MrgprA3+ Primary Sensory Neurons Mediate Acute Allergic Itch Responses in Atopic Dermatitis Model Mice.

Itch is a prominent symptom of atopic dermatitis (AD). However, the underlying mechanism remains complex and has not yet been fully elucidated. Mas-related G protein-coupled receptor A3 (MrgprA3) has emerged attention as a marker of primary sensory neurons that specifically transmit itch signals; however, its involvement in AD-related itch has not been extensively explored. In this study, we developed an AD itch mouse model by repeatedly applying house dust mite (HDM) extract to barrier-impaired skin via a special diet. To clarify the role of MrgprA3+ neurons in itch behavior in our AD model, we adopted a toxin receptor-mediated cell knockout strategy using transgenic mice in which the diphtheria toxin receptor (DTR) gene was placed under the control of the Mrgpra3 promoter. When the HDM extract was repeatedly applied to the face and back skin of special diet-fed mice, the mice exhibited AD-like dry and eczematous skin lesions accompanied by three types of itch-related behaviors:1) spontaneous scratching, 2) acute scratching after antigen challenge, and 3) light touch-evoked scratching. Upon diphtheria toxin administration, substantial depletion of DTR+/MrgprA3+ neurons was observed in the dorsal root ganglion. Ablation of MrgprA3+ neurons suppressed acute itch responses after HDM application, whereas spontaneous and touch-evoked itch behaviors remained unaffected. Our findings unequivocally demonstrate that in our AD model, MrgprA3+ primary sensory neurons mediate acute allergic itch responses, whereas these neurons are not involved in spontaneous itch or alloknesis.

Disease-relevant upregulation of P2Y1 receptor in astrocytes enhances neuronal excitability via IGFBP2.

Reactive astrocytes play a pivotal role in the pathogenesis of neurological diseases; however, their functional phenotype and the downstream molecules by which they modify disease pathogenesis remain unclear. Here, we genetically increase P2Y1 receptor (P2Y1R) expression, which is upregulated in reactive astrocytes in several neurological diseases, in astrocytes of male mice to explore its function and the downstream molecule. This astrocyte-specific P2Y1R overexpression causes neuronal hyperexcitability by increasing both astrocytic and neuronal Ca2+ signals. We identify insulin-like growth factor-binding protein 2 (IGFBP2) as a downstream molecule of P2Y1R in astrocytes; IGFBP2 acts as an excitatory signal to cause neuronal excitation. In neurological disease models of epilepsy and stroke, reactive astrocytes upregulate P2Y1R and increase IGFBP2. The present findings identify a mechanism underlying astrocyte-driven neuronal hyperexcitability, which is likely to be shared by several neurological disorders, providing insights that might be relevant for intervention in diverse neurological disorders.

IRF8 and MAFB drive distinct transcriptional machineries in different resident macrophages of the central nervous system.

The central nervous system (CNS) includes anatomically distinct macrophage populations including parenchyma microglia and CNS-associated macrophages (CAMs) localized at the interfaces like meninges and perivascular space, which play specialized roles for the maintenance of the CNS homeostasis with the help of precisely controlled gene expressions. However, the transcriptional machinery that determines their cell-type specific states of microglia and CAMs remains poorly understood. Here we show, by myeloid cell-specific deletion of transcription factors, IRF8 and MAFB, that both adult microglia and CAMs utilize IRF8 to maintain their core gene signatures, although the genes altered by IRF8 deletion are different in the two macrophage populations. By contrast, MAFB deficiency robustly affected the gene expression profile of adult microglia, whereas CAMs are almost independent of MAFB. Our data suggest that distinct transcriptional machineries regulate different macrophages in the CNS.

Spatiotemporal dynamics of the CD11c+ microglial population in the mouse brain and spinal cord from developmental to adult stages.

CD11c-positive (CD11c+) microglia have attracted considerable attention because of their potential implications in central nervous system (CNS) development, homeostasis, and disease. However, the spatiotemporal dynamics of the proportion of CD11c+ microglia in individual CNS regions are poorly understood. Here, we investigated the proportion of CD11c+ microglia in six CNS regions (forebrain, olfactory bulb, diencephalon/midbrain, cerebellum, pons/medulla, and spinal cord) from the developmental to adult stages by flow cytometry and immunohistochemical analyses using a CD11c reporter transgenic mouse line, Itgax-Venus. We found that the proportion of CD11c+ microglia in total microglia varied between CNS regions during postnatal development. Specifically, the proportion was high in the olfactory bulb and cerebellum at postnatal day P(4) and P7, respectively, and approximately half of the total microglia were CD11c+. The proportion declined sharply in all regions to P14, and the low percentage persisted over P56. In the spinal cord, the proportion of CD11c+ microglia was also high at P4 and declined to P14, but increased again at P21 and thereafter. Interestingly, the distribution pattern of CD11c+ microglia in the spinal cord markedly changed from gray matter at P4 to white matter at P21. Collectively, our findings reveal the differences in the spatiotemporal dynamics of the proportion of CD11c+ microglia among CNS regions from early development to adult stages in normal mice. These findings improve our understanding of the nature of microglial heterogeneity and its dynamics in the CNS.

