Feasibility and effectiveness of transcutaneous auricular vagus nerve stimulation (taVNS) in awake mice.

AIMS: Transcutaneous auricular vagus nerve stimulation (taVNS) is widely used to treat a variety of disorders because it is noninvasive, safe, and well tolerated by awake patients. However, long-term and repetitive taVNS is difficult to achieve in awake mice. Therefore, developing a new taVNS method that fully mimics the method used in clinical settings and is well-tolerated by awake mice is greatly important for generalizing research findings related to the effects of taVNS. The study aimed to develop a new taVNS device for use in awake mice and to test its reliability and effectiveness. METHODS: We demonstrated the reliability of this taVNS device through retrograde neurotropic pseudorabies virus (PRV) tracing and evaluated its effectiveness through morphological analysis. After 3 weeks of taVNS application, the open field test (OFT) and elevated plus maze (EPM) were used to evaluate anxiety-like behaviors, and the Y-maze test and novel object recognition test (NORT) were used to evaluate recognition memory behaviors, respectively. RESULTS: We found that repetitive taVNS was well tolerated by awake mice, had no effect on anxiety-like behaviors, and significantly improved memory. CONCLUSION: Our findings suggest that this new taVNS device for repetitive stimulation of awake mice is safe, tolerable, and effective.

Mediating Effect of the Parent-Child Relationship on the Association Between Maternal Nurturance and Early Child Development: A Longitudinal Study During the COVID-19 Pandemic.

Background: Research on the specific pathways from maternal nurturance to early child development remains limited. Grounded in transactional theory, this study is the first to examine these pathways through the parent-child relationship. Methods: This longitudinal study involved mothers of children aged 1-3 years. Data on sociodemographic characteristics, Comprehensive General Parenting Questionnaire, and Child-Parent Relationship Scale were collected at Time 1, when children were 1 year old. At Time 2, when children were 3 years old, Caregiver-Reported Early Development Instruments were measured. Structural equation modeling (SEM) was used to explore direct and indirect pathways from maternal nurturance to early child development. Results: A total of 1145 mother-child dyads participated, with children averaging 32 months (SD = 6.4) and mothers averaging 28.7 years (SD = 4.0). Maternal nurturance had significant direct (ß = 0.271), indirect (ß = 0.065), and total (ß = 0.336) effects on early child development. Direct effects accounted for 80.7% of the total effects, while indirect effects accounted for 19.3%. Maternal nurturance indirectly predicted higher early child development through increased parent-child closeness (ß = 0.048), explaining 14.3% of the total effects. Maternal nurturance indirectly promoted early child development through reduced parent-child conflict (ß = 0.017), explaining 5.1% of the total effects. Conclusion: The novelty of this study lies in its demonstration of the mediating role of the parent-child relationship in the effect of maternal nurturance on early child development. This longitudinal study provides insights for governments agencies, policymakers, and healthcare workers to develop intervention programs that enhance maternal nurturance through the parent-child relationship to promote early child development.

Divergent input patterns to the central lateral amygdala play a duet in fear memory formation.

Somatostatin (SOM)-expressing neurons in the central lateral amygdala (CeL) are responsible for fear memory learning, but the circuit and molecular mechanisms underlying this biology remain elusive. Here, we found that glutamatergic neurons in the lateral parabrachial nucleus (LPB) directly dominated the activity of CeLSOM neurons, and that selectively inhibiting the LPBGlu→CeLSOM pathway suppressed fear memory acquisition. By contrast, inhibiting CeL-projecting glutamatergic neurons in the paraventricular thalamic nucleus (PVT) interfered with consolidation-related processes. Notably, CeLSOM-innervating neurons in the LPB were modulated by presynaptic cannabinoid receptor 1 (CB1R), and knock down of CB1Rs in LPB glutamatergic neurons enhanced excitatory transmission to the CeL and partially rescued the impairment in fear memory induced by CB1R activation in the CeL. Overall, our study reveals the mechanisms by which CeLSOM neurons mediate the formation of fear memories during fear conditioning in mice, which may provide a new direction for the clinical research of fear-related disorders.

Autism-associated neuroligin 3 deficiency in medial septum causes social deficits and sleep loss in mice.

