms, 750 μA) applied to the tissue every 5 minutes and DA was detected using a triangular scanning waveform (−0.4 to +1.2 and back to −0.4 V, Ag vs AgCl) at the rate of 400 V/s. Recording electrodes were calibrated with known concentrations of DA (3 μM). After stable DA responses were obtained (3 consecutive collections with no more than 10% variation), phasic stimulation parameters (5 and 10 pulse stimulations at 5, 10, 20, and 100 Hz) were applied. All FSCV data were analyzed using a Michaelis-Menten kinetics-based algorithm [21], using Demon Voltammetry and Analysis software [72]. It is important to note that NAc shell receives both norepinephrine (NE) and DA projections and the cyclic voltammogram is indistinguishable for both. However, the density of the NE projection is significantly lower than DA [41,50] and below the detection threshold of our assay.

Statistics

Data were analyzed using Graph Pad Prism 10 software and the aforementioned R packages. For the experiments monitoring longitudinal data, we used two-way repeated-measures ANOVAs followed by Sidak’s post hoc tests. For two-factor designs, we used two-way ANOVA followed by Sidak’s post hoc test for behavioral and functional outputs. For data containing a single independent variable, we first checked for normality using the Shapiro-Wilk test. If data were normally distributed, we next used unpaired two-tailed t-tests followed by a Welch’s correction if an F-test demonstrated significantly different variances between groups. Outliers were identified by use of the Grubb’s test (α = 0.05). Error bars depict ± SEM. F and t/q values for each dataset and analysis are provided in Supplementary Tables 1 and 2.

RESULTS

Prolonged SNI Alters Transcriptional and Epigenetic Regulation of MEF2C in the Nucleus Accumbens

Our laboratory has previously characterized transcriptomic alterations in the NAc of C57BL/6J male mice 3.5 months after SNI [53]. Ingenuity Pathway Analysis (IPA) of differentially-expressed genes (DEGs; P-nominal < 0.05) from the SNI-Saline vs Sham-Saline NAc RNA-seq dataset predicted changes in several canonical pathways, including: “Synaptogenesis Signaling Pathway”, “Dopamine-DARPP32 Feedback in cAMP Signaling”, “Endocannabinoid Neuronal Synapse Pathway”, “CREB Signaling in Neurons”, “Opioid Signaling Pathway”, “Axon Guidance Signaling”, and “Synaptic Long-term Potentiation”. We were initially interested in the “Synaptic Long-term Potentiation” pathway because of clinically observed changes in the baseline activity of the NAc and other mesocorticolimbic regions in chronic pain patients [60]. In order to better define transcriptional regulators associated with the 26 DEGs from this pathway, we used the Enrichr ARCHS4 TFs Coexp gateway [12], which predicts TFs co-expressed/associated with a set of input DEGs. The top five hits included SLC4A10 (P-adj. = 9.14e-10, OR = 42.57), MYT1L (P-adj. = 1.83e-8, OR = 35.93), TRIM23 (P-adj. = 3.11e-7, OR = 30.06), RORB (P-adj. = 3.11e-7, OR = 30.06), and MEF2C (P-adj. = 4.44e-6, OR = 24.83) (Figure 1A). Of note, our group was particularly interested in MEF2C because it is regulated by HDAC1 and HDAC5 [19,46], histone deacetylases that we have shown previously are involved in the maintenance of neuropathic pain and the regulation of the anti-allodynic properties of monoamine-targeting antidepressants [17,53]. Furthermore, MEF2C is a master regulator of synaptic plasticity and neuronal activity [1,5], which are altered in the mesocorticolimbic circuit of patients suffering from conditions such as chronic pain and mood disorders [60]. Accordingly, MEF2C was also among the top 10 TFs of “Synaptogenesis Signaling”-associated DEGs (P-adj. = 3.027e-9, OR = 17.87; ARCHS4 TFs Coexp). Lastly, with relevance to the dopaminergic mesocorticolimbic circuitry, MEF2C was the third top TF associated with the Dopamine-DARPP32 Feedback in cAMP Signaling pathway (P-adj. = 8.52e-6, OR=22.61; ARCHS4 TFs Coexp).

Figure 1. Prolonged SNI reduced Mef2c expression in NAc neurons.

(A) MEF2C was a transcription factor predicted to contribute to gene expression changes observed at 3.5 months post-SNI in the NAc of C57BL/6J male mice (n = 4 mice/condition; ARCHS4 TF Coexp Enrichr Gateway). (B, C) Mef2c transcript was downregulated at week three post-SNI in combined C57BL/6J female and male mice (n = 19 mice/condition; *P < 0.05 by unpaired t-test) and trended downwards in combined DBA/2J female and male mice (n = 16-17 mice/condition). (D) Representative RNAscope images from Sham and SNI DBA/2J male mice (three weeks post-injury). (E, F) Mef2c expression was downregulated in D1R+ (n = 716-1072 cells/condition from 3 animals/condition; ****P < 0.0001 by unpaired t-test with Welch’s correction) and D2R+ (n = 333-545 cells/condition from 3 animals/condition; ****P < 0.0001 by unpaired t-test with Welch’s correction) NAc neurons at three weeks post-SNI, respectively. W3 = Week 3. Data are displayed as mean ± SEM. W = Week.