A method for selective and efficient isolation of gray matter astrocytes from the spinal cord of adult mice.

A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.

Laminar-selective spinal astrocyte population capable of converting tactile information into nociceptive in rats.

We previously identified a spinal astrocyte population that expresses hairy and enhancer of split 5 (Hes5) and is selectively present in superficial laminae in mice. However, it was unclear whether such astrocyte heterogeneity is commonly observed across species. Using adeno-associated viral (AAV) vectors incorporating a rat Hes5 promotor (AAV-Hes5P), we found that AAV-Hes5P-captured astrocytes were selectively located in the superficial laminae in rats. Furthermore, activation of AAV-Hes5P+ astrocytes elicited allodynia-like behavior and increased c-FOS+ cells in the superficial laminae. Thus, laminar-selective Hes5+ astrocytes are conserved beyond species and have the capability to convert tactile information to nociceptive.

Anterior cingulate cross-hemispheric inhibition via the claustrum resolves painful sensory conflict.

The anterior cingulate cortex (ACC) responds to noxious and innocuous sensory inputs, and integrates them to coordinate appropriate behavioral reactions. However, the role of the projections of ACC neurons to subcortical areas and their influence on sensory processing are not fully investigated. Here, we identified that ACC neurons projecting to the contralateral claustrum (ACC→contraCLA) preferentially respond to contralateral mechanical sensory stimulation. These sensory responses were enhanced during attending behavior. Optogenetic activation of ACC→contraCLA neurons silenced pyramidal neurons in the contralateral ACC by recruiting local circuit fast-spiking interneuron activation via an excitatory relay in the CLA. This circuit activation suppressed withdrawal behavior to mechanical stimuli ipsilateral to the ACC→contraCLA neurons. Chemogenetic silencing showed that the cross-hemispheric circuit has an important role in the suppression of contralateral nociceptive behavior during sensory-driven attending behavior. Our findings identify a cross-hemispheric cortical-subcortical-cortical arc allowing the brain to give attentional priority to competing innocuous and noxious inputs.

[Chronic Pain and Allostasis: Role of Glial Cells].

Lesions or diseases affecting the somatosensory system cause neuropathic pain, a debilitating chronic pain condition. From our recent study using a mouse model of neuropathic pain, CD11c+ microglia that appear in the spinal cord after nerve injury are important cells required for the pain remission. In this article, we review the transition of microglial states after nerve injury and the allostatic control mechanisms of neuropathic pain by CD11c+ microglia.

[Neuropathic pain decoded by microglial heterogeneity].

Lesion or diseases affecting the somatosensory system causes neuropathic pain, a debilitating chronic pain condition. Previous studies using its experimental models have demonstrated the critical contribution of microglia to the development of neuropathic pain. Upon sensing nerve damage, spinal cord microglia alter their morphology, gene expression and function, which lead to an increase in the excitability of pain-transmission neural pathway, causing the pain onset. Recently, newly identified CD11c-positive microglia as a subset that increases during the remission phase of neuropathic pain has been shown to be required for spontaneous remission of neuropathic pain and to play an important role in maintaining the remission state. Thus, these findings suggest that the functions and roles of microglia under neuropathic pain conditions are not one-dimensional but change during the onset, maintenance, and remission phases, and they also provide a clue to establish a new strategy to decipher neuropathic pain and other neurological diseases from the heterogeneity of microglia.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism through which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in the injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3-/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8-/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8-/- bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism through which migrating macrophages attract astrocytes and affect the pathophysiology and outcome after SCI.

Function of Glial Cells in Neuroinflammatory and Neuroimmunological Responses II.

It is now well established that glial cells play an equal, if not greater, role in regulating intricate functions of the central nervous system (CNS) compared with neurons [...].

Microglial diversity in neuropathic pain.

