Encoding of self-initiated actions in axon terminals of the mesocortical pathway.

Significance: The initiation of goal-directed actions is a complex process involving the medial prefrontal cortex and dopaminergic inputs through the mesocortical pathway. However, it is unclear what information the mesocortical pathway conveys and how it impacts action initiation. In this study, we unveiled the indispensable role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Aim: To investigate the role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Approach: We designed a lever-press task in which mice internally determine the timing of the press, receiving a larger reward for longer waiting periods. Results: Our study revealed that self-initiated actions depend on dopaminergic signaling mediated by D2 receptors, whereas sensory-triggered lever-press actions do not involve D2 signaling. Microprism-mediated two-photon calcium imaging further demonstrated ramping activity in mesocortical axon terminals approximately 0.5 s before the self-initiated lever press. Remarkably, the ramping patterns remained consistent whether the mice responded to cues immediately for a smaller reward or held their response for a larger reward. Conclusions: We conclude that mesocortical dopamine axon terminals encode the timing of self-initiated actions, shedding light on a crucial aspect of the intricate neural mechanisms governing goal-directed behavior.

Functional diversity of dopamine axons in prefrontal cortex during classical conditioning.

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

Glial senescence enhances α-synuclein pathology owing to its insufficient clearance caused by autophagy dysfunction.

Parkinson's disease (PD) is characterized by the pathological accumulation of α-synuclein (α-syn) and loss of dopaminergic neurons in the substantia nigra. Aging is a significant risk factor for PD. The accumulation of senescent glial cells in the aged brain contributes to PD progression by inducing chronic neuroinflammatory processes. However, although the insufficient degradation of α-syn aggregates results in PD deterioration, the possible alteration in the ability of α-syn clearance in senescent glia has received little attention. In this study, we investigated how aging and glial senescence affect the capacity of α-syn clearance. We found that following the intra-striatal injection of human α-syn (hu-α-syn) preformed fibril, hu-α-syn pathology persisted more in aged mice compared with younger mice and that aged microglia exhibited greater accumulation of hu-α-syn than younger microglia. Moreover, in vitro assay revealed that the clearance of hu-α-syn was primarily dependent on the autophagy-lysosome system rather than on the ubiquitin-proteasome system and that the capacity of hu-α-syn clearance was diminished in senescent glia because of autophagy-lysosome system dysfunction. Overall, this study provides new insights into the role of senescent glia in PD pathogenesis.

Functional Diversity of Dopamine Axons in Prefrontal Cortex During Classical Conditioning.

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

CXCR4 signaling regulates repair Schwann cell infiltration into the spinal cord after spinal cord injury in mice.

Schwann cells are glial cells that myelinate neuronal axons in the peripheral nervous system (PNS). When the PNS is damaged, Schwann cells de-differentiate into p75-positive "repair Schwann cells," which contribute to neural circuit regeneration. Interestingly, Schwann cells in the dorsal roots are known to be reprogrammed to repair Schwann cells even after spinal cord injury (SCI) and then migrate into the injured spinal cord. However, the molecular mechanism underlying the migration of repair Schwann cells remains unknown. Since a recent in vitro study revealed the importance of CXCR4 signaling in Schwann cell migration, we investigated whether CXCR4 signaling is involved in the PNS-to-central nervous system (CNS) migration of repair Schwann cells after SCI. We revealed that repair Schwann cells express CXCR4, and its ligand CXCL12 is upregulated in the injured spinal cord. We also found that the pharmacological inhibition of CXCR4 signaling decreased the infiltration of repair Schwann cells. Moreover, CXCR4 agonist administration effectively increased the infiltration of repair Schwann cells along with improved motor function. These findings strongly suggest the involvement of CXCR4 signaling in the PNS-to-CNS migration of repair Schwann cells after SCI.

Intravital Imaging Reveals the Ameliorating Effect of Colchicine in a Photothrombotic Stroke Model via Inhibition of Neutrophil Recruitment.