In order to investigate endogenous changes in NAc MEF2C after peripheral nerve injury, we first assessed the transcriptional regulation of MEF2C in whole NAc from male and female C57BL/6J and DBA/2J mice three weeks after SNI using RT-qPCR. We observed a moderate, significant decrease in Mef2c gene expression in C57BL/6J mice (Figure 1B; statistics available in Supplementary Tables 1 (for main figures) & 2 (supplementary figures)), and a trending decrease in DBA/2J mice (Figure 1C). As MEF2C is expressed in a variety of cell subtypes, we next used RNAscope in situ hybridization on NAc slices from male DBA/2J mice to determine whether Mef2c expression changes in the two major medium spiny neuron subtypes in this region: Drd1+ and Drd2+ neurons (Figure 1D). As seen in Figures 1E and 1F, Mef2c was downregulated at this timepoint in both Drd1+ and Drd2+ neurons, respectively, suggesting a pan-neuronal effect of peripheral nerve injury. At week seven, we observed a continued downregulation of Mef2c expression in combined male C57BL/6J and female DBA/2J mice (Figure S1A).

Furthermore, as Class IIa HDACs such as HDAC5 are potent repressors of MEF2C activity in mesolimbic regions such as the NAc, we next wanted to investigate whether prolonged nerve injury affects the expression or nuclear localization of HDAC5. Regarding the former, we did not observe any changes in Hdac5 expression in the NAc of combined male and female DBA/2J (Figure S1B), nor combined male and female C57BL/6J cohorts (Figure S1C), three weeks after SNI. Next, we measured phosphorylation levels at the Ser259 and Ser498 residues of HDAC5, which are positively associated with 14-3-3-mediated nuclear export [40]. Western blot analysis of NAc from female DBA/2J mice showed a decrease in phosphorylation levels at both HDAC5 residues at three weeks post-SNI (Figure S1D), suggesting increased nuclear localization and therefore repression of MEF2C (full Western blots available in Figure S1E).

To assess changes in Mef2c regulation at a severely prolonged timepoint after nerve injury, we measured NAc Mef2c expression at three months post-SNI. Interestingly, we did not observe any differences in NAc Mef2c expression in male DBA/2J mice (Figure S2A). As such, we performed bisulfite conversion PCR in male DBA/2J mice to assess the accessibility of the Mef2c promoter region three months post-SNI and identify any epigenetic/transcriptional regulators that might be responsible for reversing the injury-dependent downregulation observed at earlier timepoints.

Using an expanded promoter window, we used MethPrimer to predict four CpG islands (A-D) (Figure S2B). Primers were validated by PCR (Figure S2B). When comparing all successfully sequenced CG sites between SNI and Sham, we observed a significant hypomethylation of the Mef2c promoter (Figure S2C), as might be expected during a phase of expression normalization after a downregulation. SNI versus Sham ratiometric analysis of CG sites revealed overall hypomethylation at CpG Island A-C, with individually downregulated CG sites in Island A and D (Figure S2D). We identified several TFs that were predicted to target the individually downregulated CG sites above, including JUN, JUND, and FOS (Figure S2E). Other transcription factors of interest that were found to target the predicted MEF2C islands in general included NR3C1 [30,42,47], NFKB [10,33], YY1 [35,63], PAX5 [3], and GATA2 [13,34], which have been implicated to some extent in depression, anxiety, or pain. These bisulfite conversion findings suggested an epigenetic bias towards increased transcription of MEF2C several months after peripheral nerve injury, possibly reflecting a compensatory mechanism in NAc cells by which Mef2c expression can return to normal levels.

Mef2c Overexpression in the NAc Alleviates SNI-induced Sensory and Affective Behavioral Maladaptations

