Lights at night mediate depression-like behavioral and molecular phenotypes in a glucocorticoid-dependent manner in male rats.

Nocturnal light pollution, an underappreciated mood manipulator, disturbs the circadian rhythms of individuals in modern society. Preclinical and clinical studies have suggested that exposure to lights at night (LANs) results in depression-like phenotypes. However, the mechanism underlying the action of LANs remains unclear. Therefore, this study explored the potential influence of LANs on depression-related brain regions by testing brain-derived neurotrophic factor (BDNF), synaptic transmission, and plasticity in male Sprague-Dawley rats. Depression-related behavioral tests, enzyme-linked immunosorbent assays, and intracellular and extracellular electrophysiological recordings were performed. Resultantly, rats exposed to either white or blue LAN for 5 or 21 days exhibited depression-like behaviors. Both white and blue LANs reduced BDNF expression in the medial prefrontal cortex (mPFC) and ventrolateral periaqueductal gray (vlPAG). Moreover, both lights at night (LANs) elevated the plasma corticosterone levels. Pharmacologically, the activation of glucocorticoid receptors mimics the LAN-mediated effects on depression-like behaviors and reduces BDNF levels, whereas the inhibition of glucocorticoid receptors blocks LAN-mediated behavioral and molecular actions. Electrophysiologically, both LANs attenuated the stimulation-response curve, increased the paired-pulse ratio, and decreased the frequency and amplitude of miniature excitatory postsynaptic currents in the vlPAG. In the mPFC, LANs attenuate long-term potentiation and long-term depression. Collectively, these results suggested that white and blue LANs disturbed BDNF expression, synaptic transmission, and plasticity in the vlPAG and mPFC in a glucocorticoid-dependent manner. The results of the present study provide a theoretical basis for understanding the effects of nocturnal light exposure on depression-like phenotypes.

Blue light at night produces stress-evoked heightened aggression by enhancing brain-derived neurotrophic factor in the basolateral amygdala.

Light is an underappreciated mood manipulator. People are often exposed to electronic equipment, which results in nocturnal blue light exposure in modern society. Light pollution drastically shortens the night phase of the circadian rhythm. Preclinical and clinical studies have reported that nocturnal light exposure can influence mood, such as depressive-like phenotypes. However, the effects of blue light at night (BLAN) on other moods and how it alters mood remain unclear. Here, we explored the impact of BLAN on stress-provoked aggression in male Sprague‒Dawley rats, focusing on its influence on basolateral amygdala (BLA) activity. Resident-intruder tests, extracellular electrophysiological recordings, and enzyme-linked immunosorbent assays were performed. The results indicated that BLAN produces stress-induced heightened aggressive and anxiety-like phenotypes. Moreover, BLAN not only potentiates long-term potentiation and long-term depression in the BLA but also results in stress-induced elevation of brain-derived neurotrophic factor (BDNF), mature BDNF, and phosphorylation of tyrosine receptor kinase B expression in the BLA. Intra-BLA microinfusion of BDNF RNAi, BDNF neutralizing antibody, K252a, and rapamycin blocked stress-induced heightened aggressive behavior in BLAN rats. In addition, intra-BLA application of BDNF and 7,8-DHF caused stress-induced heightened aggressive behavior in naïve rats. Collectively, these results suggest that BLAN results in stress-evoked heightened aggressive phenotypes, which may work by enhancing BLA BDNF signaling and synaptic plasticity. This study reveals that nocturnal blue light exposure may have an impact on stress-provoked aggression. Moreover, this study provides novel insights into the BLA BDNF-dependent mechanism underlying the impact of the BLAN on mood.

Protein Arginine Methyltransferase 5 Contributes to Paclitaxel-Induced Neuropathic Pain by Activating Transient Receptor Potential Vanilloid 1 Epigenetic Modification in Dorsal Root Ganglion.