Patients with autism spectrum disorder (ASD) frequently experience sleep disturbance. Genetic mutations in the neuroligin 3 (NLG3) gene are highly correlative with ASD and sleep disturbance. However, the cellular and neural circuit bases of this correlation remain elusive. Here, we found that the conditional knockout of Nlg3 (Nlg3-CKO) in the medial septum (MS) impairs social memory and reduces sleep. Nlg3 CKO in the MS caused hyperactivity of MSGABA neurons during social avoidance and wakefulness. Activation of MSGABA neurons induced social memory deficits and sleep loss in C57BL/6J mice. In contrast, inactivation of these neurons ameliorated social memory deficits and sleep loss in Nlg3-CKO mice. Sleep deprivation led to social memory deficits, while social isolation caused sleep loss, both resulting in a reduction in NLG3 expression and an increase in activity of GABAergic neurons in the MS from C57BL/6J mice. Furthermore, MSGABA-innervated CA2 neurons specifically regulated social memory without impacting sleep, whereas MSGABA-innervating neurons in the preoptic area selectively controlled sleep without affecting social behavior. Together, these findings demonstrate that the hyperactive MSGABA neurons impair social memory and disrupt sleep resulting from Nlg3 CKO in the MS, and achieve the modality specificity through their divergent downstream targets.

Acute Ongoing Nociception Delays Recovery of Consciousness from Sevoflurane Anesthesia via a Midbrain Circuit.

Although anesthesia provides favorable conditions for surgical procedures, recent studies have revealed that the brain remains active in processing noxious signals even during anesthesia. However, whether and how these responses affect the anesthesia effect remains unclear. The ventrolateral periaqueductal gray (vlPAG), a crucial hub for pain regulation, also plays an essential role in controlling general anesthesia. Hence, it was hypothesized that the vlPAG may be involved in the regulation of general anesthesia by noxious stimuli. Here, we found that acute noxious stimuli, including capsaicin-induced inflammatory pain, acetic acid-induced visceral pain, and incision-induced surgical pain, significantly delayed recovery from sevoflurane anesthesia in male mice, whereas this effect was absent in the spared nerve injury-induced chronic pain. Pretreatment with peripheral analgesics could prevent the delayed recovery induced by acute nociception. Furthermore, we found that acute noxious stimuli, induced by the injection of capsaicin under sevoflurane anesthesia, increased c-Fos expression and activity in the GABAergic neurons of the ventrolateral periaqueductal gray. Specific reactivation of capsaicin-activated vlPAGGABA neurons mimicked the effect of capsaicin and its chemogenetic inhibition prevented the delayed recovery from anesthesia induced by capsaicin. Finally, we revealed that the vlPAGGABA neurons regulated the recovery from anesthesia through the inhibition of ventral tegmental area dopaminergic neuronal activity, thus decreasing dopamine (DA) release and activation of DA D1-like receptors in the brain. These findings reveal a novel, cell- and circuit-based mechanism for regulating anesthesia recovery by nociception, and it is important to provide new insights for guiding the management of the anesthesia recovery period.

Midbrain glutamatergic circuit mechanism of resilience to socially transferred allodynia in male mice.

The potential brain mechanism underlying resilience to socially transferred allodynia remains unknown. Here, we utilize a well-established socially transferred allodynia paradigm to segregate male mice into pain-susceptible and pain-resilient subgroups. Brain screening results show that ventral tegmental area glutamatergic neurons are selectively activated in pain-resilient mice as compared to control and pain-susceptible mice. Chemogenetic manipulations demonstrate that activation and inhibition of ventral tegmental area glutamatergic neurons bi-directionally regulate resilience to socially transferred allodynia. Moreover, ventral tegmental area glutamatergic neurons that project specifically to the nucleus accumbens shell and lateral habenula regulate the development and maintenance of the pain-resilient phenotype, respectively. Together, we establish an approach to explore individual variations in pain response and identify ventral tegmental area glutamatergic neurons and related downstream circuits as critical targets for resilience to socially transferred allodynia and the development of conceptually innovative analgesics.

Transcranial Direct Current Stimulation for Anxiety During Laparoscopic Colorectal Cancer Surgery: A Randomized Clinical Trial.

