Nuclear modifier YARS2 allele correction restored retinal ganglion cells-specific deficiencies in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted eye disease due to the degeneration of retinal ganglion cells (RGCs). Mitochondrial 11778G > A mutation is the most common LHON-associated mitochondrial DNA (mtDNA) mutation. Our recent studies demonstrated some LHON families manifested by synergic interaction between m.11778G > A mutation and YARS2 allele (c.572G > T, p.Gly191Val) encoding mitochondrial tyrosyl-tRNA synthetase. However, the RGC-specific effects of LHON-associated mtDNA mutations remain elusive and there is no highly effective therapy for LHON. Here, we generated patients-derived induced pluripotent stem cells (iPSCs) from fibroblasts derived from a Chinese LHON family (both m.11778G > A and c.572G > T mutations, only m.11778G > A mutation, and control subject). The c.572G > T mutation in iPSC lines from a syndromic individual was corrected by CRISPR/Cas9. Those iPSCs were differentiated into neural progenitor cells and subsequently induced RGC-like cells using a stepwise differentiation procedure. Those RGC-like cells derived from symptomatic individual harboring both m.11778G > A and c.572G > T mutations exhibited greater defects in neuronal differentiation, morphology including reduced area of soma, numbers of neurites and shortened length of axons, electrophysiological properties than those in cells bearing only m.11778G > A mutation. Furthermore, these RGC-like cells revealed more drastic reductions in oxygen consumption rates, levels of mitochondrial ATP and increasing productions of reactive oxygen species than those in other cell models. These mitochondrial dysfunctions promoted the apoptotic process for RGC degenerations. Correction of YARS2 c.572G > T mutation rescued deficiencies of patient-derived RGC-like cells. These findings provide new insights into pathophysiology of LHON arising from RGC-specific mitochondrial dysfunctions and step toward therapeutic intervention for this disease.

Epitranscriptomic profiling of N4-acetylcytidine-related RNA acetylation in the spinal dorsal horn of rat with cancer-induced bone pain.

Recently, epigenetics involved in the regulation of gene expression has become a research hotspot. This study evaluated N4-acetylcytidine (ac4c) RNA acetylation in the spinal dorsal horn (SDH) of rats with cancer-induced bone pain (CIBP). The ac4C-specific RIP sequencing and NAT10-specific RIP sequencing were performed to identify the differences in ac4C acetylation and gene expression in the SDH between CIBP and sham groups, the relationship with the acetylation-modifying enzyme NAT10, and association analysis was performed. By interfering with the NAT10 expression, the relationship between some up-regulated genes and ac4C acetylation in CIBP was verified. In this study, we demonstrated that bone cancer increases the levels of NAT10 and the overall acetylation, inducing differential ac4C patterns in the SDH of rats. Through verification experiments, it was found that ac4C acetylation of some genes is regulated by NAT10, and differential ac4C patterns in RNA determine the expression of this RNA. We exposed that some CIBP-related gene expression was altered in the SDH of rats, which was regulated by differentially expressed ac4C acetylation.

Activation of the Intrinsic Pain Inhibitory Circuit from the Midcingulate Cg2 to Zona Incerta Alleviates Neuropathic Pain.