We next aimed to investigate if the promotion of MEF2C expression/action in the NAc after SNI through neuron-specific viral overexpression (AAV8-hSyn-Mef2c-Gfp or AAV8-hSyn-Gfp, encoding enhanced green fluorescent protein (GFP)) affects the induction or maintenance of sensory hypersensitivity in the SNI model. Successful infection was validated with qPCR of Mef2c (Figure S3A) and GFP Western blots (Figure S3B; Week 16 post-SNI/Sham). We first stereotaxically infected the NAc of DBA/2J male mice with either the Mef2c or Gfp vectors and then allowed for four weeks of viral expression, followed by SNI or Sham surgeries and behavioral assays (Figure 2A). Of note, Mef2c overexpression did not affect locomotor activity in Sham or SNI animals (Figure S3D). Despite successful overexpression by day 12 after stereotaxic surgery (Figure S3A) and validation of vector expression in the core and shell of the NAc by week 4 after stereotaxic surgery (Figure S3C), we did not observe any effects of NAc Mef2c overexpression on sensory behaviors, such as mechanical allodynia, until four weeks post-SNI. At this timepoint, we observed a significant amelioration of punctate allodynia in the von Frey assay, which persisted through week 11 (Figure 2B). This effect was also confirmed in male C57BL/6J mice (Figure S3E). Mef2c-overexpressing mice that underwent SNI (Mef2c-SNI) also demonstrated reduced dynamic allodynia compared to Gfp-infected counterparts (Gfp-SNI) in the brush assay of dynamic allodynia (Figure 2C), but no virus group difference was observed in the pin prick assay. Notably, SNI led to punctate hyperalgesia in the form of “Strong Responses” (see Methods) in both Mef2c and Gfp infected groups (Figure 2D).

Figure 2. Mef2c overexpression in the NAc before injury reversed SNI-induced behavioral maladaptations in DBA/2J male mice.

(A) Diagram of the experimental process. (B) Mef2c overexpression persistently reduced mechanical allodynia in the von Frey assay after week four post-SNI (n = 10-12 animals/condition; **P < 0.01, ****P < 0.0001 by Sidak’s post hoc). (C) Mef2c overexpression reduced dynamic allodynia in the brush assay at week five post-SNI (n = 10-12 animals/condition; **P < 0.01, ****P < 0.0001 by Sidak’s post hoc). (D) No differences in the number of “Strong Responses” after pin pricks were observed between Gfp and Mef2c overexpressing SNI animals at 12 weeks post-injury, though both SNI groups were more sensitive to noxious stimuli than their Sham counterparts (n = 10-12 animals/condition; *P < 0.05, **P < 0.01 by Tukey’s post hoc). (E) Mef2c overexpression attenuated thermal allodynia in SNI animals (n = 10-12 animals/condition; ***P < 0.001 by Sidak’s post hoc). (F, G) Mef2c overexpression reduced cold hyperalgesia in SNI animals as measured in the 0°C cold plate (week six post-SNI) and plantar dry ice (week 16 post-SNI), respectively (n = 10-12 animals/condition; *P < 0.05, **P < 0.01, ****P < 0.0001 by Sidak’s post hoc). (H) SNI-induced elevated hindlimb guarding behavior was reduced by Mef2c overexpression at week two post-SNI (n = 10-12 animals/condition; *P < 0.05, ***P < 0.001 by Sidak’s post hoc). (I) Mef2c-overexpressing SNI animals buried fewer marbles than their Gfp counterparts on week 10 post-SNI (n = 10-12 animals/condition; **P < 0.01 by Sidak’s post hoc). (J) SNI-induced increased wheel-running activity at week 14 were reversed by Mef2c overexpression (n = 7-10 animals/condition; *P < 0.05, **P < 0.01 by Sidak’s post hoc). Data are displayed as mean ± SEM. W = Week.

Similar therapeutic-like results in Mef2c-SNI groups of mice were observed with behavioral assays of temperature hypersensitivity, including thermal allodynia in the 42°C hot plate assay (Figure 2E) and cold hyperalgesia in the 0°C cold plate (Figure 2F) and plantar dry ice (Figure 2G) assays. Reversal of cold hyperalgesia was also confirmed in male C57BL/6J mice at week 8 post-SNI (Figure S3F).

We hypothesized that the interruption of sensory hypersensitivity maintenance would also disrupt the onset or maintenance of affective abnormalities, such as anxiety-like behaviors. In order to assess the effects on the induction of early anxiety-like behavior, we used an adapted guarding assay two weeks post-SNI/Sham surgeries. Mef2c-SNI mice guarded their injured hindlimb significantly less than Gfp-SNI mice, which in turn guarded significantly more than Gfp-Sham mice (Figure 2H). To assess sustained anxiety-like behaviors, we performed a marble burying assay and voluntary running wheel paradigm on weeks 10 and 14 after SNI/Sham, respectively. Mef2c-SNI mice buried significantly fewer marbles than their Gfp-SNI counterparts, though we only observed a trending increase in marbles buried between Gfp-Sham and Gfp-SNI groups (Figure 2I). Mef2c-SNI mice also ran significantly less on a wheel over a one-hour window than their Gfp-SNI counterparts, which in turn ran more than the Gfp-Sham group (Figure 2J).