Although post-stroke neutrophil recruitment is known to be deleterious to neural tissues in the peri-infarct area, the precise behavior of recruited neutrophils remains elusive. In this study, potential therapeutic agents for modifying neutrophil behavior in the peri-infarct area were explored through intravital imaging of an experimental stroke mouse model. By applying in vivo 2-photon imaging to study a tightly controlled photothrombotic stroke mouse model, we established a highly sensitive and reproducible method for investigating the temporal dynamics of ischemic brain lesions. Taking advantage of this system, we revealed that neutrophil depletion by a neutrophil-specific antibody ameliorated the expansion of the infarct area, confirming the deleterious effect of neutrophils in the peri-infarct cortex. To identify neutrophil-targeted therapeutic approaches, we screened various agents and found that colchicine and an anti-P-selectin antibody were the most effective in inhibiting neutrophil attachment to the vessel wall in the early phase (6 h post-infarction). Interestingly, further investigation in the later phase (16 h post-infarction) revealed that colchicine potently inhibited neutrophil infiltration into the peri-infarct cortex; however, the anti-P-selectin antibody did not. Subsequent analysis revealed that the effect of the anti-P-selectin antibody against neutrophil attachment to the vessel wall was transient and thus insufficient for mitigating neutrophil infiltration. Finally, we revealed that colchicine treatment effectively ameliorated infarct expansion. In conclusion, we have established an intravital strategy to directly investigate pathophysiology in the ischemic border zone, and found that colchicine administration in the acute phase of ischemic stroke is a potential novel therapeutic strategy.

A novel aquaporin-4-associated optic neuritis rat model with severe pathological and functional manifestations.

BACKGROUND: Optic neuritis (ON) is a common manifestation of aquaporin-4 (AQP4) antibody seropositive neuromyelitis optica (NMO). The extent of tissue damage is frequently severe, often leading to loss of visual function, and there is no curative treatment for this condition. To develop a novel therapeutic strategy, elucidating the underlying pathological mechanism using a clinically relevant experimental ON model is necessary. However, previous ON animal models have only resulted in mild lesions with limited functional impairment. In the present study, we attempted to establish a feasible ON model with severe pathological and functional manifestations using a high-affinity anti-AQP4 antibody. Subsequently, we aimed to address whether our model is suitable for potential drug evaluation by testing the effect of minocycline, a well-known microglia/macrophage inhibitor. METHODS: AQP4-immunoglobulin G (IgG)-related ON in rats was induced by direct injection of a high-affinity anti-AQP4 monoclonal antibody, E5415A. Thereafter, the pathological and functional characterizations were performed, and the therapeutic potential of minocycline was investigated. RESULTS: We established an experimental ON model that reproduces the histological characteristics of ON in seropositive NMO, such as loss of AQP4/glial fibrillary acidic protein immunoreactivity, immune cell infiltration, and extensive axonal damage. We also observed that our rat model exhibited severe visual dysfunction. The histological analysis showed prominent accumulation of macrophages/activated microglia in the lesion site in the acute phase. Thus, we investigated the possible effect of the pharmacological inhibition of macrophages/microglia activation by minocycline and revealed that it effectively ameliorated axonal damage and functional outcome. CONCLUSIONS: We established an AQP4-IgG-induced ON rat model with severe functional impairments that reproduce the histological characteristics of patients with NMO. Using this model, we revealed that minocycline treatment ameliorates functional and pathological outcomes, highlighting the usefulness of our model for evaluating potential therapeutic drugs for ON in NMO.

Humanized Anti-RGMa Antibody Treatment Promotes Repair of Blood-Spinal Cord Barrier Under Autoimmune Encephalomyelitis in Mice.

