Piwi-Interacting RNA (piRNA) is the largest class of small noncoding RNA and is involved in various physiological and pathological processes. However, whether it has a role in pain modulation remains unknown. In the present study, we found that spinal piRNA-DQ541777 (piR-DQ541777) was significantly increased in the male mouse model of sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain. Knockdown of spinal piR-DQ541777 alleviated CCI-induced thermal hyperalgesia and mechanical allodynia and spinal neuronal sensitization. However, the overexpression of spinal piR-DQ541777 in naive mice produced pain behaviors and increased spinal neuron sensitization. Furthermore, we found that piR-DQ541777 regulates pain behaviors by targeting CDK5 regulatory subunit-associated protein 1 (Cdk5rap1). CCI increased the methylation level of CpG islands in the cdk5rap1 promoter and consequently reduced the expression of Cdk5rap1, which was reversed by the knockdown of piR-DQ541777 and mimicked by the overexpression of piR-DQ541777 in naive mice. Finally, piR-DQ541777 increased the methylation level of CpG islands by recruiting DNA methyltransferase 3A (DNMT3a) to cdk5rap1 promoter. In conclusion, this study represents a novel role of piR-DQ541777 in the regulation of neuropathic pain through the methylation of cdk5rap1.
Dynamin 1 (dyn1) is required for clathrin-mediated endocytosis in most secretory (neuronal and neuroendocrine) cells. There are two modes of Ca2+ -dependent catecholamine release from single dense-core vesicles: full-quantal (quantal) and subquantal in adrenal chromaffm cells, but their relative occurrences and impacts on total secretion remain unclear. To address this fundamental question in neurotransmission area using both sexes of animals, here we report the following: (1) dynl-KO increased quantal size (QS, but not vesicle size/content) by >= 250% in dynl -KO mice; (2) the KO-increased QS was rescued by dynl (but not its deficient mutant or dyn2); (3) the ratio of quantal versus subquantal events was increased by KO; (4) following a release event, more protein contents were retained in WT versus KO vesicles; and (5) the fusion pore size (d(p)) was increased from <= 9 to >= 9 nm by KO. Therefore, Ca2+-induced exocytosis is generally a subquantal release in sympathetic adrenal chromaffm cells, implying that neurotransmitter release is generally regulated by dynamin in neuronal cells.
Vestibular compensation is responsible for the spontaneous recovery of postural, locomotor, and oculomotor dysfunctions in patients with peripheral vestibular lesion or posterior circulation stroke. Mechanism investigation of vestibular compensation is of great importance in both facilitating recovery of vestibular function and understanding the postlesion functional plasticity in the adult CNS. Here, we report that postsynaptic histamine H1 receptor contributes greatly to facilitating vestibular compensation. The expression of H1 receptor is restrictedly increased in the ipsilesional rather than contralesional GABAergic projection neurons in the medial vestibular nucleus (MVN), one of the most important centers for vestibular compensation, in unilateral labyrinthectomized male rats. Furthermore, H1 receptor mediates an asymmetric excitation of the commissural GABAergic but not glutamatergic neurons in the ipsilesional MVN, which may help to rebalance bilateral vestibular systems and promote vestibular compensation. Selective blockage of H1 receptor in the MVN significantly retards the recovery of both static and dynamic vestibular symptoms following unilateral labyrinthectomy, and remarkably attenuates the facilitation of betahistine, whose effect has traditionally been attributed to its antagonistic action on the presynaptic H3 receptor, on vestibular compensation. These results reveal a previously unknown role for histamine H1 receptor in vestibular compensation and amelioration of vestibular motor deficits, as well as an involvement of H1 receptor in potential therapeutic effects of betahistine. The findings provide not only a new insight into the postlesion neuronal circuit plasticity and functional recovery in the CNS, but also a novel potential therapeutic target for vestibular disorders.
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 Cg2(Glu)) 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 (ZI(GABA)) mediated the pain inhibitory role. However, MCC Cg2(Glu) became hypoactive after nerve injury. Although a brief activation of the MCC Cg2(Glu) to ZI(GABA) 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 Cg2(Glu) constitute a component of intrinsic pain inhibitory circuitry and their hypoactivity underlies neuropathic pain. We propose that selective and persistent activation of the MCC Cg2(Glu) to ZI(GABA) circuit may serve as a potential therapeutic strategy for this disease.