Neuropathic pain is one of the most common and notorious neurological diseases. The changes in cerebral structures after nerve injury and the corresponding contributions to neuropathic pain are not well understood. Here we found that the majority of glutamatergic neurons in the area 2 of midcingulate cortex (MCC Cg2Glu) were inhibited by painful stimulation in male mice. Optogenetic manipulation revealed that these neurons were tonically involved in the inhibitory modulation of multimodal nociception. We further identified the projections to GABAergic neurons in the zona incerta (ZIGABA) mediated the pain inhibitory role. However, MCC Cg2Glu became hypoactive after nerve injury. Although a brief activation of the MCC Cg2Glu to ZIGABA circuit was able to relieve the aversiveness associated with spontaneous ongoing pain, consecutive activation of the circuit was required to alleviate neuropathic allodynia. In contrast, glutamatergic neurons in the area 1 of MCC played opposite roles in pain modulation. They became hyperactive after nerve injury and only consecutive inhibition of their activity relieved allodynia. These results demonstrate that MCC Cg2Glu constitute a component of intrinsic pain inhibitory circuitry and their hypoactivity underlies neuropathic pain. We propose that selective and persistent activation of the MCC Cg2Glu to ZIGABA circuit may serve as a potential therapeutic strategy for this disease.SIGNIFICANCE STATEMENT Glutamatergic neurons in the area 2 of midcingulate cortex (MCC Cg2Glu) are tonically involved in the intrinsic pain inhibition via projecting to GABAergic neurons in the zona incerta. They are hypoactive after nerve injury. Selective activation of the circuit compensates the reduction of its analgesic strength and relieves neuropathic pain. Therefore, MCC Cg2Glu and the related analgesic circuit may serve as therapeutic targets for neuropathic pain. In contrast, MCC Cg1Glu have an opposite role in pain modulation and become hyperactive after nerve injury. The present study provides novel evidence for the concept that neuropathic pain is associated with the dysfunction of endogenous pain modulatory system and new perspective on the treatment of neuropathic pain.

Knowing the Neuronal Mechanism of Spontaneous Pain to Treat Chronic Pain in the Future.

Spontaneous pain is the major complain for the patients to see a doctor. Human imaging studies presented that spontaneous pain is mainly associated with activity changes in medial pain pathway, while broader brain regions were activated by allodynia pain. On behavioral level, temporally disassociation between the evoked pain and spontaneous pain was observed; these data gave a hint that the spontaneous pain and evoked pain may be mediated by different neuronal mechanisms. And more attentions should be paid to the spontaneous pain to treat the chronic pain in the future.

The Analgesic Effects of (5R,6R)6-(3-Propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1] Octane on a Mouse Model of Neuropathic Pain.

BACKGROUND: Both pharmacologic and genetic approaches have been used to study the involvement of the muscarinic acetylcholine system in the regulation of chronic pain. Previous studies suggest that the M2 and M4 subtypes of muscarinic acetylcholine receptors (mAChRs) are important targets for the development of chronic pain. (5R,6R)6-(3-Propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1] octane (PTAC) has agonist effects on muscarinic M2 and M4 receptors and antagonist effects on muscarinic M1, M3, and M5 receptors. However, its analgesic effects have been less studied. METHODS: Male C57B L/6 mice were anesthetized, and left common peroneal nerve (CPN) ligation was performed to induce neuropathic pain. Before and after the application of PTAC systemically or specifically to the anterior cingulate cortex (ACC), the withdrawal thresholds to mechanical stimulation and static weight balance were measured, and the effects of PTAC on the conditioned place preference (CPP) were further evaluated. Western blotting was used to examine the expression of M1 and M2 in the striatum, ACC, and ventral tegmental area. RESULTS: The application of PTAC ([i.p.] intraperitoneal injection) increased the paw withdraw threshold in both the early (0.05 mg/kg, mean difference [95% confidence interval, CI]: 0.19 [0.05-0.32]; 0.10 mg/kg: mean difference [95% CI]: 0.34 [0.22-0.46]) and the late phases (0.05 mg/kg: mean difference [95% CI]: 0.45 [0.39-0.50]; 0.1 mg/kg: mean difference [95% CI]: 0.44 [0.37-0.51]) after nerve injury and rebalanced the weight distribution on the hind paws of mice (L/R ratio: before, 0.56 ± 0.03. 0.05 mg/kg, 1.00 ± 0.04, 0.10 mg/kg, 0.99 ± 0.03); however, it failed to induce place preference in the CPP (0.05 mg/kg, 2-way analysis of variance, P > .05; 0.2 mg/kg, 2-way analysis of variance, P > .05,). At the same doses, the analgesic effects at D3-5 lasted longer than the effects at D14-16. This may be due to the down-regulation of the M2 and M1 in tested brain regions. CONCLUSIONS: These observations suggested that PTAC has analgesic effects on the neuropathic pain induced by nerve injury.

Long-term temporal imprecision of information coding in the anterior cingulate cortex of mice with peripheral inflammation or nerve injury.