The lack of established biomarkers which reflect dynamic neuropathological alterations in multiple sclerosis (MS) makes it difficult to determine the therapeutic response to the tested drugs and to identify the key biological process that mediates the beneficial effect of them. In the present study, we applied high-field MR imaging in locally-induced experimental autoimmune encephalomyelitis (EAE) mice to evaluate dynamic changes following treatment with a humanized anti-repulsive guidance molecule-a (RGMa) antibody, a potential drug for MS. Based on the longitudinal evaluation of various MRI parameters including white matter, axon, and myelin integrity as well as blood-spinal cord barrier (BSCB) disruption, anti-RGMa antibody treatment exhibited a strong and prompt therapeutic effect on the disrupted BSCB, which was paralleled by functional improvement. The antibody's effect on BSCB repair was also suggested via GeneChip analysis. Moreover, immunohistochemical analysis revealed that EAE-induced vascular pathology which is characterized by aberrant thickening of endothelial cells and perivascular type I/IV collagen deposits were attenuated by anti-RGMa antibody treatment, further supporting the idea that the BSCB is one of the key therapeutic targets of anti-RGMa antibody. Importantly, the extent of BSCB disruption detected by MRI could predict late-phase demyelination, and the predictability of myelin integrity based on the extent of acute-phase BSCB disruption was compromised following anti-RGMa antibody treatment. These results strongly support the concept that longitudinal MRI with simultaneous DCE-MRI and DTI analysis can be used as an imaging biomarker and is useful for unbiased prioritization of the key biological process that mediates the therapeutic effect of tested drugs.

Neuroplasticity related to chronic pain and its modulation by microglia.

Neuropathic pain is often chronic and can persist after overt tissue damage heals, suggesting that its underlying mechanism involves the alteration of neuronal function. Such an alteration can be a direct consequence of nerve damage or a result of neuroplasticity secondary to the damage to tissues or to neurons. Recent studies have shown that neuroplasticity is linked to causing neuropathic pain in response to nerve damage, which may occur adjacent to or remotely from the site of injury. Furthermore, studies have revealed that neuroplasticity relevant to chronic pain is modulated by microglia, resident immune cells of the central nervous system (CNS). Microglia may directly contribute to synaptic remodeling and altering pain circuits, or indirectly contribute to neuroplasticity through property changes, including the secretion of growth factors. We herein highlight the mechanisms underlying neuroplasticity that occur in the somatosensory circuit of the spinal dorsal horn, thalamus, and cortex associated with chronic pain following injury to the peripheral nervous system (PNS) or CNS. We also discuss the dynamic functions of microglia in shaping neuroplasticity related to chronic pain. We suggest further understanding of post-injury ectopic plasticity in the somatosensory circuits may shed light on the differential mechanisms underlying nociceptive, neuropathic, and nociplastic-type pain. While one of the prominent roles played by microglia appears to be the modulation of post-injury neuroplasticity. Therefore, future molecular- or genetics-based studies that address microglia-mediated post-injury neuroplasticity may contribute to the development of novel therapies for chronic pain.

RGMa Signal in Macrophages Induces Neutrophil-Related Astrocytopathy in NMO.

OBJECTIVE: Repulsive guidance molecule-a (RGMa) is a glycosylphosphatidylinositol-linked glycoprotein which has multiple functions including axon growth inhibition and immune regulation. However, its role in the pathophysiology of neuromyelitis optica (NMO) is poorly understood. Perivascular astrocytopathy, which is induced by the leakage of aquaporin-4 (AQP4)-specific IgG into the central nervous system parenchyma, is a key feature of NMO pathology. We investigated the RGMa involvement in the pathology of NMO astrocytopathy, and tested a therapeutic potential of humanized anti-RGMa monoclonal antibody (RGMa-mAb). METHODS: Using a clinically relevant NMO rat model, we evaluated the therapeutic effect of a RGMa-mAb by behavioral testing, immunohistochemistry, and gene expression assay. We further performed in vitro experiments to address the RGMa-signaling in macrophages. RESULTS: In both NMO rats and an NMO-autopsied sample, RGMa was expressed by the spared neurons and astrocytes, whereas its receptor neogenin was expressed by infiltrating macrophages. AQP4-IgG-induced astrocytopathy and clinical exacerbation in NMO rats were ameliorated by RGMa-mAb treatment. RGMa-mAb treatment significantly suppressed neutrophil infiltration, and decreased the expression of neutrophil chemoattractants. Interestingly, neogenin-expressing macrophages accumulated in the lesion expressed CXCL2, a strong neutrophil chemoattractant, and further analysis revealed that RGMa directly regulated CXCL2 expression in macrophages. Finally, we found that our NMO rats developed neuropathic pain, and RGMa-mAb treatment effectively ameliorated the severity of neuropathic pain. INTERPRETATION: RGMa signaling in infiltrated macrophages is a critical driver of neutrophil-related astrocytopathy in NMO lesions, and RGMa-mAb may provide an efficient therapeutic strategy for NMO-associated neuropathic pain and motor deficits in patients with NMO. ANN NEUROL 2022;91:532-547.