Importance: Perioperative anxiety is prevalent among patients undergoing surgical treatment of cancer and often influences their prognosis. Transcranial direct current stimulation (tDCS) has shown potential in the treatment of various anxiety-related disorders, but data on the impact of tDCS on perioperative anxiety are limited. Objective: To evaluate the effect of tDCS in reducing perioperative anxiety among patients undergoing laparoscopic colorectal cancer (CRC) resection. Design, Setting, And Participants: This randomized clinical trial was conducted from March to August 2023 at the Affiliated Hospital of Xuzhou Medical University. Patients aged 18 years or older undergoing elective laparoscopic radical resection for CRC were randomly assigned to either the active tDCS group or the sham tDCS group. Intention-to-treat data analysis was performed in September 2023. Interventions: Patients were randomly assigned to receive 2 sessions of either active tDCS or sham tDCS over the left dorsolateral prefrontal cortex on the afternoon of the day before the operation and in the morning of the day of operation. Main Outcomes and Measures: The main outcome was the incidence of perioperative anxiety from the day of the operation up to 3 days after the procedure, as measured using the Hospital Anxiety and Depression Scale-Anxiety (HADS-A) subscale (range: 0-21, with higher scores indicating more anxiety). Secondary outcomes included postoperative delirium (assessed by the Confusion Assessment Method or Confusion Assessment Method intensive care unit scale); pain (assessed by the 10-point Numeric Rating Scale [NRS], with scores ranging from 0 [no pain] to 10 [worst pain]); frailty (assessed by the Fatigue, Resistance, Ambulation, Illness and Loss of Weight [FRAIL] Index, with scores ranging from 0 [most robust] to 5 [most frail]; and sleep quality (assessed by the Pittsburgh Sleep Quality Index [PSQI], with scores ranging from 0 to 21 and higher scores indicating worse sleep quality) after the 2 sessions of the tDCS intervention. Results: A total of 196 patients (mean [SD] age, 63.5 [11.0] years; 124 [63.3%] men) were recruited and randomly assigned to the active tDCS group (98 patients) or the sham tDCS group (98 patients). After the second tDCS intervention on the day of the operation, the incidence of perioperative anxiety was 38.8% in the active tDCS group and 70.4% in the sham tDCS group (relative risk, 0.55 [95% CI, 0.42-0.73]; P < .001). Patients in the active tDCS group vs the sham tDCS group were less likely to have postoperative delirium (8.2% vs 25.5%) and, at 3 days after the operation, had lower median (IQR) pain scores (NRS, 1.0 [1.0-1.0] vs 2.0 [2.0-2.0]), better median (IQR) sleep quality scores (PSQI, 10.5 [10.0-11.0] vs 12.0 [11.0-13.0]), and lower median (IQR) FRAIL Index (2.0 [1.0-2.0] vs 2.0 [2.0-3.0]). Conclusions and Relevance: Findings of this randomized clinical trial indicate that administration of 2 preoperative sessions of tDCS was associated with a decreased incidence of perioperative anxiety in patients undergoing elective CRC resection. Active tDCS was also associated with better anxiety scores, pain levels, and sleep quality as well as reduced postoperative delirium and frailty. The findings suggest that tDCS may be a novel strategy for improving perioperative anxiety in patients undergoing CRC resection. Trial Registration: Chinese Clinical Trial Register Identifier: ChiCTR2300068859.

CRF regulates pain sensation by enhancement of corticoaccumbal excitatory synaptic transmission.

Both peripheral and central corticotropin-releasing factor (CRF) systems have been implicated in regulating pain sensation. However, compared with the peripheral, the mechanisms underlying central CRF system in pain modulation have not yet been elucidated, especially at the neural circuit level. The corticoaccumbal circuit, a structure rich in CRF receptors and CRF-positive neurons, plays an important role in behavioral responses to stressors including nociceptive stimuli. The present study was designed to investigate whether and how CRF signaling in this circuit regulated pain sensation under physiological and pathological pain conditions. Our studies employed the viral tracing and circuit-, and cell-specific electrophysiological methods to label the CRF-containing circuit from the medial prefrontal cortex to the nucleus accumbens shell (mPFCCRF-NAcS) and record its neuronal propriety. Combining optogenetic and chemogenetic manipulation, neuropharmacological methods, and behavioral tests, we were able to precisely manipulate this circuit and depict its role in regulation of pain sensation. The current study found that the CRF signaling in the NAc shell (NAcS), but not NAc core, was necessary and sufficient for the regulation of pain sensation under physiological and pathological pain conditions. This process was involved in the CRF-mediated enhancement of excitatory synaptic transmission in the NAcS. Furthermore, we demonstrated that the mPFCCRF neurons monosynaptically connected with the NAcS neurons. Chronic pain increased the protein level of CRF in NAcS, and then maintained the persistent NAcS neuronal hyperactivity through enhancement of this monosynaptic excitatory connection, and thus sustained chronic pain behavior. These findings reveal a novel cell- and circuit-based mechanistic link between chronic pain and the mPFCCRF → NAcS circuit and provide a potential new therapeutic target for chronic pain.