Microglia play pivotal roles in controlling CNS functions in diverse physiological and pathological contexts, including neuropathic pain, a chronic pain condition caused by lesions or diseases of the somatosensory nervous system. In this review article, we summarize evidence primarily from basic research on the role of microglia in the development and remission of neuropathic pain. The identification of a subset of microglia that emerged after pain development and that was necessary for remission of neuropathic pain highlights the highly divergent and dynamic nature of microglia in the course of neuropathic pain. Understanding microglial diversity in terms of gene expression, physiological states, and functional roles could lead to new strategies that aid in the diagnosis and management of neuropathic pain, and that may not have been anticipated from the viewpoint of targeting all microglia uniformly.

The Functions and Phenotypes of Microglia in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease worldwide, but therapeutic strategies to slow down AD pathology and symptoms have not yet been successful. While attention has been focused on neurodegeneration in AD pathogenesis, recent decades have provided evidence of the importance of microglia, and resident immune cells in the central nervous system. In addition, new technologies, including single-cell RNA sequencing, have revealed heterogeneous cell states of microglia in AD. In this review, we systematically summarize the microglial response to amyloid-ß and tau tangles, and the risk factor genes expressed in microglia. Furthermore, we discuss the characteristics of protective microglia that appear during AD pathology and the relationship between AD and microglia-induced inflammation during chronic pain. Understanding the diverse roles of microglia will help identify new therapeutic strategies for AD.

Voltage-gated calcium channel subunit α2δ-1 in spinal dorsal horn neurons contributes to aberrant excitatory synaptic transmission and mechanical hypersensitivity after peripheral nerve injury.

Neuropathic pain, an intractable pain symptom that occurs after nerve damage, is caused by the aberrant excitability of spinal dorsal horn (SDH) neurons. Gabapentinoids, the most commonly used drugs for neuropathic pain, inhibit spinal calcium-mediated neurotransmitter release by binding to α2δ-1, a subunit of voltage-gated calcium channels, and alleviate neuropathic pain. However, the exact contribution of α2δ-1 expressed in SDH neurons to the altered synaptic transmission and mechanical hypersensitivity following nerve injury is not fully understood. In this study, we investigated which types of SDH neurons express α2δ-1 and how α2δ-1 in SDH neurons contributes to the mechanical hypersensitivity and altered spinal synaptic transmission after nerve injury. Using in situ hybridization technique, we found that Cacna2d1, mRNA coding α2δ-1, was mainly colocalized with Slc17a6, an excitatory neuronal marker, but not with Slc32a1, an inhibitory neuronal marker in the SDH. To investigate the role of α2δ-1 in SDH neurons, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system and showed that SDH neuron-specific ablation of Cacna2d1 alleviated mechanical hypersensitivity following nerve injury. We further found that excitatory post-synaptic responses evoked by electrical stimulation applied to the SDH were significantly enhanced after nerve injury, and that these enhanced responses were significantly decreased by application of mirogabalin, a potent α2δ-1 inhibitor, and by SDH neuron-specific ablation of Cacna2d1. These results suggest that α2δ-1 expressed in SDH excitatory neurons facilitates spinal nociceptive synaptic transmission and contributes to the development of mechanical hypersensitivity after nerve injury.

GPR55 contributes to neutrophil recruitment and mechanical pain induction after spinal cord compression in mice.

Pain transmission and processing in the nervous system are modulated by various biologically active substances, including lysophospholipids, through direct and indirect actions on the somatosensory pathway. Lysophosphatidylglucoside (LysoPtdGlc) was recently identified as a structurally unique lysophospholipid that exerts biological actions via the G protein-coupled receptor GPR55. Here, we demonstrated that GPR55-knockout (KO) mice show impaired induction of mechanical pain hypersensitivity in a model of spinal cord compression (SCC) without the same change in the models of peripheral tissue inflammation and peripheral nerve injury. Among these models, only SCC recruited peripheral inflammatory cells (neutrophils, monocytes/macrophages, and CD3+ T-cells) in the spinal dorsal horn (SDH), and GPR55-KO blunted these recruitments. Neutrophils were the first cells recruited to the SDH, and their depletion suppressed the induction of SCC-induced mechanical hypersensitivity and inflammatory responses in compressed SDH. Furthermore, we found that PtdGlc was present in the SDH and that intrathecal administration of an inhibitor of secretory phospholipase A2 (an enzyme required for producing LysoPtdGlc from PtdGlc) reduced neutrophil recruitment to compressed SDH and suppressed pain induction. Finally, by screening compounds from a chemical library, we identified auranofin as a clinically used drug with an inhibitory effect on mouse and human GPR55. Systemically administered auranofin to mice with SCC effectively suppressed spinal neutrophil infiltration and pain hypersensitivity. These results suggest that GPR55 signaling contributes to the induction of inflammatory responses and chronic pain after SCC via the recruitment of neutrophils and may provide a new target for reducing pain induction after spinal cord compression, such as spinal canal stenosis.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism by which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3 -/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8 -/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8 -/ bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism in which migrating macrophages attracted astrocytes and affected the pathophysiology and outcome after SCI.