BACKGROUND: Paclitaxel (PTX), which is a first-line chemotherapy drug used to treat various types of cancers, exhibits peripheral neuropathy as a common side effect that is difficult to treat. Protein arginine methyltransferase 5 (PRMT 5) is a key regulator of the chemotherapy response, as chemotherapy drugs induce PRMT5 expression. However, little is known about the PRMT5-mediated epigenetic mechanisms involved in PTX-induced neuropathic allodynia. METHODS: Sprague-Dawley rats were intraperitoneally given PTX to induce neuropathic pain. Biochemical analyses were conducted to measure the protein expression levels in the dorsal root ganglion (DRG) of the animals. The von Frey test and hot plate test were used to evaluate nociceptive behaviors. RESULTS: PTX increased the PRMT5 (mean difference [MD]: 0.68, 95% confidence interval [CI], 0.88-0.48; P < .001 for vehicle)-mediated deposition of histone H3R2 dimethyl symmetric (H3R2me2s) at the transient receptor potential vanilloid 1 ( Trpv1 ) promoter in the DRG. PRMT5-induced H3R2me2s recruited WD repeat domain 5 (WDR5) to increase trimethylation of lysine 4 on histone H3 (H3K4me3) at Trpv1 promoters, thus resulting in TRPV1 transcriptional activation (MD: 0.65, 95% CI, 0.82-0.49; P < .001 for vehicle) in DRG in PTX-induced neuropathic pain. Moreover, PTX increased the activity of NADPH oxidase 4 (NOX4) (MD: 0.66, 95% CI, 0.81-0.51; P < .001 for vehicle), PRMT5-induced H3R2me2s, and WDR5-mediated H3K4me3 in the DRG in PTX-induced neuropathic pain. Pharmacological antagonism and the selective knockdown of PRMT5 in DRG neurons completely blocked PRMT5-mediated H3R2me2s, WDR5-mediated H3K4me3, or TRPV1 expression and neuropathic pain development after PTX injection. Remarkably, NOX4 inhibition not only attenuated allodynia behavior and reversed the above-mentioned signaling but also reversed NOX4 upregulation via PTX. CONCLUSIONS: Thus, the NOX4/PRMT5-associated epigenetic mechanism in DRG has a dominant function in the transcriptional activation of TRPV1 in PTX-induced neuropathic pain.

Melatonin Relieves Paclitaxel-Induced Neuropathic Pain by Regulating pNEK2-Dependent Epigenetic Pathways in DRG Neurons.

The neurohormone melatonin (MLT) demonstrates promising potential in ameliorating neuropathic pain induced by paclitaxel (PTX) chemotherapy. However, little is known about its protective effect on dorsal root ganglion (DRG) neurons in neuropathic pain resulting from the chemotherapeutic drug PTX. Here, PTX-treated rats revealed that intrathecal administration of MLT dose-dependently elevated hind paw withdrawal thresholds and latency, indicating that MLT significantly reversed PTX-induced neuropathic pain. Mechanistically, the analgesic effects of MLT were found to be mediated via melatonin receptor 2 (MT2), as pretreatment with an MT2 receptor antagonist inhibited these effects. Moreover, intrathecal MLT injection reversed the pNEK2-dependent epigenetic program induced by PTX. All of the effects caused by MLT were blocked by pretreatment with an MT2 receptor-selective antagonist, 4P-PDOT. Remarkably, multiple MLT administered during PTX treatment (PTX+MLTs) exhibited not only rapid but also lasting reversal of allodynia/hyperalgesia compared to single-bolus MLT administered after PTX treatment (PTX+MLT). In addition, PTX+MLTs exhibited greater efficacy in reversing PTX-induced alterations in pRSK2, pNEK2, JMJD3, H3K27me3, and TRPV1 expression and interaction in DRG neurons than PTX+MLT. These results indicated that MLT administered during PTX treatment reduced the incidence and/or severity of neuropathy and had a better inhibitory effect on the pNEK2-dependent epigenetic program compared to MLT administered after PTX treatment. In conclusion, MLT/MT2 is a promising therapy for the treatment of pNEK2-dependent painful neuropathy resulting from PTX treatment. MLT administered during PTX chemotherapy may be more effective in the prevention or reduction of PTX-induced neuropathy and maintaining quality.

Pressure-Volume Loop Analysis of Voiding Workload: An Application in Trans-Vaginal Mesh-Repaired Pelvic Organ Prolapse Patients.