The functional role of the visual and olfactory modalities in the development of socially transferred mechanical hypersensitivity in male C57BL/6J mice.

An increasing body of evidence suggests that the state of hyperalgesia could be socially transferred from one individual to another through a brief empathetic social contact. However, how the social transfer of pain develops during social contact is not well-known. Utilizing a well-established mouse model, the present study aims to study the functional role of visual and olfactory cues in the development of socially-transferred mechanical hypersensitivity. Behavioral tests demonstrated that one hour of brief social contact with a conspecific mouse injected with complete Freund's adjuvant (CFA) was both sufficient and necessary for developing socially-transferred mechanical hypersensitivity. One hour of social contact with visual deprivation could not prevent the development of socially-transferred mechanical hypersensitivity, and screen observation of a CFA cagemate was not sufficient to develop socially-transferred mechanical hypersensitivity in bystanders. Methimazole-induced olfactory deprivation, a compound with reversible toxicity on the nasal olfactory epithelium, was sufficient to prevent the development of socially-transferred mechanical hypersensitivity. Intriguingly, repeated but not acute olfactory exposure to the CFA mouse bedding induced a robust decrease in 50 % paw withdrawal thresholds (50 %PWTs) to mechanical stimuli, an effect returned to the baseline level after two days of washout with clean bedding. The findings strongly indicate that the normal olfactory function is crucial for the induction of mechanical hypersensitivity through brief empathetic contact, offering valuable insights for animal housing in future pain research.

Neuronal and Molecular Mechanisms Underlying Chronic Pain and Depression Comorbidity in the Paraventricular Thalamus.

Patients with chronic pain often develop comorbid depressive symptoms, which makes the pain symptoms more complicated and refractory. However, the underlying mechanisms are poorly known. Here, in a repeated complete Freund's adjuvant (CFA) male mouse model, we reported a specific regulatory role of the paraventricular thalamic nucleus (PVT) glutamatergic neurons, particularly the anterior PVT (PVA) neurons, in mediating chronic pain and depression comorbidity (CDC). Our c-Fos protein staining observed increased PVA neuronal activity in CFA-CDC mice. In wild-type mice, chemogenetic activation of PVA glutamatergic neurons was sufficient to decrease the 50% paw withdrawal thresholds (50% PWTs), while depressive-like behaviors evaluated with immobile time in tail suspension test (TST) and forced swim test (FST) could only be achieved by repeated chemogenetic activation. Chemogenetic inhibition of PVA glutamatergic neurons reversed the decreased 50% PWTs in CFA mice without depressive-like symptoms and the increased TST and FST immobility in CFA-CDC mice. Surprisingly, in CFA-CDC mice, chemogenetically inhibiting PVA glutamatergic neurons failed to reverse the decrease of 50% PWTs, which could be restored by rapid-onset antidepressant S-ketamine. Further behavioral tests in chronic restraint stress mice and CFA pain mice indicated that PVA glutamatergic neuron inhibition and S-ketamine independently alleviate sensory and affective pain. Molecular profiling and pharmacological studies revealed the 5-hydroxytryptamine receptor 1D (Htr1d) in CFA pain-related PVT engram neurons as a potential target for treating CDC. These findings identified novel CDC neuronal and molecular mechanisms in the PVT and provided insight into the complicated pain neuropathology under a comorbid state with depression and related drug development.

Efficacy of transcranial direct current stimulation for improving postoperative quality of recovery in elderly patients undergoing lower limb major arthroplasty: a randomized controlled substudy.