Complement cascade functions during brain development and neurodegeneration.

The complement system, an essential tightly regulated innate immune system, is a key regulator of normal central nervous system (CNS) development and function. However, aberrant complement component expression and activation in the brain may culminate into marked neuroinflammatory response, neurodegenerative processes and cognitive impairment. Over the years, complement-mediated neuroinflammatory responses and complement-driven neurodegeneration have been increasingly implicated in the pathogenesis of a wide spectrum of CNS disorders. This review describes how complement system contributes to normal brain development and function. We also discuss how pathologic insults such as misfolded proteins, lipid droplet/lipid droplet-associated protein or glycosaminoglycan accumulation could trigger complement-mediated neuroinflammatory responses and neurodegenerative process in neurodegenerative proteinopathies, age-related macular degeneration and neurodegenerative lysosomal storage disorders.

Interleukin-17A regulates ependymal cell proliferation and functional recovery after spinal cord injury in mice.

Ependymal cells have been suggested to act as neural stem cells and exert beneficial effects after spinal cord injury (SCI). However, the molecular mechanism underlying ependymal cell regulation after SCI remains unknown. To examine the possible effect of IL-17A on ependymal cell proliferation after SCI, we locally administrated IL-17A neutralizing antibody to the injured spinal cord of a contusion SCI mouse model, and revealed that IL-17A neutralization promoted ependymal cell proliferation, which was paralleled by functional recovery and axonal reorganization of both the corticospinal tract and the raphespinal tract. Further, to test whether ependymal cell-specific manipulation of IL-17A signaling is enough to affect the outcomes of SCI, we generated ependymal cell-specific conditional IL-17RA-knockout mice and analyzed their anatomical and functional response to SCI. As a result, conditional knockout of IL-17RA in ependymal cells enhanced both axonal growth and functional recovery, accompanied by an increase in mRNA expression of neurotrophic factors. Thus, Ependymal cells may enhance the regenerative process partially by secreting neurotrophic factors, and IL-17A stimulation negatively regulates this beneficial effect. Molecular manipulation of ependymal cells might be a viable strategy for improving functional recovery.

Arid5a Promotes Immune Evasion by Augmenting Tryptophan Metabolism and Chemokine Expression.

The acquisition of mesenchymal traits leads to immune evasion in various cancers, but the underlying molecular mechanisms remain unclear. In this study, we found that the expression levels of AT-rich interaction domain-containing protein 5a (Arid5a), an RNA-binding protein, were substantially increased in mesenchymal tumor subtypes. The deletion of Arid5a in tumor cell lines enhanced antitumor immunity in immunocompetent mice, but not in immunodeficient mice, suggesting a role for Arid5a in immune evasion. Furthermore, an Arid5a-deficient tumor microenvironment was shown to have robust antitumor immunity, as manifested by suppressed infiltration of granulocytic myeloid-derived suppressor cells and regulatory T cells. In addition, infiltrated T cells were more cytotoxic and less exhausted. Mechanistically, Arid5a stabilized Ido1 and Ccl2 mRNAs and augmented their expression, resulting in enhanced tryptophan catabolism and an immunosuppressive tumor microenvironment. Thus, our findings demonstrate the role of Arid5a beyond inflammatory diseases and suggest Arid5a as a promising target for the treatment of immunotolerant malignant tumors.See related Spotlight by Van den Eynde, p. 854.