Although trans-vaginal mesh (TVM) offers a successful anatomical reconstruction and can subjectively relieve symptoms/signs in pelvic organ prolapse (POP) patients, its objective benefits to the voiding function of the bladder have not been well established. In this study, we investigated the therapeutic advantage of TVM on bladder function by focusing on the thermodynamic workload of voiding. The histories of 31 POP patients who underwent TVM repair were retrospectively reviewed. Cystometry and pressure volume analysis (PVA) of the patients performed before and after the operation were analyzed. TVM postoperatively decreased the mean voiding resistance (mRv, p < 0.05, N = 31), reduced the mean and peak voiding pressure (mPv, p < 0.05 and pPv, p < 0.01, both N = 31), and elevated the mean flow rate (mFv, p < 0.05, N = 31) of voiding. While displaying an insignificant effect on the voided volume (Vv, p < 0.05, N = 31), TVM significantly shortened the voiding time (Tv, p < 0.05, N = 31). TVM postoperatively decreased the loop-enclosed area (Apv, p < 0.05, N = 31) in the PVA, indicating that TVM lessened the workload of voiding. Moreover, in 21 patients who displayed postvoiding urine retention before the operation, TVM decreased the residual volume (Vr, p < 0.01, N = 21). Collectively, our results reveal that TVM postoperatively lessened the workload of bladder voiding by diminishing voiding resistance, which reduced the pressure gradient required for driving urine flow.

BTRX-246040 Acts Through the Ventrolateral Periaqueductal Gray to Exert Antidepressant-Relevant Actions in Mice.

BACKGROUND: BTRX-246040, a nociceptin/orphanin FQ peptide receptor antagonist, is being developed for the treatment of depressive patients. However, the underlying mechanism of this potential antidepressant is still largely unclear. Here, we studied the antidepressant-related actions of BTRX-246040 in the ventrolateral periaqueductal gray (vlPAG). METHODS: The tail suspension test, forced swim test, female urine sniffing test, sucrose preference test, and learned helplessness (LH) combined with pharmacological approaches were employed to examine the antidepressant-like effects and drug effects on LH-induced depressive-like behavior in C57BL/6J mice. Electrophysiological recordings in vlPAG neurons were used to study synaptic activity. RESULTS: Intraperitoneal administration of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Systemic BTRX-246040 (10 mg/kg) resulted in an increased frequency and amplitude of miniature excitatory postsynaptic currents (EPSCs) in the vlPAG. Moreover, slice perfusion of BTRX-246040 directly elevated the frequency and amplitude of miniature EPSCs and enhanced the evoked EPSCs in the vlPAG, which were blocked by pretreatment with the nociceptin/orphanin FQ peptide receptor agonist Ro 64-6198. In addition, intra-vlPAG application of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Moreover, intra-vlPAG pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione reversed both systemic and local BTRX-246040-mediated antidepressant-like behavioral effects. Furthermore, both systemic and local BTRX-246040 decreased the LH phenotype and reduced LH-induced depressive-like behavior. CONCLUSIONS: The results suggested that BTRX-246040 may act through the vlPAG to exert antidepressant-relevant actions. The present study provides new insight into a vlPAG-dependent mechanism underlying the antidepressant-like actions of BTRX-246040.

Phosphorylated Upstream Frameshift 1-dependent Nonsense-mediated µ-Opioid Receptor mRNA Decay in the Spinal Cord Contributes to the Development of Neuropathic Allodynia-like Behavior in Rats.

BACKGROUND: Nonsense-mediated messenger RNA (mRNA) decay increases targeted mRNA degradation and has been implicated in the regulation of gene expression in neurons. The authors hypothesized that nonsense-mediated µ-opioid receptor mRNA decay in the spinal cord is involved in the development of neuropathic allodynia-like behavior in rats. METHODS: Adult Sprague-Dawley rats of both sexes received spinal nerve ligation to induce neuropathic allodynia-like behavior. The mRNA and protein expression contents in the dorsal horn of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by the von Frey test and the burrow test. RESULTS: On Day 7, spinal nerve ligation significantly increased phosphorylated upstream frameshift 1 (UPF1) expression in the dorsal horn (mean ± SD; 0.34 ± 0.19 in the sham ipsilateral group vs. 0.88 ± 0.15 in the nerve ligation ipsilateral group; P < 0.001; data in arbitrary units) and drove allodynia-like behaviors in rats (10.58 ± 1.72 g in the sham ipsilateral group vs. 1.19 ± 0.31 g in the nerve ligation ipsilateral group, P < 0.001). No sex-based differences were found in either Western blotting or behavior tests in rats. Eukaryotic translation initiation factor 4A3 (eIF4A3) triggered SMG1 kinase (0.06 ± 0.02 in the sham group vs. 0.20 ± 0.08 in the nerve ligation group, P = 0.005, data in arbitrary units)-mediated UPF1 phosphorylation, leading to increased nonsense-mediated mRNA decay factor SMG7 binding and µ-opioid receptor mRNA degradation (0.87 ± 0.11-fold in the sham group vs. 0.50 ± 0.11-fold in the nerve ligation group, P = 0.002) in the dorsal horn of the spinal cord after spinal nerve ligation. Pharmacologic or genetic inhibition of this signaling pathway in vivo ameliorated allodynia-like behaviors after spinal nerve ligation. CONCLUSIONS: This study suggests that phosphorylated UPF1-dependent nonsense-mediated µ-opioid receptor mRNA decay is involved in the pathogenesis of neuropathic pain.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