Background: Previous studies have demonstrated improvements in motor, behavioral, and emotional areas following transcranial direct current stimulation (tDCS), but no published studies have reported the efficacy of tDCS on postoperative recovery quality in patients undergoing lower limb major arthroplasty. We hypothesized that tDCS might improve postoperative recovery quality in elderly patients undergoing lower limb major arthroplasty. Methods: Ninety-six patients (≥65 years) undergoing total hip arthroplasty (THA) or total knee arthroplasty (TKA) were randomized to receive 2 mA tDCS for 20 min active-tDCS or sham-tDCS. The primary outcome was the 15-item quality of recovery (QoR-15) score on postoperative day one (Т2). Secondary outcomes included the QoR-15 scores at the 2nd hour (T1), the 1st month (Т3), and the 3rd month (Т4) postoperatively, numeric rating scale scores, and fatigue severity scale scores. Results: Ninety-six elderly patients (mean age, 71 years; 68.7% woman) were analyzed. Higher QoR-15 scores were found in the active-tDCS group at T2 (123.0 [114.3, 127.0] vs. 109.0 [99.3, 115.3]; median difference, 13.0; 95% CI, 8.0 to 17.0; p < 0.001). QoR-15 scores in the active-tDCS group were higher at T1 (p < 0.001), T3 (p = 0.001), and T4 (p = 0.001). The pain scores in the active-tDCS group were lower (p < 0.001 at motion; p < 0.001 at rest). The fatigue degree scores were lower in the active-tDCS group at T1 and T2 (p < 0.001 for each). Conclusion: tDCS may help improve the quality of early recovery in elderly patients undergoing lower limb major arthroplasty. Clinical trial registration: The trial was registered at the China Clinical Trial Center (ChiCTR2200057777, https://www.chictr.org.cn/showproj.html?proj=162744).

The anterior cingulate cortex controls the hyperactivity in subthalamic neurons in male mice with comorbid chronic pain and depression.

Neurons in the subthalamic nucleus (STN) become hyperactive following nerve injury and promote pain-related responses in mice. Considering that the anterior cingulate cortex (ACC) is involved in pain and emotion processing and projects to the STN, we hypothesize that ACC neurons may contribute to hyperactivity in STN neurons in chronic pain. In the present study, we showed that ACC neurons enhanced activity in response to noxious stimuli and to alterations in emotional states and became hyperactive in chronic pain state established by spared nerve injury of the sciatic nerve (SNI) in mice. In naïve mice, STN neurons were activated by noxious stimuli, but not by alterations in emotional states. Pain responses in STN neurons were attenuated in both naïve and SNI mice when ACC neurons were inhibited. Furthermore, optogenetic activation of the ACC-STN pathway induced bilateral hyperalgesia and depression-like behaviors in naive mice; conversely, inhibition of this pathway is sufficient to attenuate hyperalgesia and depression-like behaviors in SNI mice and naïve mice subjected to stimulation of STN neurons. Finally, mitigation of pain-like and depression-like behaviors in SNI mice by inhibition of the ACC-STN projection was eliminated by activation of STN neurons. Our results demonstrate that hyperactivity in the ACC-STN pathway may be an important pathophysiology in comorbid chronic pain and depression. Thus, the ACC-STN pathway may be an intervention target for the treatment of the comorbid chronic pain and depression.

DNA N6-methyladenine methylase N6AMT1 controls neuropathic pain through epigenetically modifying Kcnj16 in dorsal horn neurons.

ABSTRACT: Nerve injury-induced aberrant changes in gene expression in spinal dorsal horn neurons are critical for the genesis of neuropathic pain. N6-methyladenine (m 6 A) modification of DNA represents an additional layer of gene regulation. Here, we report that peripheral nerve injury significantly decreased the level of m 6 A-specific DNA methyltransferase 1 ( N6amt1 ) in dorsal horn neurons. This decrease was attributed, at least partly, to a reduction in transcription factor Nr2f6 . Rescuing the decrease in N6amt1 reversed the loss of m 6 A at the promoter for inwardly rectifying potassium channel subfamily J member 16 ( Kcnj16 ), mitigating the nerve injury-induced upregulation of Kcnj16 expression in the dorsal horn and alleviating neuropathic pain hypersensitivities. Conversely, mimicking the downregulation of N6amt1 in naive mice erased DNA m 6 A at the Kcnj16 promoter, elevated Kcnj16 expression, and led to neuropathic pain-like behaviors. Therefore, decreased N6amt1 caused by NR2F6 is required for neuropathic pain, likely through its regulation of m 6 A-controlled KCNJ16 in dorsal horn neurons, suggesting that DNA m 6 A modification may be a potential new target for analgesic and treatment strategies.

An Ascending Excitatory Circuit from the Dorsal Raphe for Sensory Modulation of Pain.