Mef2c Overexpression Alters Synaptic Gene Expression

We next investigated the mechanisms underlying these behavioral ameliorations of Mef2c overexpression through RNA-seq. In generating the mouse cohort for this experiment, we also wanted to determine whether viral overexpression of Mef2c one month after SNI produced anti-allodynic and anxiolytic effects, despite the prior induction of chronic pain-like behaviors (Figure 3A). Indeed, we observed a significant reduction in mechanical hypersensitivity in male DBA/2J SNI-Mef2c mice by two weeks post-stereotaxic surgery (Figure 3B). We also noted a significant reduction in marbles buried in SNI-Mef2c mice compared to SNI-Gfp mice by five weeks after viral injection (Figure 3C). Of note, we also validated the impact of Mef2c overexpression on sensory hypersensitivity after prolonged nerve injury in female C57BL/6J mice, this time performing stereotaxic surgery on week 10 post-SNI. As seen in Figure S4A, Mef2c overexpressors displayed reduced mechanical allodynia by week 2 after stereotaxic surgery, which persisted through week 5. The Mef2c overexpressors in this cohort also demonstrated an increased frequency of entering the center area of an open field, suggesting reduced anxiety-like behavior (Figure S4B). Figure S4C shows representative viral expression of the Mef2c vector at five weeks post-stereotaxic surgery.

Figure 3. Mef2c overexpression in the NAc of male DBA/2J mice after injury reversed SNI-induced behavioral and transcriptional maladaptations.

(A) Diagram of the experimental process. (B) Mef2c overexpression four weeks post-SNI led to a reduction in mechanical allodynia by two weeks post-stereotaxic surgery (n = 9-12 animals/condition; **P < 0.01, ****P < 0.0001 by Sidak’s post hoc). (C) Mef2c overexpression one-month post-SNI led to a reduction in marble burying compared to SNI-Gfp animals at week five post-stereotaxic surgery (n = 9-12 animals/condition; ***P < 0.001, ****P < 0.0001 by Sidak’s post hoc). (D) Petal diagram of DEGs from RNA-seq of NAc from Sham-Gfp, Sham-Mef2c, SNI-Gfp, and SNI-Mef2c groups at week five post-stereotaxic surgery (p-nom < 0.05; n = 6 animals/condition). The green section of the diagram represents the DEGs used for Enrichr and IPA analyses. (E) DEGs unique to Mef2c overexpression under SNI conditions were associated with the synaptic compartment (Jensen COMPARTMENTS Enrichr gateway; −log₁₀(p-adj. > 1.3)). (F) Top 10 canonical pathways associated with Mef2c overexpression under SNI conditions (IPA; −log₁₀(p > 1.3)). (G) Predicted changes in the “Synaptic Long-term Potentiation Pathway” due to Mef2c overexpression under SNI conditions (IPA; orange = increased predicted activity, blue = decreased predicted activity). (H) qPCR validation of DEGs related to synaptic plasticity and potentiation (n = 7-9 animals/condition; *P < 0.05 by unpaired t-test (Welch’s correction for Sncg)). (I) RNA-seq deconvolution comparison of SNI-Gfp and SNI-Mef2c groups (n = 6 animals/condition; *P < 0.05 by multiple t-tests). OPC = oligodendrocyte precursor cells, OLG = oligodendrocytes, OEG = olfactory ensheathing glia, NSC = neural stem cells, ARP = astrocyte-restricted precursors, ImmN = immature neurons, mNEUR = mature neurons, NendC = neuroendocrine cells, EPC = ependymocytes, EC = endothelial cells, VSMC = vascular smooth muscle cells, VLMC = vascular and leptomeningeal cells, ABC = arachnoid barrier cells, MG = microglia, MAC = macrophages, PC = pericytes, ASC = astrocytes. Data are displayed as mean ± SEM.

Figure 3D shows a petal diagram that depicts shared and unique differentially expressed genes (DEGs; P-nominal < 0.05) from our RNA-seq analysis across viral and surgical conditions. No log₂(Fold Change) threshold and a nominal p-value cutoff were applied at this step to account for elevated variability from combined viral vector expression and peripheral nerve injury. We were particularly interested in DEGs shared across the following comparisons, as they represented the effects of Mef2c overexpression in neuropathic injury states: a) SNI-Mef2c vs SNI-Gfp, and b) SNI-Mef2c vs Sham-Mef2c. Cross-referencing the resulting 1316 DEGs (P-nom < 0.05) with the Enrichr Jensen COMPARTMENTS gateway highlighted an enrichment of genes associated with the post-synaptic compartment of excitatory synapses in this DEG group (Figure 3E).