BACKGROUND: The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. METHODS: Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. RESULTS: PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. CONCLUSIONS: pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

(2R,6R)-hydroxynorketamine targeting the basolateral amygdala regulates fear memory.

(2R,6R)-Hydroxynorketamine (HNK), a ketamine metabolite, has been proposed as an ideal next-generation antidepressant due to its rapid-acting and long-lasting antidepression-relevant actions. Interestingly, recent studies have shown that (2R,6R)-HNK may have diverse impacts on memory formation. However, its effect on fear memory extinction is still unknown. In the present study, we assessed the effects of (2R,6R)-HNK on synaptic transmission and plasticity in the basolateral amygdala (BLA) and explored its actions on auditory fear memory extinction. Adult male C57BL/6J mice were used in this study. The extracellular electrophysiological recording was conducted to assay synaptic transmission and plasticity. The auditory fear conditioning paradigm was performed to test fear extinction. The results showed that (2R,6R)-HNK at 30 mg/kg increased the number of c-fos-positive cells in the BLA. Moreover, (2R,6R)-HNK enhanced the induction and maintenance of long-term potentiation (LTP) in the BLA in a dose-dependent manner (at 1, 10, and 30 mg/kg). In addition, (2R,6R)-HNK at 30 mg/kg and directly slice perfusion of (2R,6R)-HNK enhanced BLA synaptic transmission. Furthermore, intra-BLA application and systemic administration of (2R,6R)-HNK reduced the retrieval of recent fear memory and decreased the retrieval of remote fear memory. Both local and systemic (2R,6R)-HNK also inhibited the spontaneous recovery of remote fear memory. Taken together, these results indicated that (2R,6R)-HNK could regulate BLA synaptic transmission and plasticity and act through the BLA to modulate fear memory. The results revealed that (2R,6R)-HNK may be a potential drug to treat posttraumatic stress disorder (PTSD) patients.

Modification of bladder thermodynamics in stress urinary incontinence patients submitted to trans-obturator tape: A retrospective study based on urodynamic assessment.

Importance: It needs to be clarified whether trans-obturator tape (TOT)-enhanced urethral resistance could impact the voiding function. Objective: Although TOT has been well-recognized for enhancing urethral resistance to restore continence in stress urinary incontinence (SUI) patients, whether the bladder's voiding functions adapt to the TOT-enhanced resistance has not been adequately investigated. This study thereby aimed to investigate whether TOT impacts the bladder's thermodynamic efficacy during the voiding phase. Design: A retrospective analysis of urodynamics performed before and after TOT was assessed. Setting: A tertiary referral hospital in Taiwan. Participants: A total of 26 female SUI patients who underwent urodynamic investigations before and after TOT. Main outcomes and measures: The area enclosed by the pressure-volume loop (Apv), which represents the work performed by the bladder during voiding, in a pressure-volume analysis established by plotting the detrusor pressure versus intra-vesical volume was retrospectively analyzed. Paired Student's t-tests were employed to assess the difference in values before and after the operation. Significance in difference was set at p < 0.05. Results: TOT increased Apv in 20 of 26 (77%) patients and significantly increased the mean Apv compared to the preoperative control (2.17 ± 0.18 and 1.51 ± 0.13 × 103 cmH2O-ml, respectively p < 0.01). TOT also increased the mean urethral resistance (1.03 ± 0.30 vs. 0.29 ± 0.05 cmH2O-sec/ml, p < 0.01) and mean voiding pressure (25.87 ± 1.72 and 19.30 ± 1.98 cmH2O p < 0.01) but did not affect the voided volume and voiding time. Moreover, the TOT-induced Apv increment showed a moderate correlation with the changes in urethral resistance and voiding pressure (both r > 0.5) but no correlation with changes in voided volume or voiding time. It is remarkable that the TOT-induced urethral resistance increment showed a strong correlation with changes in voiding pressure (r > 0.7). Conclusion and Relevance: The bladder enhances thermodynamic efficacy by adapting the voiding mechanism to increased urethral resistance caused by TOT. Further studies with higher case series and longer follow-ups should assess whether this effect could be maintained over time or expire in a functional detrusor decompensation, to define diagnostic criteria that allow therapeutic interventions aimed at its prevention during the follow-up. Clinical Trial Registration: (clinicaltrials.gov), identifier (NCT05255289).