The dorsal raphe nucleus (DRN) is an important nucleus in pain regulation. However, the underlying neural pathway and the function of specific cell types remain unclear. Here, we report a previously unrecognized ascending facilitation pathway, the DRN to the mesoaccumbal dopamine (DA) circuit, for regulating pain. Chronic pain increased the activity of DRN glutamatergic, but not serotonergic, neurons projecting to the ventral tegmental area (VTA) (DRNGlu-VTA) in male mice. The optogenetic activation of DRNGlu-VTA circuit induced a pain-like response in naive male mice, and its inhibition produced an analgesic effect in male mice with neuropathic pain. Furthermore, we discovered that DRN ascending pathway regulated pain through strengthened excitatory transmission onto the VTA DA neurons projecting to the ventral part of nucleus accumbens medial shell (vNAcMed), thereby activated the mesoaccumbal DA neurons. Correspondingly, optogenetic manipulation of this three-node pathway bilaterally regulated pain behaviors. These findings identified a DRN ascending excitatory pathway that is crucial for pain sensory processing, which can potentially be exploited toward targeting pain disorders.

Pulmonary vagus nerve transection for chronic cough after video-assisted lobectomy: a randomized controlled trial.

BACKGROUND: Chronic cough is common after lobectomy. Vagus nerves are part of the cough reflex. Accordingly, transection of the pulmonary branches of vagus nerve may prevent chronic cough. And there are no clear recommendations on the management of the pulmonary branches of vagus in any thoracic surgery guidelines. METHODS: This is a single-center, randomized controlled trial. Adult patients undergoing elective video-assisted thoracoscopic lobectomy and lymphadenectomy were randomized at a 1:1 ratio to undergo a sham procedure (control group) or transection of the pulmonary branches of the vagus nerve that innervate the bronchial stump plus the caudal-most large pulmonary branch of the vagus nerve. The primary outcome was the rate of chronic cough, as assessed at 3 months after surgery in the intent-to-treat population. RESULTS: Between 1 February 2020 and 1 August 2020, 116 patients (59.6±10.1 years of age; 45 men) were randomized (58 in each group). All patients received designated intervention. The rate of chronic cough at 3 months was 19.0% (11/58) in the vagotomy group versus 41.4% (24/58) in the control group (OR=0.332, 95% CI: 0.143-0.767; P =0.009). In the 108 patients with 2-year assessment, the rate of persistent cough was 12.7% (7/55) in the control and 1.9% (1/53) in the vagotomy group ( P =0.032). The two groups did not differ in postoperative complications and key measures of pulmonary function, for example, maximal voluntary ventilation, diffusing capacity of the lungs for carbon monoxide, and forced expiratory volume. CONCLUSION: Transecting the pulmonary branches of vagus nerve that innervate the bronchial stump plus the caudal-most large pulmonary branch decreased the rate of chronic cough without affecting pulmonary function in patients undergoing video-assisted lobectomy and lymphadenectomy.

Multiple integrated social stress induces depressive-like behavioral and neural adaptations in female C57BL/6J mice.

Despite women representing most of those affected by major depression, preclinical studies have focused almost exclusively on male subjects, partially due to a lack of ideal animal paradigms. As the persistent need regarding the sex balance of neuroscience research and female-specific pathology of mental disorders surges, the establishment of natural etiology-based and systematically validated animal paradigms for depression with female subjects becomes an urgent scientific problem. This study aims to establish, characterize, and validate a "Multiple Integrated Social Stress (MISS)" model of depression in female C57BL/6J mice by manipulating and integrating daily social stressors that females are experiencing. Female C57BL/6J mice randomly experienced social competition failure in tube test, modified vicarious social defeat stress, unescapable overcrowding stress followed by social isolation on each day, for ten consecutive days. Compared with their controls, female MISS mice exhibited a relatively decreased preference for social interaction and sucrose, along with increased immobility in the tail suspension test, which could last for at least one month. These MISS mice also exhibited increased levels of blood serum corticosterone, interleukin-6 L and 1ß. In the pharmacological experiment, MISS-induced dysfunctions in social interaction, sucrose preference, and tail suspension tests were amended by systematically administrating a single dose of sub-anesthetic ketamine, a rapid-onset antidepressant. Compared with controls, MISS females exhibited decreased c-Fos activation in their anterior cingulate cortex, prefrontal cortex, nucleus accumbens and some other depression-related brain regions. Furthermore, 24 h after the last exposure to the paradigm, MISS mice demonstrated a decreased center zone time in the open field test and decreased open arm time in the elevated plus-maze test, indicating anxiety-like behavioral phenotypes. Interestingly, MISS mice developed an excessive nesting ability, suggesting a likely behavioral phenotype of obsessive-compulsive disorder. These data showed that the MISS paradigm was sufficient to generate pathological profiles in female mice to mimic core symptoms, serum biochemistry and neural adaptations of depression in clinical patients. The present study offers a multiple integrated natural etiology-based animal model tool for studying female stress susceptibility.