We have identified a member of the growth arrest and DNA damage (Gadd45) protein family, Gadd45 gamma, which is known to be critically involved in DNA repair, as a key player in the regulation of immediate early gene (IEG) expression underlying the consolidation of associative fear memory in adult male C57BL/6 mice. Gadd45 gamma temporally influences learning-induced IEG expression in the prelimbic prefrontal cortex (PLPFC) through its interaction with DNA double-strand break (DSB)-mediated changes in DNA methylation. Our findings suggest a two-hit model of experience-dependent IEG activity and learning that comprises (1) a first wave of IEG expression governed by DSBs and followed by a rapid increase in DNA methylation, and (2) a second wave of IEG expression associated with the recruitment of Gadd45 gamma and active DNA demethylation at the same site, which is necessary for memory consolidation.
Serotonin (5-hydroxytryptamine, 5-HT) receptor agonists are neuroprotective in CNS injury models. However, the neuroprotective functional implications and synaptic mechanism of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a serotonin receptor (5-HT1A) agonist, in an adult male Wistar rat model of chronic glaucoma model remain unknown. We found that ocular hypertension decreased 5-HT1A receptor expression in rat retinas because the number of retinal ganglion cells (RGCs) was significantly reduced in rats with induced ocular hypertension relative to that in control retinas and 8-OH-DPAT enhanced the RGC viability. The protective effects of 8-OH-DPAT were blocked by intravitreal administration of the selective 5-HT1A antagonist WAY-100635 or the selective GABA(A) receptor antagonist SR95531. Using patch-clamp techniques, spontaneous and miniature GABAergic IPSCs (sIPSCs and mIPSCs, respectively) of RGCs in rat retinal slices were recorded. 8-OH-DPAT significantly increased the frequency and amplitude of GABAergic sIPSCs and mIPSCs in ON-and OFF-type RGCs. Among the signaling cascades mediated by the 5-HT1A receptor, the role of cAMP-protein kinase A (PKA) signaling was investigated. The 8-OH-DPAT-induced changes at the synaptic level were enhanced by PKA inhibition by H-89 and blocked by PKA activation with bucladesine. Furthermore, the density of phosphorylated PKA (p-PKA)/PKA was significantly increased in glaucomatous retinas and 8-OH-DPAT significantly decreased p-PKA/PKA expression, which led to the inhibition of PKA phosphorylation upon relieving neurotransmitter GAB Arelease. These results showed that the activation of 5-HT1A receptors in retinas facilitated presynaptic GABA release functions by suppressing cAMP-PKA signaling and decreasing PKA phosphorylation, which could lead to the de-excitation of RGC circuits and suppress excitotoxic processes in glaucoma.
Dysfunctions of gene transcription and translation in the nociceptive pathways play the critical role in development and maintenance of chronic pain. Circular RNAs (circRNAs) are emerging as new players in regulation of gene expression, but whether and how circRNAs are involved in chronic pain remain elusive. We showed here that complete Freund's adjuvant-induced chronic inflammation pain significantly increased circRNA-Filipi1l (filamin A interacting protein 1-like) expression in spinal neurons of mice. Blockage of this increase attenuated complete Freund's adjuvant-induced nociceptive behaviors, and overexpression of spinal circRNA-Filipi1l in naive mice mimicked the nociceptive behaviors as evidenced by decreased thermal and mechanical nociceptive threshold. Furthermore, we found that mature circ RNA-Filipllexpression was negatively regulated by miRNA-1224 via binding and splicing of precursor of circRNA-Filipi1l (pre-circRNA-Filipi1l) in the Argonaute-2 (Ago2)-dependent manner. Increase of spinal circRNA-Filipi1l expression resulted from the decrease of m iRNA-1224 expression under chronic inflammation pain state. miRNA-1224 knockdown or Ago2 overexpression induced nociceptive behaviors in naive mice, which was prevented by the knockdown of spinal circRNA-Filipi1l. Finally, we demonstrated that a ubiquitin protein ligase E3 component n-recognin 5 (Ubr5), validated as a target of circRNA-Filipi1l, plays a pivotal role in regulation of nociception by spinal circRNA-Filipi1l. These data suggest that miRNA-1224-mediated and Ago2-dependent modulation of spinal circRNA-Filipi1l expression regulates nociception via targeting Ubr5, revealing a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.
Primary cilia are microtubule-based protuberances that project from the eukaryotic cell body to sense the extracellular environment. Ciliogenesis is closely correlated to the cell cycle and defects of cilia are related to human systemic diseases such as primary ciliary dyskinesia. However, the role of ciliogenesis in cortical development remains unclear. Here, we demonstrate that Cenpj, a protein that is required for centriole biogenesis, plays a role in regulating cilium disassembly in vivo. Depletion of Cenpj in neural progenitor cells results in long cilia and abnormal cilia disassembly. Radial glial cells Cenpj depletion exhibit uncompleted cell division, reduced cell proliferation, and increased cell apoptosis in the developing mouse cerebrum cortex, leading to microcephaly. In addition, Cenpj depletion causes long and thin primary cilia and motile cilia in adult neural stem cells and reduced cell proliferation in the subventricular zone. Furthermore, we show that Cenpj regulates cilia disassembly and neurogenesis through Kif2a, a plus-end-directed motor protein. These data collected from mice of both sexes provide insights into how ciliogenesis plays roles in cortical development and how primary microcephaly is induced by Cenpj mutations in humans.