Ingenuity Pathway Analysis (IPA) of the aforementioned DEGs identified several top canonical pathways that were predicted to change with Mef2c overexpression in nerve-injured states, including “G-Protein Coupled Receptor Signaling”, “Synaptic Long-term Potentiation”, and “Synaptogenesis Signaling” (Figure 3F; −log₁₀(p-value) > 1.3). Analysis of the “Synaptic Long-term Potentiation” (LTP) pathway predicted that Mef2c overexpression after SNI increased LTP through activation of GIRK channels, cAMP/PKA signaling, MAP2K/ERK signaling, CAMK signaling, and CREB/CREB-binding protein function (Figure 3G). We also validated differential expression of two LTP- and synaptic function-associated DEGs in NAc, Ephb2 [25,70] and Sncg [9], between SNI-Gfp and SNI-Mef2c animals (Figure 3H).

While viral overexpression of Mef2c was limited to neurons in our model, we examined if the transcriptional changes in our RNA-seq data were due to contributions from non-neuronal cell subtypes. Using RNA-seq deconvolution against annotations from Ximerakis et al. [71], we identified a significant increase in the transcriptional contribution of mature neurons (mNEUR) in NAc from SNI-Mef2c animals compared to those of SNI-Gfp animals, as well as an increase in vascular smooth muscle cell (VSMC) contribution (Figure 3I). We also noted a significant reduction in the transcriptional contribution of astrocyte-restricted precursors (ARP), as well as trending reductions in neuroendocrine cells (NendC) and oligodendrocytes (OLG) (Figure 3I). While we identified several changes in cell subtype transcriptional contributions when comparing Sham-Gfp and Sham-Mef2c animals, we did not observe a difference in mNEUR contributions, suggesting an SNI-specific transcriptional response in Mef2c overexpressors (Figure S5A). However, we observed a similar reduction in ARP contribution, suggesting a conserved effect of neuronal Mef2c overexpression on astrocyte development/interactions. When comparing Sham-Gfp to SNI-Gfp, we primarily found changes in immune-related cell populations (Figure S5B), but the presence of viral vectors in these tissues must be taken into consideration when interpreting this data.

Mef2c Overexpression Reverses SNI-induced Changes in Neuronal Physiology

In order to determine how the aforementioned transcriptional changes associated with Mef2c overexpression affected neuronal activity and signaling, we performed whole cell recordings in neurons from both the core and shell of NAc slices prepared from SNI or Sham male C57BL/6J mice that expressed GFP after viral infection with AAV8-hSyn-Gfp or AAV8-hSyn-Mef2c-Gfp (Figure 4A). After at least one month of SNI, neurons from SNI-Gfp animals were significantly hyperexcitable relative to Sham-Gfp neurons, showing higher peak firing rates in response to depolarizing current injection (Figure 4B). While no differences were observed between Sham-Mef2c and Sham-Gfp neurons (Figure 4C), overexpression of Mef2c in neurons of SNI animals reversed hyperexcitability associated with nerve injury (Figure 4D). Figure 4E depicts representative action potential traces from all four injury-virus conditions in response to 170 pA of injected current.

Figure 4. Mef2c overexpression in the NAc of male C57BL/6J mice after injury reversed SNI-induced physiological maladaptations in neurons.

(A) Diagram of the experimental process. (B) Prolonged SNI induces neuron hyperexcitability. (C) Mef2c overexpression did not affect excitability in neurons of Sham animals. (D) Mef2c overexpression reversed SNI-induced neuron hyperexcitability. N = 9-24 cells/condition from n = 3-4 animals/condition for excitability data; *P < 0.05 by Sidak’ post hoc for B-D. (E) Representative action potential traces of individual neurons from all injury-virus conditions at 170 pA of injected current. (F) Representative mEPSC traces from all injury-virus conditions. (G, H) Mef2c overexpression increased mEPSC frequency in only SNI animals. (I, J) Mef2c overexpression decreased the mean mEPSC amplitude per cell regardless of injury condition. N = 15-20 cells/condition from 3-4 animals/condition for amplitude and IEI; *P < 0.05, **P < 0.01, ***P < 0.001 by Sidak’s post hoc for H and J. Longitudinal data is depicted as mean ± SEM. Mean per cell data is depicted by box and whisker plots depicting the minimum, maximum, upper/lower quartiles, median, and mean.

We hypothesized that this reversal of SNI-induced neuronal hyperexcitability could result from postsynaptic upregulation in potassium channel expression following Mef2c overexpression. As seen below, of the potassium channel DEGs we observed through RNA-seq, there was a bias towards upregulation (SNI-Mef2c vs SNI-Gfp; 83.3% upregulated; gene (log2FC, p-nom value)): Kcnj4 (+0.552, 0.0271), Kcns3 (−0.717, 0.0446), Kcnip3 (+0.380, 0.0467), Kcnf1 (+0.438, 0.00291), Kcnq3 (+0.294, 0.00618), Kcns2 (+0.392, 0.00710), Kcnj10 (−0.308, 0.00743), Kcnj3 (+0.686, 0.0174), Kcnk15 (+2.216, 0.0228), Kcnv1 (+0.678, 0.0228), Kcnb1 (+0.318, 0.0232), Kcnma1 (+0.397, 0.0237).