Temporal properties of spike firing in the central nervous system (CNS) are critical for neuronal coding and the precision of information storage. Chronic pain has been reported to affect cognitive and emotional functions, in addition to trigger long-term plasticity in sensory synapses and behavioral sensitization. Less is known about the possible changes in temporal precision of cortical neurons in chronic pain conditions. In the present study, we investigated the temporal precision of action potential firing in the anterior cingulate cortex (ACC) by using both in vivo and in vitro electrophysiological approaches. We found that peripheral inflammation caused by complete Freund's adjuvant (CFA) increased the standard deviation (SD) of spikes latency (also called jitter) of ∼51% of recorded neurons in the ACC of adult rats in vivo. Similar increases in jitter were found in ACC neurons using in vitro brain slices from adult mice with peripheral inflammation or nerve injury. Bath application of glutamate receptor antagonists CNQX and AP5 abolished the enhancement of jitter induced by CFA injection or nerve injury, suggesting that the increased jitter depends on the glutamatergic synaptic transmission. Activation of adenylyl cyclases (ACs) by bath application of forskolin increased jitter, whereas genetic deletion of AC1 abolished the change of jitter caused by CFA inflammation. Our study provides strong evidence for long-term changes of temporal precision of information coding in cortical neurons after peripheral injuries and explains neuronal mechanism for chronic pain caused cognitive and emotional impairment.

Sleep deprivation impairs cAMP signalling in the hippocampus.

Millions of people regularly obtain insufficient sleep. Given the effect of sleep deprivation on our lives, understanding the cellular and molecular pathways affected by sleep deprivation is clearly of social and clinical importance. One of the major effects of sleep deprivation on the brain is to produce memory deficits in learning models that are dependent on the hippocampus. Here we have identified a molecular mechanism by which brief sleep deprivation alters hippocampal function. Sleep deprivation selectively impaired 3', 5'-cyclic AMP (cAMP)- and protein kinase A (PKA)-dependent forms of synaptic plasticity in the mouse hippocampus, reduced cAMP signalling, and increased activity and protein levels of phosphodiesterase 4 (PDE4), an enzyme that degrades cAMP. Treatment of mice with phosphodiesterase inhibitors rescued the sleep-deprivation-induced deficits in cAMP signalling, synaptic plasticity and hippocampus-dependent memory. These findings demonstrate that brief sleep deprivation disrupts hippocampal function by interfering with cAMP signalling through increased PDE4 activity. Thus, drugs that enhance cAMP signalling may provide a new therapeutic approach to counteract the cognitive effects of sleep deprivation.

Huperzine A Alleviates Mechanical Allodynia but Not Spontaneous Pain via Muscarinic Acetylcholine Receptors in Mice.

Chronic pain is a major health issue and most patients suffer from spontaneous pain. Previous studies suggest that Huperzine A (Hup A), an alkaloid isolated from the Chinese herb Huperzia serrata, is a potent analgesic with few side effects. However, whether it alleviates spontaneous pain is unclear. We evaluated the effects of Hup A on spontaneous pain in mice using the conditioned place preference (CPP) behavioral assay and found that application of Hup A attenuated the mechanical allodynia induced by peripheral nerve injury or inflammation. This effect was blocked by atropine. However, clonidine but not Hup A induced preference for the drug-paired chamber in CPP. The same effects occurred when Hup A was infused into the anterior cingulate cortex. Furthermore, ambenonium chloride, a competitive inhibitor of acetylcholinesterase, also increased the paw-withdrawal threshold but failed to induce place preference in CPP. Therefore, our data suggest that acetylcholinesterase in both the peripheral and central nervous systems is involved in the regulation of mechanical allodynia but not the spontaneous pain.

Coordination between midcingulate cortex and retrosplenial cortex in pain regulation.