The transition of apical progenitors (APs) to basal progenitors (BPs) is an important neurogenic process during cortical expansion. Presenilin enhancer 2 (Pen-2, also named as Psenen) is a key subunit of gamma-secretase and has been implicated in neurodevelopmental disease. However, it remains unknown how Pen-2 may regulate the maintenance of APs. To address this question, we generated a conditional KO (cKO) mouse in which Pen-2 is specifically inactivated in neural progenitor cells in the telencephalon. Both male and female embryos were used. We show that Pen-2 cKO cortices display remarkable depletion of Aps, but transient increase on BPs, compared with controls. We demonstrate that the proliferation rate of APs or BPs is not changed, but the switch of APs to BPs is dramatically accelerated in Pen-2 cKO cortices. Molecular analyses reveal decreased levels of Hes1 and Hes5 but increased levels of Ngn2 and NeuroD1 in Pen-2 KO cells. We report that expression of Notch1 intracellular domain in Pen-2 cKO cortices restores the population of APs and BPs. In summary, these findings highlight a central role of the Notch signaling in Pen-2-dependent maintenance of neural stem cells in the developing neocortex.
In awake rodents, the neural representation of olfactory information in the olfactory bulb is largely dependent on brain state and behavioral context. Learning-modified neural plasticity has been observed in mitral/tufted cells, the main output neurons of the olfactory bulb. Here, we propose that the odor information encoded by mitral/tufted cell responses in awake mice is highly dependent on the behavioral task demands. We used fiber photometry to record calcium signals from the mitral/tufted cell population in awake, head-fixed male mice under different task demands. We found that the mitral/tufted cell population showed similar responses to two distinct odors when the odors were presented in the context of a go/go task, in which the mice received a water reward regardless of the identity of the odor presented. However, when the same odors were presented in a go/no-go task, in which one odor was rewarded and the other was not, then the mitral cell population responded very differently to the two odors, characterized by a robust reduction in the response to the nonrewarded odor. Thus, the representation of odors in the mitral/tufted cell population depends on whether the task requires discrimination of the odors. Strikingly, downstream of the olfactory bulb, pyramidal neurons in the posterior piriform cortex also displayed a task-demand-dependent neural representation of odors, but the anterior piriform cortex did not, indicating that these two important higher olfactory centers use different strategies for neural representation.
Studying the mismatch between perception and reality helps us better understand the constructive nature of the visual brain. The Pinna-Brelstaff motion illusion is a compelling example illustrating how a complex moving pattern can generate an illusory motion perception. When an observer moves toward (expansion) or away (contraction) from the Pinna-Brelstaff figure, the figure appears to rotate. The neural mechanisms underlying the illusory complex-flow motion of rotation, expansion, and contraction remain unknown. We studied this question at both perceptual and neuronal levels in behaving male macaques by using carefully parametrized PinnaBrelstaff figures that induce the above motion illusions. Wefirst demonstrate that macaques perceive illusory motion in a manner similar to that of human observers. Neurophysiological recordings were subsequently performed in the middle temporal area (MT) and the dorsal portion of the medial superior temporal area (MSTd). We find that subgroups of MSTd neurons encoding a particular global pattern of real complex-flow motion (rotation, expansion, contraction) also represent illusory motion patterns of the same class. They require an extra 15 ms to reliably discriminate the illusion. In contrast, MT neurons encode both real and illusory local motions with similar temporal delays. These findings reveal that illusory complex-flow motion is first represented in MSTd by the same neurons that normally encode real complex-flow motion. However, the extraction of global illusory motion in MSTd from other classes of real complex-flow motion requires extra processing time. Our study illustrates a cascaded integration mechanism from MT to MSTd underlying the transformation from external physical to internal nonveridical flow-motion perception.