IP3R1-dependent astrocyte calcium signaling in chronic itch.

Astrocytes, the most abundant type of glial cell, are electrically non-excitable cells that use intracellular calcium (Ca2+) for functional regulation. Changes in intracellular Ca2+ concentration play important roles in the central nervous system (CNS), as they are involved in the release of gliotransmitters and the control of extracellular ion concentrations, thereby affecting the regulation of neuronal excitability, CNS homeostasis, and behavior. Intracellular calcium mobilization in astrocytes is known to be mediated via inositol 1,4,5-trisphosphate receptors (IP3Rs), particularly IP3R2, and its association with CNS pathogenesis has been widely reported. In addition, the existence of IP3R2-independent calcium signaling has recently been postulated; however, the detailed mechanisms and its role in astrocyte functions and CNS pathogenesis are still poorly understood. In this paper, we describe the putative mechanisms underlying IP3R1-dependent calcium signaling in astrocytes and its effects on the reactive state, compare this signaling with IP3R2-dependent calcium signaling, and discuss its contribution to chronic itch-like behavior.

Identification of Spinal Inhibitory Interneurons Required for Attenuating Effect of Duloxetine on Neuropathic Allodynia-like Signs in Rats.

Neuropathic pain is a chronic pain condition that occurs after nerve damage; allodynia, which refers to pain caused by generally innocuous stimuli, is a hallmark symptom. Although allodynia is often resistant to analgesics, the antidepressant duloxetine has been used as an effective therapeutic option. Duloxetine increases spinal noradrenaline (NA) levels by inhibiting its transporter at NAergic terminals in the spinal dorsal horn (SDH), which has been proposed to contribute to its pain-relieving effect. However, the mechanism through which duloxetine suppresses neuropathic allodynia remains unclear. Here, we identified an SDH inhibitory interneuron subset (captured by adeno-associated viral (AAV) vectors incorporating a rat neuropeptide Y promoter; AAV-NpyP+ neurons) that is mostly depolarized by NA. Furthermore, this excitatory effect was suppressed by pharmacological blockade or genetic knockdown of α1B-adrenoceptors (ARs) in AAV-NpyP+ SDH neurons. We found that duloxetine suppressed Aß fiber-mediated allodynia-like behavioral responses after nerve injury and that this effect was not observed in AAV-NpyP+ SDH neuron-selective α1B-AR-knockdown. These results indicate that α1B-AR and AAV-NpyP+ neurons are critical targets for spinal NA and are necessary for the therapeutic effect of duloxetine on neuropathic pain, which can support the development of novel analgesics.

Selective Involvement of a Subset of Spinal Dorsal Horn Neurons Operated by a Prodynorphin Promoter in Aß Fiber-Mediated Neuropathic Allodynia-Like Behavioral Responses in Rats.

Mechanical allodynia (pain produced by innocuous stimuli such as touch) is the main symptom of neuropathic pain. Its underlying mechanism remains to be elucidated, but peripheral nerve injury (PNI)-induced malfunction of neuronal circuits in the central nervous system, including the spinal dorsal horn (SDH), is thought to be involved in touch-pain conversion. Here, we found that intra-SDH injection of adeno-associated viral vectors including a prodynorphin promoter (AAV-PdynP) captured a subset of neurons that were mainly located in the superficial laminae, including lamina I, and exhibited mostly inhibitory characteristics. Using transgenic rats that enable optogenetic stimulation of touch-sensing Aß fibers, we found that the light-evoked paw withdrawal behavior and aversive responses after PNI were attenuated by selective ablation of AAV-PdynP-captured SDH neurons. Notably, the ablation had no effect on withdrawal behavior from von Frey filaments. Furthermore, Aß fiber stimulation did not excite AAV-PdynP+ SDH neurons under normal conditions, but after PNI, this induced excitation, possibly due to enhanced Aß fiber-evoked excitatory synaptic inputs and elevated resting membrane potentials of these neurons. Moreover, the chemogenetic silencing of AAV-PdynP+ neurons of PNI rats attenuated the Aß fiber-evoked paw withdrawal behavior and c-FOS expression in superficial SDH neurons. Our findings suggest that PNI renders AAV-PdynP-captured neurons excitable to Aß fiber stimulation, which selectively contributes to the conversion of Aß fiber-mediated touch signal to nociceptive. Thus, reducing the excitability of AAV-PdynP-captured neurons may be a new option for the treatment of neuropathic allodynia.