Introduction: The cingulate cortex, with its subregions ACC, MCC, and RSC, is key in pain processing. However, the detailed interactions among these regions in modulating pain sensation have remained unclear. Methods: In this study, chemogenetic tools were employed to selectively activate or inhibit neuronal activity in the MCC and RSC of rodents to elucidate their roles in pain regulation.Results: Our results showed that chemogenetic activation in both the RSC and MCC heightened pain sensitivity. Suppression of MCC activity disrupted the RSC's regulation of both mechanical and thermal pain, while RSC inhibition specifically affected the MCC's regulation of thermal pain. Discussion: The findings indicate a complex interplay between the MCC and RSC, with the MCC potentially governing the RSC's pain regulatory mechanisms. The RSC, in turn, is crucial for the MCC's control over thermal sensation, revealing a collaborative mechanism in pain processing. Conclusion: This study provides evidence for the MCC and RSC's collaborative roles in pain regulation, highlighting the importance of their interactions for thermal and mechanical pain sensitivity. Understanding these mechanisms could aid in developing targeted therapies for pain disorders.

Predicting aversive events and terminating fear in the mouse anterior cingulate cortex during trace fear conditioning.

A variety of studies have implicated the anterior cingulate cortex (ACC) in fear, including permanent storage of fear memory. Recent pharmacological and genetic studies indicate that early synaptic plasticity in the ACC may also contribute to certain forms of fear memory at early time points. However, no study has directly examined the possible changes in neuronal activity of ACC neurons in freely behaving mice during early learning. In the present study, we examined the neural responses of the ACC during trace fear conditioning. We found that ACC putative pyramidal and nonpyramidal neurons were involved in the termination of fear behavior ("un-freezing"), and the spike activity of these neurons was reduced during freezing. Some of the neurons were also found to acquire un-freezing locked activity and change their tuning. The results implicate the ACC neurons in fear learning and controlling the abolition of fear behavior. We also show that the ACC is important for making cue-related fear memory associations in the trace fear paradigm as measured with tone-evoked potentials and single-unit activity. Collectively, our findings indicate that the ACC is involved in predicting future aversive events and terminating fear during trace fear.

Post-translational modification of NMDA receptor GluN2B subunit and its roles in chronic pain and memory.

N-methyl-d-aspartate receptors (NMDA receptors) play critical roles in brain functions and diseases. The expression, trafficking, synaptic location and function of different NMDA receptor subtypes are not static, but regulated dynamically in a cell-specific and synapse-specific manner during physiological and pathological conditions. In this review, we will examine recent evidence on the post-translational modulation of NMDA receptors subunit, in particular GluN2B subunit, such as phosphorylation, palmitoylation, and ubiquitination. In parallel, we will overview the roles of these modifications of GluN2B-NMDA receptor subtype in physiological functions, such as learning and memory, and pathophysiological conditions, such as chronic pain, ischemia and neurodegenerative diseases.

Cortical GluK1 kainate receptors modulate scratching in adult mice.

Recent investigations into the mechanisms mediating itch transmission have focused on spinal mechanisms, whereas few studies have investigated the role of the cerebral cortex in itch-related behaviors. Human imaging studies show that several cortical regions are active in correspondence with itch, including the anterior cingulate cortex (ACC). We present here evidence of cortical modulation of pruritogen-induced scratching behavior. We combine pharmacological, genetic, and electrophysiological approaches to show that cortical GluK1-containing kainate (KA) receptors are involved in scratching induced by histamine and non-histamine-dependent itching stimuli. We further show that scratching corresponds with enhanced excitatory transmission in the ACC through KA receptor modulation of inhibitory circuitry. In addition, we found that inhibiting GluK1-containing KA receptors in the ACC also reduced behavioral nociceptive responses induced by formalin. Our results reveal a new role of the cortex in pruritogen-induced scratching.

Cingulate protein arginine methyltransferases 1 regulates peripheral hypersensitivity via fragile X messenger ribonucleoprotein.