MicroRNA-489-3p attenuates neuropathic allodynia by regulating oncoprotein DEK/TET1-dependent epigenetic modification in the dorsal horn.

Originally characterized as an oncoprotein overexpressed in many forms of cancer that participates in numerous cellular pathways, DEK has since been well described regarding the regulation of epigenetic markers and transcription factors in neurons. However, its role in neuropathic allodynia processes remain elusive and intriguingly complex. Here, we show that DEK, which is induced in spinal dorsal horn neurons after spinal nerve ligation (SNL), is regulated by miR-489-3p. Moreover, SNL-induced decrease in miR-489-3p expression increased the expression of DEK, which recruited TET1 to the promoter fragments of the Bdnf, Grm5, and Stat3 genes, thereby enhancing their transcription in the dorsal horn. Remarkably, these effects were also induced by intrathecally administering naïve animals with miR-489-3p inhibitor, which could be inhibited by knockdown of TET1 siRNA or DEK siRNA. Conversely, delivery of intrathecal miR-489-3p-mimic into SNL rats attenuated allodynia behavior and reversed protein expression coupled to the promoter segments in the dorsal horn. Thus, a spinal miR-489-3p/DEK/TET1 transcriptional axis may contribute to neuropathic allodynia. These results may provide a new target for treating neuropathic allodynia.

High-density genetic map and genome-wide association studies of aesthetic traits in Phalaenopsis orchids.

Phalaenopsis spp. represent the most popular orchids worldwide. Both P. equestris and P. aphrodite are the two important breeding parents with the whole genome sequence available. However, marker-trait association is rarely used for floral traits in Phalaenopsis breeding. Here, we analyzed markers associated with aesthetic traits of Phalaenopsis orchids by using genome-wide association study (GWAS) with the F1 population P. Intermedia of 117 progenies derived from the cross between P. aphrodite and P. equestris. A total of 113,517 single nucleotide polymorphisms (SNPs) were identified in P. Intermedia by using genotyping-by-sequencing with the combination of two different restriction enzyme pairs, Hinp1 I/Hae III and Apek I/Hae III. The size-related traits from flowers were negatively related to the color-related traits. The 1191 SNPs from Hinp1 I/ Hae III and 23 simple sequence repeats were used to establish a high-density genetic map of 19 homolog groups for P. equestris. In addition, 10 quantitative trait loci were highly associated with four color-related traits on chromosomes 2, 5 and 9. According to the sequence within the linkage disequilibrium regions, 35 candidate genes were identified and related to anthocyanin biosynthesis. In conclusion, we performed marker-assisted gene identification of aesthetic traits with GWAS in Phalaenopsis orchids.

Solifenacin/Mirabegron Induces an Acute Compliance Increase in the Filling Phase of the Capacity-Reduced Urinary Bladder: A Pressure-Volume Analysis in Rats.