Mef2c overexpression also affected excitatory input to neurons, as shown by the analysis of miniature excitatory postsynaptic current (mEPSC) traces in Figure 4F. Specifically, Mef2c overexpression increased mEPSC frequency only in SNI animals (Figure 4G,H). Furthermore, Mef2c overexpression decreased mEPSC amplitude regardless of the injury state (Figure 4I,J). The altered frequency finding in neurons from SNI-Mef2c animals suggested an increase in presynaptic neurotransmitter release, specifically in the NAc. As such, we set out to investigate whether neurotransmission from other mesolimbic regions that innervate the NAc was altered in this model.

Mef2c Overexpression in the NAc Affects Signaling in the Broader Mesolimbic Circuitry

Other groups have demonstrated altered dopamine (DA) neurotransmission in VTA-to-NAc projecting neurons after acute peripheral injuries [24,55], although the effects of prolonged peripheral nerve injuries are poorly understood. As such, we performed ex vivo fast scan cyclic voltammetry to measure DA in the NAc core and shell subregions from male C57BL/6J animals that had undergone one month of SNI/Sham followed by at least three weeks of expression of AAV8-hSyn-Gfp or AAV8-hSyn-Mef2c-Gfp (Figure 5A). This analysis was segregated into shell and core due to geographical variability of innervating brain regions and neuronal cell subtypes. Figure 5B depicts representative transient DA traces from the four injury-virus conditions.

Figure 5. Mef2c overexpression after injury reversed SNI-induced augmentation of DA release after phasic stimulation in the NAc shell.

(A) Diagram of the experimental process. (B) Representative traces of dopamine release in the NAc core (left) and shell (right) across injury-viral conditions (atlas images adapted from Allen Mouse Brain Atlas). (C) Mef2c overexpression did not affect DA release after tonic stimulation in the NAc core. (D-F) Mef2c overexpression did not affect DA release after phasic stimulation in the NAc core. N = 7-8 animals per group for C-F. (G) Mef2c overexpression did not affect DA release after tonic stimulation in the NAc shell. (H) SNI induced an increase in DA release after phasic stimulation in the NAc shell. (I) Mef2c overexpression did not affect phasic-stimulated DA release in the NAc shell. (J) Mef2c overexpression reversed SNI-induced DA release augmentation after phasic stimulation in the NAc shell. N = 7-8 animals per group for G-J; *P < 0.05, **P < 0.01 by Sidak’s post hoc. Longitudinal data is depicted as mean ± SEM. Tonic stimulation data is depicted by box and whisker plots depicting the minimum, maximum, upper/lower quartiles, median, and mean.

As seen in Figure 5C, Mef2c overexpression did not affect DA release in the NAc core after tonic stimulation, regardless of injury condition. We also observed no differences in DA release after phasic stimulation in the NAc core of Mef2c-overexpressing animals, regardless of injury condition, frequency, and pulse number (Figures 5, D–F). However, from a quality control perspective, all conditions demonstrated significant increases in DA release upon increasing the stimulation frequency and pulse number (Supplementary Table 1).

As with the core, we did not observe changes in tonic-evoked DA release in the shell under any condition (Figure 5G). However, we observed an increase in phasic-evoked DA release in the shell of SNI-Gfp animals, specifically at 20 Hz with 10 pulses (Figure 5H). There was no difference in phasic-evoked DA release between Sham-Mef2c and Sham-Gfp neurons (Figure 5I). However, Mef2c overexpression in SNI animals reversed the maladaptive increase in phasic-evoked DA release observed in SNI-Gfp animals (Figure 5J).

Establishing a Context for Mef2c Amongst Existing Therapeutic Substances

Considering the antiallodynic effectiveness of Mef2c overexpression in a specific brain region, and the likely toxicity of any systemic pharmacological intervention due to MEF2C’s critical role in cardiac and immune function, we wanted to determine whether Mef2c overexpression in NAc mirrors the transcriptional mechanisms of currently-available pain therapeutics that affect the reward pathway, such as monoamine-targeting antidepressants. In a prior study [59], our group performed RNA-seq on whole NAc tissue from C57BL/6J male mice seven weeks after SNI or Sham surgery and after three weeks of bi-daily treatment with saline or desipramine (DMI; 15 mg/kg i.p.). We compared the transcriptomic effects of DMI (which did not alter Mef2c expression in peripheral nerve injury conditions) versus Mef2c overexpression (Figure 6A) in the context of SNI-specific gene expression changes by selecting DEGs from the SNI vs Sham (saline) comparison in the prior study (log₂FC > |0.32|, p-nom < 0.05). While the timepoint of intervention initiation between studies was the same (4 weeks), we allowed for two additional weeks of viral expression than in our desipramine cohort to account for the fact that AAV8 vector expression is normally not robust enough to elicit a phenotype until approximately two weeks post-injection. As seen in the heat map in Figure 6B, DMI and Mef2c counter-regulate SNI-specific genes in a markedly different pattern, with a relative upregulation signature in DMI-treated SNI animals and a relative downregulation signature in Mef2c-overexpressing SNI animals. When quantified, DMI counter-regulated 126 genes, while Mef2c counter-regulated 129 genes (p-nom < 0.05). Of these genes, DMI and Mef2c only counter-regulated four shared genes: Nos2, Rhbdf2, Gm2395, and Gm3534.