The deficit of fragile X messenger ribonucleoprotein (FMRP) leads to intellectual disability in human and animal models, which also leads to desensitization of pain after nerve injury. Recently, it was shown that the protein arginine methyltransferases 1 (PRMT1) regulates the phase separation of FMRP. However, the role of PRMT1 in pain regulation has been less investigated. Here we showed that the downregulation of PRMT1 in the anterior cingulate cortex (ACC) contributes to the development of peripheral pain hypersensitivity. We observed that the peripheral nerve injury decreased the expression of PRMT1 in the ACC; knockdown of the PRMT1 via shRNA in the ACC decreased the paw withdrawal thresholds (PWTs) of naïve mice. Moreover, the deficits of FMRP abolished the effects of PRMT1 on pain sensation. Furthermore, overexpression of PRMT1 in the ACC increased the PWTs of mice with nerve injury. These observations indicate that the downregulation of cingulate PRMT1 was necessary and sufficient to develop peripheral hypersensitivity after nerve injury. Thus, we provided evidence that PRMT1 is vital in regulating peripheral pain hypersensitivity after nerve injury via the FMRP.

Single-cell RNA sequencing uncovers the cell type-dependent transcriptomic changes in the retrosplenial cortex after peripheral nerve injury.

The retrosplenial cortex (RSC) is a vital area for storing remote memory and has recently been found to undergo broad changes after peripheral nerve injury. However, little is known about the role of RSC in pain regulation. Here, we examine the involvement of RSC in the pain of mice with nerve injury. Notably, reducing the activities of calcium-/calmodulin-dependent protein kinase type II-positive splenial neurons chemogenetically increases paw withdrawal threshold and extends thermal withdrawal latency in mice with nerve injury. The single-cell or single-nucleus RNA-sequencing results predict enhanced excitatory synaptic transmissions in RSC induced by nerve injury. Local infusion of 1-naphthyl acetyl spermine into RSC to decrease the excitatory synaptic transmissions relieves pain and induces conditioned place preference. Our data indicate that RSC is critical for regulating physiological and neuropathic pain. The cell type-dependent transcriptomic information would help understand the molecular basis of neuropathic pain.

Effects of elevation of brain magnesium on fear conditioning, fear extinction, and synaptic plasticity in the infralimbic prefrontal cortex and lateral amygdala.

Anxiety disorders, such as phobias and posttraumatic stress disorder, are among the most common mental disorders. Cognitive therapy helps in treating these disorders; however, many cases relapse or resist the therapy, which justifies the search for cognitive enhancers that might augment the efficacy of cognitive therapy. Studies suggest that enhancement of plasticity in certain brain regions such as the prefrontal cortex (PFC) and/or hippocampus might enhance the efficacy of cognitive therapy. We found that elevation of brain magnesium, by a novel magnesium compound [magnesium-l-threonate (MgT)], enhances synaptic plasticity in the hippocampus and learning and memory in rats. Here, we show that MgT treatment enhances retention of the extinction of fear memory, without enhancing, impairing, or erasing the original fear memory. We then explored the molecular basis of the effects of MgT treatment on fear memory and extinction. In intact animals, elevation of brain magnesium increased NMDA receptors (NMDARs) signaling, BDNF expression, density of presynaptic puncta, and synaptic plasticity in the PFC but, interestingly, not in the basolateral amygdala. In vitro, elevation of extracellular magnesium concentration increased synaptic NMDAR current and plasticity in the infralimbic PFC, but not in the lateral amygdala, suggesting a difference in their sensitivity to elevation of brain magnesium. The current study suggests that elevation of brain magnesium might be a novel approach for enhancing synaptic plasticity in a regional-specific manner leading to enhancing the efficacy of extinction without enhancing or impairing fear memory formation.

Characterization of neuronal intrinsic properties and synaptic transmission in layer I of anterior cingulate cortex from adult mice.

The neurons in neocortex layer I (LI) provide inhibition to the cortical networks. Despite increasing use of mice for the study of brain functions, few studies were reported about mouse LI neurons. In the present study, we characterized intrinsic properties of LI neurons of the anterior cingulate cortex (ACC), a key cortical area for sensory and cognitive functions, by using whole-cell patch clamp recording approach. Seventy one neurons in LI and 12 pyramidal neurons in LII/III were recorded. Although all of the LI neurons expressed continuous adapting firing characteristics, the unsupervised clustering results revealed five groups in the ACC, including: Spontaneous firing neurons; Delay-sAHP neurons, Delay-fAHP neurons, and two groups of neurons with ADP, named ADP1 and ADP2, respectively. Using pharmacological approaches, we found that LI neurons received both excitatory (mediated by AMPA, kainate and NMDA receptors), and inhibitory inputs (which were mediated by GABA(A) receptors). Our studies provide the first report characterizing the electrophysiological properties of neurons in LI of the ACC from adult mice.