Aims: Pressure in the bladder, which is a high compliance organ, is only slightly elevated to a considerable filling volume during storage. Although cystometry off-line offers mean compliance, no protocol is available for real-time assays of the dynamics of bladder compliance, and the potential impact of solifenacin and mirabegron on dynamic bladder compliance has not been established. Methods: Along with constantly infused cystometry, a pressure-volume analysis (PVA) was performed by plotting intra-vesical volume against pressure in Sprague-Dawley rats. The instant compliance was assayed as the slope of the trajectory, and the mean compliance (Cm) was determined by the slope of the line produced by regression of the data points at the end of the first, second, and third quarters of the filling phase. Results: Under a steady-state, the PVA trajectory moved clockwise which shaped coincident enclosed loops with stable compliance. Though administering to naïve animals solifenacin, but not mirabegron (both 1 × 10-5-1 × 10-1 mg/kg, i.a.) decreased the peak pressure, both of these reagents exhibited acute increments in the trajectory slope and Cm of the filling phase in a dose-dependent manner (ED50 = 1.4 × 10-4 and 2.2 × 10-5 mg/kg, respectively). Resembling urine frequency/urgency in OAB patients, the voiding frequency of a capacity-reduced bladder was increased in association with decreased compliance which was ameliorated by both acute solifenacin and mirabegron injections (both 1 × 10-1 mg/kg). Conclusion: In addition to their well-known anti-inotropic/relaxative effects, solifenacin, and mirabegron induce an acute increase in bladder compliance to ameliorate OAB-like syndromes. Together with time-domain cystometry, PVA offers a platform for investigating the physiology/pathophysiology/pharmacology of bladder compliance which is crucial for urine storage.

Blocking the Spinal Fbxo3/CARM1/K+ Channel Epigenetic Silencing Pathway as a Strategy for Neuropathic Pain Relief.

Many epigenetic regulators are involved in pain-associated spinal plasticity. Coactivator-associated arginine methyltransferase 1 (CARM1), an epigenetic regulator of histone arginine methylation, is a highly interesting target in neuroplasticity. However, its potential contribution to spinal plasticity-associated neuropathic pain development remains poorly explored. Here, we report that nerve injury decreased the expression of spinal CARM1 and induced allodynia. Moreover, decreasing spinal CARM1 expression by Fbxo3-mediated CARM1 ubiquitination promoted H3R17me2 decrement at the K+ channel promoter, thereby causing K+ channel epigenetic silencing and the development of neuropathic pain. Remarkably, in naïve rats, decreasing spinal CARM1 using CARM1 siRNA or a CARM1 inhibitor resulted in similar epigenetic signaling and allodynia. Furthermore, intrathecal administration of BC-1215 (a novel Fbxo3 inhibitor) prevented CARM1 ubiquitination to block K+ channel gene silencing and ameliorate allodynia after nerve injury. Collectively, the results reveal that this newly identified spinal Fbxo3-CARM1-K+ channel gene functional axis promotes neuropathic pain. These findings provide essential insights that will aid in the development of more efficient and specific therapies against neuropathic pain.

NMDA receptor partial agonist GLYX-13 alleviates chronic stress-induced depression-like behavior through enhancement of AMPA receptor function in the periaqueductal gray.

Depression is a common mental disorder affecting more than 300 million people worldwide and is one of the leading causes of disability among all medical illnesses. The accumulation of preclinical data has fueled the revival of interest in targeting glutamatergic neurotransmission for the treatment of major depressive disorder. GLYX-13, a glutamatergic compound that acts as an N-methyl-d-aspartate (NMDA) modulator with glycine-site partial agonist properties, produces rapid and long-lasting antidepressant effects in both animal models and patients. However, the mechanisms underlying the antidepressant actions of GLYX-13 have not been fully characterized, especially in the midbrain ventrolateral periaqueductal gray (vlPAG), a brain stem area that controls stress-associated depression-like behavior. Here, we use a combination of electrophysiological recordings, behavioral tests, and pharmacological manipulations to study the antidepressant actions of GLYX-13 in the vlPAG. A single intravenous injection of a GLYX-13 rapidly mitigated footshock stress (FS)-induced depression-like behavior in rats. The FS-induced diminished glutamatergic transmission in the vlPAG was also reversed by a single GLYX-13 intravenous injection. Moreover, intra-vlPAG GLYX-13 microinjection produced a long-lasting antidepressant effect; however, this effect was prevented by the intra-vlPAG microinjection of tropomyosin-related kinase B (TrkB) receptor antagonist ANA-12, a selective mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin, and CNQX, an AMPA receptor antagonist. Additionally, a bath application of GLYX-13 enhanced glutamatergic transmission in vlPAG neurons; however, this enhancement effect was blocked by the co-application of ANA-12 and rapamycin. These results demonstrate that BDNF-TrkB-mTORC1 signaling in the vlPAG is required for the sustained antidepressant effects of GLYX-13.

Inhibiting MLL1-WDR5 interaction ameliorates neuropathic allodynia by attenuating histone H3 lysine 4 trimethylation-dependent spinal mGluR5 transcription.