Figure 6. Mef2c overexpression induced transcriptional changes in the NAc that were distinct from those triggered by systemic administration of tricyclic antidepressants.

(A) Diagram of comparison between C57BL/6J males that received DMI or Saline versus DBA/2J males that received stereotaxic surgery with AAV8-hSyn-Mef2c-Gfp or AAV8-hSyn-Gfp (n = 4-6 animals/condition). (B) Union heatmap depicting the effects of DMI or Mef2c overexpression compared to SNI-induced DEGs in the NAc. (C-D) GO: Biological Process hits for unique contra-regulation of SNI-induced DEGs by DMI or Mef2c overexpression, respectively.

Using the GO: Biological Process Enrichr gateway, we analyzed the pathways associated with genes uniquely counter-regulated by DMI versus Mef2c overexpression (−log₁₀(p-value) > 1.3). As seen in Figure 6C, DMI effects on transcription were primarily associated with vascularization/angiogenesis. Conversely, Mef2c overexpression was associated with neurotransmission and cation-mediated signal transduction (Figure 6D). This reinforced that Mef2c overexpression alters pathways dissimilar to tricyclic antidepressants.

DISCUSSION

We utilized mouse models of peripheral neuropathy, transcriptomics, whole cell recordings and voltammetry to characterize NAc mechanisms underlying the maintenance of sensory hypersensitivity and emotional aspects of chronic pain. From a broader perspective, we found that after prolonged nerve injury, NAc neurons have altered electrophysiological characteristics, such as hyperexcitability, and that dopamine neurotransmission onto the NAc is sensitized to stimulation. Bioinformatic analysis of datasets from mouse NAc tissue collected at 3.5 months post-SNI [53] guided us towards MEF2C as a potential regulator of maladaptive changes in neuronal activity and synaptic plasticity under prolonged nerve injury states. Indeed, we found that MEF2C was molecularly repressed between three weeks and at least seven weeks after peripheral nerve injury. Overexpression of Mef2c transcript in NAc neurons partially reversed abnormal somatosensory and affective behaviors in SNI mice. Of note, anti-allodynic effects were observed with both pre- and post-injury vector administration, suggesting Mef2c overexpression can at least partially prevent pain chronification and attenuate existing chronic pain signs. Furthermore, this overexpression resulted in the normalization of neuronal physiology and DA neurotransmission in the NAc of SNI animals. Transcriptional analysis pointed to gene expression changes that affected pathways such as GPCR signaling, long-term potentiation, and synaptogenesis. These transcriptional effects of Mef2c overexpression differed from those induced by DMI, a commonly prescribed tricyclic antidepressant for neuropathic pain.

Future work will focus on a deeper characterization of injury-induced mechanisms that lead to the long-term repression of MEF2C, such as changes in the nuclear presence of negative regulators such as HDAC1/5 and chromatin accessibility changes that might contribute to reduced Mef2c transcription. Our deconvolution results also emphasize the need for further study into cell subtype-specific regulation of MEF2C, both from the perspective of neuronal subclusters and glial cells such as microglia, which express MEF2C and have been implicated in chronic pain and depression [28,61,69]. Given prior literature demonstrating the differential contribution of NAc D1R and D2R+ medium spiny neurons to pain maintenance [58,65], the effects of selective Mef2c overexpression in various neuronal subtypes will also provide further clarity on how local NAc interventions affect the broader mesocorticolimbic circuitry and behavioral phenotypes. Notably, most information on the role of different cell neuronal subtypes or NAc subregions in pain maintenance drives predominantly from acute timepoints, and therefore it is not possible to make comparisons with findings from our study [56,58,65]. Furthermore, it will be interesting to further investigate the role of NAc MEF2C in regulating the persistence of pain using longer timepoints and additional nerve injury paradigms, given the emergence of factors such as sex-specific phenotypes [43].

Methylation analysis of the Mef2c promoter revealed hypomethylation, which might represent a homeostatic attempt by the affected neurons to return MEF2C levels to baseline values. LaPlant et al. previously showed that broadly blocking NAc DNA methylation in the chronic social defeat stress model resulted in antidepressant-like effects [36]. Prior work by our group has demonstrated a large transcriptional overlap between stress models, such as chronic unpredictable stress, and peripheral nerve injury [17]. This further suggests that hypomethylation at the Mef2c promoter region is a compensatory epigenetic response after prolonged nerve injury.