The nucleus tractus solitarii (NTS) is implicated in the control of breathing, but the neuronal phenotype and circuit mechanism involved in such a physiological function remain incompletely understood. This study focused on the respiratory role of paired-like homeobox 2b gene (Phox2b)-expressing NTS neurons and sought to determine whether selective stimulation of this set of neurons activates breathing in male mice. A Cre-dependent vector encoding a Gq-coupled human M3 muscarinic receptor (hM3Dq) was microinjected into the NTS of Phox2b-Cre transgenic mice. The hM3Dq-transduced neurons were pharmacologically activated in conscious mice while respiratory effects were measured by plethysmography. We demonstrate that chemogenetic stimulation of Phox2b-expressing NTS neurons significantly increased baseline minute volume via an increase in respiratory frequency rather than tidal volume. Chemogenetic stimulation also synergized with moderate CO2 stimulation to enhance pulmonary ventilatory response. Selective ablation of Phox2b-expressing NTS neurons notably attenuated a hypercapnic ventilatory response. Moreover, histological evidence revealed that stimulation of Phox2b-expressing NTS neurons increased neuronal activity of the preBotzinger complex. Finally, we presented the neuroanatomical evidence of direct projection of Phox2b-expressing NTS neurons to putative respiratory central pattern generator. Overall, these findings suggest that selective activation of Phox2b-expressing NTS neurons potentiates baseline pulmonary ventilation via an excitatory drive to respiratory central pattern generator and this group of neurons is also required for the hypercapnic ventilatory response.
Calpains are calcium-dependent, cytosolic proteinases active at neutral pH. They do not degrade but cleave substrates at limited sites. Calpains are implicated in various pathologies, such as ischemia, injuries, muscular dystrophy, and neurodegeneration. Despite so, the physiological function of calpains remains to be clearly defined. Using the neuromuscular junction of Drosophila of both sexes as a model, we performed RNAi screening and uncovered that calpains negatively regulated protein levels of the glutamate receptor GluRIIA but not GluRIIB. We then showed that calpains enrich at the postsynaptic area, and the calcium-dependent activation of calpains induced cleavage of GluRIIA at Q788 of its C terminus. Further genetic and biochemical experiments revealed that different calpains genetically and physically interact to form a protein complex. The protein complex was required for the proteinase activation to downregulate GluRIIA. Our data provide a novel insight into the mechanisms by which different calpains act together as a complex to specifically control GluRIIA levels and consequently synaptic function.
Major depressive disorder is the most common mental illness. Mounting evidence indicates that astrocytes play a crucial role in the pathophysiology of depression; however, the underlying molecular mechanisms remain elusive. Compared with other neuronal cell types, astrocytes are enriched for arachidonic acid metabolism. Herein, we observed brain-region-specific alterations of epoxyeicosatrienoic acid (EET) signaling, which is an arachidonic acid metabolic pathway, in both a mouse model of depression and postmortem samples from patients with depression. The enzymatic activity of soluble epoxide hydrolase (sEH), the key enzyme in EET signaling, was selectively increased in the mPFC of susceptible mice after chronic social defeated stress and was negatively correlated with the social interaction ratio, which is an indicator of depressive-like behavior. The specific deletion of Ephx2 (encode sEH) in adult astrocytes induced resilience to stress, whereas the impaired EET signaling in the mPFC evoked depressive-like behaviors in response to stress. sEH was mainly expressed on lysosomes of astrocytes. Using pharmacological and genetic approaches performed on C57BL/6J background adult male mice, we found that EET signaling modulated astrocytic ATP release in vitro and in vivo. Moreover, astrocytic ATP release was required for the antidepressant-like effect of Ephx2 deletion in adult astrocytes. In addition, sEH inhibitors produced rapid antidepressant-like effects in multiple animal models of depression, including chronic social defeated stress and chronic mild stress. Together, our results highlight that EET signaling in astrocytes in the mPFC is essential for behavioral adaptation in response to psychiatric stress.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the production of granulocyte and macrophage populations from the hematopoietic progenitor cells; it is one of the most common growth factors in the blood. GM-CSF is also involved in bone cancer pain development by regulating tumor-nerve interactions, remodeling of peripheral nerves, and sensitization of damage-sensing (nociceptive) nerves. However, the precise mechanism for GM-CSF-dependent pain is unclear. In this study, we found that GM-CSF is highly expressed in human malignant osteosarcoma. Female Sprague Dawley rats implanted with bone cancer cells develop mechanical and thermal hyperalgesia, but antagonizing GM-CSF in these animals significantly reduced such hypersensitivity. The voltage-gated Na+ channels Nav1.7, Nav1.8, and Nav1.9 were found to be selectively upregulated in rat DRG neurons treated with GM-CSF, which resulted in enhanced excitability. GM-CSF activated the Janus kinase 2 (Jak2)-signal transducer and activator of transcription protein 3 (Stat3) signaling pathway, which promoted the transcription of Nav1.7-1.9 in DRG neurons. Accordingly, targeted knocking down of either Nav1.7-1.9 or Jak2/Stat3 in DRG neurons in vivo alleviated the hyperalgesia in male Sprague Dawley rats. Our findings describe a novel bone cancer pain mechanism and provide a new insight into the physiological and pathological functions of GM-CSF.