ABSTRACT: Mixed lineage leukemia 1 (MLL1)-mediated histone H3 lysine 4 trimethylation (H3K4me3) of a subset of genes has been linked to the transcriptional activation critical for synaptic plasticity, but its potential contribution to neuropathic allodynia development remains poorly explored. Here, we show that MLL1, which is induced in dorsal horn neuron after spinal nerve ligation (SNL), is responsible for mechanical allodynia and increased H3K4me3 at metabotropic glutamate receptor subtype 5 (mGluR5) promoter. Moreover, SNL induced WD (Trp-Asp) repeat domain 5 subunit (WDR5) expression as well as the MLL1-WDR5 interaction accompany with H3K4me3 enrichment and transcription of mGluR5 gene in the dorsal horn in neuropathic allodynia progression. Conversely, WDR5-0103, a novel inhibitor of the MLL1-WDR5 interaction, reversed SNL-induced allodynia and inhibited SNL-enhanced mGluR5 transcription/expression as well as MLL1, WDR5, and H3K4me3 at the mGluR5 promoter in the dorsal horn. Furthermore, disrupting the expression of MLL1 or WDR5 using small interfering RNA attenuated mechanical allodynia and reversed protein transcription/expression and complex localizing at mGluR5 promoter in the dorsal horn induced by SNL. This finding revealed that MLL1-WDR5 complex integrity regulates MLL1 and WDR5 recruitment to H3K4me3 enrichment at mGluR5 promoter in the dorsal horn underlying neuropathic allodynia. Collectively, our findings indicated that SNL enhances the MLL1-WDR5 complex, which facilitates MLL1 and WDR5 recruitment to H3K4me3 enrichment at mGluR5 promoter in spinal plasticity contributing to neuropathic allodynia pathogenesis.

Pressure-volume analysis of rat's micturition cycles in vivo.

AIMS: Though the pressure-volume analysis (PVA), a method based on thermodynamics, is broadly used for assaying cardiac functions, its potential application on the physiology/pathophysiology of the urinary bladder, which processes resemble thermodynamic cycles to the heart, has not been established. METHODS: Cystometry recording intravesical pressure (IVP) and intravesical volume (IVV) of rhythmic voiding contractions caused by a constant saline infusion (0.04 mL/min) were carried out in forty urethane-anesthetized female Sprague-Dawley rats, and the PVA was established by plotting IVP against IVV. RESULTS: Pressure-volume points shaped coincident enclosed loops, and loop-associated urodynamic parameters kept stable under a constant infusion rate (0.04 mL/min). Enhancing preload (by elevating infusion rates to 0.08 and 0.12 mL/min) increased the area enclosed by the loop (Apv) and shifted loops to the right and slightly upward. Augmenting afterload (by enhancing resistances using 1/4 and 1/2 urethra clamping) increased Apv and shifted loops markedly to the right and upward. Without affecting Apv, muscarine (0.01 and 0.1 mM)-induced inotropic states shifted loop to the left and upward that was as opposed to the atropine (0.01 and 0.1 mM)-induced anti-inotropic state. CONCLUSIONS: Not only consistently assayed baseline bladder functions, PVA but also validly measured modified bladder functions due to altered extrinsic environment and intrinsic contractility of the bladder itself. In accompanied by cystometry, PVA could provide a clear concept about the relationship between time, pressure, and volume in the voiding activity.

Transcription Repressor Hes1 Contributes to Neuropathic Pain Development by Modifying CDK9/RNAPII-Dependent Spinal mGluR5 Transcription.

Diverse transcriptional controls in the dorsal horn have been observed in pain hypersensitivity. However, the understanding of the exact causes and mechanisms of neuropathic pain development is still fragmentary. Here, the results demonstrated nerve injury decreased the expression of spinal hairy and enhancer of split 1 (Hes1), a transcriptional repressor, and enhanced metabotropic glutamate receptor subtype 5 (mGluR5) transcription/expression, which was accompanied with behavioral allodynia. Moreover, nerve injury decreased Hes1 levels and reciprocally increased cyclin dependent kinase-9 (CDK9) levels and recruited CDK9 to phosphorylate RNA polymerase II (RNAPII) in the promoter fragments of mGluR5, thereby enhancing mGluR5 transcription/expression in the dorsal horn. These effects were also induced by intrathecally administering naïve rats with Hes1 small interfering RNA (siRNA). Conversely, Hes1 overexpression using intrathecal lentiviral vectors in nerve injury rats produced reversal of pain behavior and reversed protein expressions, phosphorylation, and coupling to the promoter segments in the dorsal horn. Collectively, the results in this study indicated nerve injury diminishes spinal Hes1-dependent suppression of CDK9-dependent RNAPII phosphorylation on the mGluR5 promoter that possibly enhances mGluR5 transcription/expression for neuropathic pain development.