Our electrophysiology findings demonstrated that Mef2c overexpression is capable of reducing SNI-induced NAc neuron hyperexcitability, while also reducing mEPSC amplitude in an injury-independent fashion and increasing mEPSC frequency in an SNI-dependent fashion. We found it interesting that SNI alone did not significantly affect mEPSC amplitude or frequency. This may suggest that prolonged injury states can serve as a priming mechanism that allow further interventions, such as intentional alterations of transcription factor activity, to have specific therapeutic effects as seen with mEPSC frequency. We speculate that the underlying mechanism might include injury-induced chromatin remodeling or an altered epigenetic regulator landscape. While no studies to our knowledge have broadly assessed NAc neuron electrophysiology at this time point post-SNI, Guida et al. found circuit-dependent changes in NAc LTP at 1 and 12 months post-SNI [27]. From a translational perspective, several human studies have documented increases in resting-state NAc activity in conditions such as chronic pain and major depressive disorder, which aligns with our hyperexcitability findings [60]. These changes in neuron electrophysiological properties are interesting in light of our transcriptional bioinformatic predictions, such as the activation of synaptic LTP pathways. Our RNA-seq deconvolution analysis also identified potential alterations in astrocyte precursor and vascular cell regulation in the NAc by Mef2c overexpression under nerve injury, which may contribute to synaptic regulation along with endogenous changes in neurons. While future single-cell RNA sequencing analysis will further elucidate the cell type-specific role of MEF2C, so far, these findings align with a prior study in which reduced MEF2C levels resulted in impaired long-term potentiation in the hippocampus [66]. Future work will also focus on the effects of prolonged SNI and Mef2c overexpression on the density and morphology of dendritic spines of NAc neurons, which are known to be influenced by MEF2C under other conditions [1,5,54].

Our findings regarding the normalization of phasic DA neurotransmission in the NAc shell are particularly interesting in light of existing literature and suggest that an increase in evoked DA release at NAc shell synapses is maladaptive. Recently, Ren et al. showed that SNI reduced extracellular levels of DA in the NAc shell, suggestive of a hypodopaminergic basal state, and that L-DOPA normalized SNI-induced hyperexcitability [55]. Taken together, one interpretation might be that SNI causes hypodopaminergic synapses that in turn sensitize neural circuitry to stimulation, as found by Mathews et al. in ethanol studies [39]. Notably, the study by Ren et al. performed measurements at acute time points (<1 week). In order to confirm this theory, a comprehensive study should examine both extracellular and evoked DA in the NAc at a long-term timepoint after nerve injury. Gee et al. used an in vivo corneal capsaicin model to demonstrate that an acute insult can alter NAc tonic- and phasic-evoked DA neurotransmission [24]. Of note, their study suggested that pain induced reductions in extracellular DA (using fast-scan controlled-adsorption voltammetry), but augmented phasic-evoked release, similar to our observations with the prolonged SNI model. These data suggest that salient external stimuli produce heightened responsivity, possibly contributing to the greater pain perception observed in this model. From a chronic pain and depression perspective, clinical studies suggest that postsynaptic GPCR signaling and pre-synaptic uptake and metabolism are altered in ways that impair DA signaling, possibly causing a compensatory response in the form of augmented phasic-evoked DA release. Further work must be done on the regulation of DA signaling in neuron subtypes in order to better understand whether neuronal maladaptations in the NAc after prolonged nerve injury are due to circuit-level changes and/or intrinsic molecular responses within the NAc.

Our transcriptional comparison between Mef2c overexpression and DMI sought to determine whether existing therapeutics target the MEF2C pathway. This is important to understand because systemic inhibition of MEF2C is unlikely to be feasible given the protein’s ubiquity and role in cardiac and hematological processes. Our comparison to DMI demonstrated little overlap between potential transcriptional mechanisms underlying the therapeutic effects of these two approaches. It is important to note that different mouse strains and post-injury timepoints were used in this meta-analysis, which may have contributed to the lack of transcriptional overlap. Regardless, analysis of the NAc for changes in the MEF2C pathway after treatment with other systemic pain therapeutics may provide further insights into their mechanism of action and may help identify MEF2C-regulated pathways for therapeutic targeting.

Taken together, this study furthers our understanding of transcriptional, electrophysiological, and cross-regional effects of prolonged peripheral nerve injury on the NAc. Methods of reversing SNI-induced changes in Mef2c expression in the ventral striatum hold promise for functionally reversing NAc maladaptations caused by peripheral nerve injuries, both physiologically and behaviorally.

Data Availability:

RNA-seq data is available at NCBI GEO (ID: TBD after acceptance). All other data will be provided upon reasonable request.