Targeting long-term depression of excitatory synaptic transmission for the treatment of neuropathic pain.

Injury or disease in the somatosensory nervous system may cause broad molecular changes and lead to neuropathic pain. Excitatory synaptic transmission in somatosensory pathways conveys the somatosensory information from the peripheral to the central nervous system. Long-term effects of excitatory synaptic transmission on the pain pathway contribute to neuropathic pain hypersensitivity. Synaptic strength is dynamically regulated and undergoes bidirectional changes, manifested by two primary forms of synaptic plasticity, long-term potentiation and long-term depression (LTD), which are mediated by insertion and endocytosis of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), respectively. Molecular mechanisms of LTP have been extensively studied; on the other hand, the role of AMPAR endocytosis in the pain-related synaptic enhancement is less well known. Recent research in the anterior cingulate cortex reveals that loss of LTD contributes to the maintenance of neuropathic pain, which provides the novel perspective of the mechanism of LTD also being critical for maintaining neuropathic pain. More importantly, exploring the molecular mechanism of LTD may help with the development of novel analgesic strategies to manage neuropathic pain.

NMDA receptor hypofunction underlies deficits in parvalbumin interneurons and social behavior in neuroligin 3 R451C knockin mice.

Neuroligins (NLs), a family of postsynaptic cell-adhesion molecules, have been associated with autism spectrum disorder. We have reported that dysfunction of the medial prefrontal cortex (mPFC) leads to social deficits in an NL3 R451C knockin (KI) mouse model of autism. However, the underlying molecular mechanism remains unclear. Here, we find that N-methyl-D-aspartate receptor (NMDAR) function and parvalbumin-positive (PV+) interneuron number and expression are reduced in the mPFC of the KI mice. Selective knockdown of NMDAR subunit GluN1 in the mPFC PV+ interneuron decreases its intrinsic excitability. Restoring NMDAR function by its partial agonist D-cycloserine rescues the PV+ interneuron dysfunction and social deficits in the KI mice. Interestingly, early D-cycloserine administration at adolescence prevents adult KI mice from social deficits. Together, our results suggest that NMDAR hypofunction and the resultant PV+ interneuron dysfunction in the mPFC may constitute a central node in the pathogenesis of social deficits in the KI mice.

Restoration of FMRP expression in adult V1 neurons rescues visual deficits in a mouse model of fragile X syndrome.

Many people affected by fragile X syndrome (FXS) and autism spectrum disorders have sensory processing deficits, such as hypersensitivity to auditory, tactile, and visual stimuli. Like FXS in humans, loss of Fmr1 in rodents also cause sensory, behavioral, and cognitive deficits. However, the neural mechanisms underlying sensory impairment, especially vision impairment, remain unclear. It remains elusive whether the visual processing deficits originate from corrupted inputs, impaired perception in the primary sensory cortex, or altered integration in the higher cortex, and there is no effective treatment. In this study, we used a genetic knockout mouse model (Fmr1KO), in vivo imaging, and behavioral measurements to show that the loss of Fmr1 impaired signal processing in the primary visual cortex (V1). Specifically, Fmr1KO mice showed enhanced responses to low-intensity stimuli but normal responses to high-intensity stimuli. This abnormality was accompanied by enhancements in local network connectivity in V1 microcircuits and increased dendritic complexity of V1 neurons. These effects were ameliorated by the acute application of GABAA receptor activators, which enhanced the activity of inhibitory neurons, or by reintroducing Fmr1 gene expression in knockout V1 neurons in both juvenile and young-adult mice. Overall, V1 plays an important role in the visual abnormalities of Fmr1KO mice and it could be possible to rescue the sensory disturbances in developed FXS and autism patients.