Spinal RNF20-Mediated Histone H2B Monoubiquitylation Regulates mGluR5 Transcription for Neuropathic Allodynia.

To date, histone H2B monoubiquitination (H2Bub), a mark associated with transcriptional elongation and ongoing transcription, has not been linked to the development or maintenance of neuropathic pain states. Here, using male Sprague Dawley rats, we demonstrated spinal nerve ligation (SNL) induced behavioral allodynia and provoked ring finger protein 20 (RNF20)-dependent H2Bub in dorsal horn. Moreover, SNL provoked RNF20-mediated H2Bub phosphorylated RNA polymerase II (RNAPII) in the promoter fragments of mGluR5, thereby enhancing mGluR5 transcription/expression in the dorsal horn. Conversely, focal knockdown of spinal RNF20 expression reversed not only SNL-induced allodynia but also RNF20/H2Bub/RNAPII phosphorylation-associated spinal mGluR5 transcription/expression. Notably, TNF-α injection into naive rats and specific neutralizing antibody injection into SNL-induced allodynia rats revealed that TNF-α-associated allodynia involves the RNF20/H2Bub/RNAPII transcriptional axis to upregulate mGluR5 expression in the dorsal horn. Collectively, our findings indicated TNF-α induces RNF20-drived H2B monoubiquitination, which facilitates phosphorylated RNAPII-dependent mGluR5 transcription in the dorsal horn for the development of neuropathic allodynia.SIGNIFICANCE STATEMENT Histone H2B monoubiquitination (H2Bub), an epigenetic post-translational modification, positively correlated with gene expression. Here, TNF-α participated in neuropathic pain development by enhancing RNF20-mediated H2Bub, which facilitates phosphorylated RNAPII-dependent mGluR5 transcription in dorsal horn. Our finding potentially identified neuropathic allodynia pathophysiological processes underpinning abnormal nociception processing and opens a new avenue for the development of novel analgesics.

Spinal TNF-α impedes Fbxo45-dependent Munc13-1 ubiquitination to mediate neuropathic allodynia in rats.

Presynaptic active zone proteins play a crucial role in regulating synaptic plasticity. Although the ubiquitin-proteasome system underlying the degradation of the presynaptic active zone protein is well established, the contribution of this machinery to regulating spinal plasticity during neuropathic pain development remains unclear. Here, using male Sprague Dawley rats, we demonstrated along with behavioral allodynia, neuropathic injury induced a marked elevation in the expression levels of an active zone protein Munc13-1 in the homogenate and synaptic plasma membrane of the ipsilateral dorsal horn. Moreover, nerve injury-increased Munc13-1 expression was associated with an increase in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in ipsilateral dorsal horn neurons. This neuropathic injury-induced accumulation of Munc13-1 colocalized with synaptophysin but not homer1 in the dorsal horn. Focal knockdown of spinal Munc13-1 expression attenuated behavioral allodynia and the increased frequency, not the amplitude, of mEPSCs in neuropathic rats. Remarkably, neuropathic injury decreased spinal Fbxo45 expression, Fbxo45-Munc13-1 co-precipitation, and Munc13-1 ubiquitination in the ipsilateral dorsal horn. Conversely, focal knockdown of spinal Fbxo45 expression in naive animals resulted in behavioral allodynia in association with similar protein expression and ubiquitination in the dorsal horn as observed with neuropathic injury rats. Furthermore, both neuropathic insults and intrathecal injection of tumor necrosis factor-α (TNF-α) impeded spinal Fbxo45-dependent Munc13-1 ubiquitination, which was reversed by intrathecal TNF-α-neutralizing antibody. Our data revealed that spinal TNF-α impedes Fbxo45-dependent Munc13-1 ubiquitination that accumulates Munc13-1 in the presynaptic area and hence facilitates the synaptic excitability of nociceptive neurotransmission underlying neuropathic pain.