CWC22-Mediated Alternative Splicing of Spp1 Regulates Nociception in Inflammatory Pain.

Increasing evidence suggests that alternative splicing plays a critical role in pain, but its underlying mechanism remains elusive. Herein, we employed complete Freund's adjuvant (CFA) to induce inflammatory pain in mice. A combination of genomics research techniques, lentivirus-based genetic manipulations, behavioral tests, and molecular biological technologies confirmed that splicing factor Cwc22 mRNA and CWC22 protein were elevated in the spinal dorsal horn at 3 days after CFA injection. Knockdown of spinal CWC22 by lentivirus transfection (lenti-shCwc22) reversed CFA-induced thermal hyperalgesia and mechanical allodynia, whereas upregulation of spinal CWC22 (lenti-Cwc22) in naïve mice precipitated pain. Comprehensive transcriptome and genome analysis identified the secreted phosphoprotein 1 (Spp1) as a potential gene of CWC22-mediated alternative splicing, however, only Spp1 splicing variant 4 (Spp1 V4) was involved in thermal and mechanical nociceptive regulation. In conclusion, our findings demonstrate that spinal CWC22 regulates Spp1 V4 to participate in CFA-induced inflammatory pain. Blocking CWC22 or CWC22-mediated alternative splicing may provide a novel therapeutic target for the treatment of persistent inflammatory pain.

Spinal-Specific Super Enhancer in Neuropathic Pain.

Dysfunctional gene expression in nociceptive pathways plays a critical role in the development and maintenance of neuropathic pain. Super enhancers (SEs), composed of a large cluster of transcriptional enhancers, are emerging as new players in the regulation of gene expression. However, whether SEs participate in nociceptive responses remains unknown. Here, we report a spinal-specific SE (SS-SE) that regulates chronic constriction injury (CCI)-induced neuropathic pain by driving Ntmt1 and Prrx2 transcription in dorsal horn neurons. Peripheral nerve injury significantly enhanced the activity of SS-SE and increased the expression of NTMT1 and PRRX2 in the dorsal horn of male mice in a bromodomain-containing protein 4 (BRD4)-dependent manner. Both intrathecal administration of a pharmacological BRD4 inhibitor JQ1 and CRISPR-Cas9-mediated SE deletion abolished the increased NTMT1 and PRRX2 in CCI mice and attenuated their nociceptive hypersensitivities. Furthermore, knocking down Ntmt1 or Prrx2 with siRNA suppressed the injury-induced elevation of phosphorylated extracellular-signal-regulated kinase (p-ERK) and glial fibrillary acidic protein (GFAP) expression in the dorsal horn and alleviated neuropathic pain behaviors. Mimicking the increase in spinal Ntmt1 or Prrx2 in naive mice increased p-ERK and GFAP expression and led to the genesis of neuropathic pain-like behavior. These results redefine our understanding of the regulation of pain-related genes and demonstrate that BRD4-driven increases in SS-SE activity is responsible for the genesis of neuropathic pain through the governance of NTMT1 and PRRX2 expression in dorsal horn neurons. Our findings highlight the therapeutic potential of BRD4 inhibitors for the treatment of neuropathic pain.SIGNIFICANCE STATEMENT SEs drive gene expression by recruiting master transcription factors, cofactors, and RNA polymerase, but their role in the development of neuropathic pain remains unknown. Here, we report that the activity of an SS-SE, located upstream of the genes Ntmt1 and Prrx2, was elevated in the dorsal horn of mice with neuropathic pain. SS-SE contributes to the genesis of neuropathic pain by driving expression of Ntmt1 and Prrx2 Both inhibition of SS-SE with a pharmacological BRD4 inhibitor and genetic deletion of SS-SE attenuated pain hypersensitivities. This study suggests an effective and novel therapeutic strategy for neuropathic pain.