Microglia as therapeutic target in central nervous system disorders.

Chronic microglial activation is associated with the pathogenesis of several CNS disorders. Microglia show phenotypic diversity and functional complexity in diseased CNS. Thus, understanding the pathology-specific heterogeneity of microglial behavior is crucial for the future development of microglia-modulating therapy for variety of CNS disorders. This review summarizes up-to-date knowledge on how microglia contribute to CNS homeostasis during development and throughout adulthood. We discuss the heterogeneity of microglial phenotypes in the context of CNS disorders with an emphasis on neurodegenerative diseases, demyelinating diseases, CNS trauma, and epilepsy. We conclude this review with a discussion about the disease-specific heterogeneity of microglial function and how it could be exploited for therapeutic intervention.

Immunotherapies in Huntington's disease and α-Synucleinopathies.

Modulation of immune activation using immunotherapy has attracted considerable attention for many years as a potential therapeutic intervention for several inflammation-associated neurodegenerative diseases. However, the efficacy of single-target immunotherapy intervention has shown limited or no efficacy in alleviating disease burden and restoring functional capacity. Marked immune system activation and neuroinflammation are important features and prodromal signs in polyQ repeat disorders and α-synucleinopathies. This review describes the current status and future directions of immunotherapies in proteinopathy-induced neurodegeneration with emphasis on preclinical and clinical efficacies of several anti-inflammatory compounds and antibody-based therapies for the treatment of Huntington's disease and α-synucleinopathies. The review concludes with how disease modification and functional restoration could be achieved by using targeted multimodality therapy to target multiple factors.

Microglial depletion under thalamic hemorrhage ameliorates mechanical allodynia and suppresses aberrant axonal sprouting.

Central poststroke pain (CPSP) is one of the neuropathic pain syndromes that can occur following stroke involving the somatosensory system. However, the underlying mechanism of CPSP remains largely unknown. Here, we established a CPSP mouse model by inducing a focal hemorrhage in the thalamic ventrobasal complex and confirmed the development of mechanical allodynia. In this model, microglial activation was observed in the somatosensory cortex, as well as in the injured thalamus. By using a CSF1 receptor inhibitor, we showed that microglial depletion effectively prevented allodynia development in our CPSP model. In the critical phase of allodynia development, c-fos-positive neurons increased in the somatosensory cortex, accompanied by ectopic axonal sprouting of the thalamocortical projection. Furthermore, microglial ablation attenuated both neuronal hyperactivity in the somatosensory cortex and circuit reorganization. These findings suggest that microglia play a crucial role in the development of CPSP pathophysiology by promoting sensory circuit reorganization.

Interhemispherically dynamic representation of an eye movement-related activity in mouse frontal cortex.

Cortical plasticity is fundamental to motor recovery following cortical perturbation. However, it is still unclear how this plasticity is induced at a functional circuit level. Here, we investigated motor recovery and underlying neural plasticity upon optogenetic suppression of a cortical area for eye movement. Using a visually-guided eye movement task in mice, we suppressed a portion of the secondary motor cortex (MOs) that encodes contraversive eye movement. Optogenetic unilateral suppression severely impaired contraversive movement on the first day. However, on subsequent days the suppression became inefficient and capability for the movement was restored. Longitudinal two-photon calcium imaging revealed that the regained capability was accompanied by an increased number of neurons encoding for ipsiversive movement in the unsuppressed contralateral MOs. Additional suppression of the contralateral MOs impaired the recovered movement again, indicating a compensatory mechanism. Our findings demonstrate that repeated optogenetic suppression leads to functional recovery mediated by the contralateral hemisphere.

Microglia suppress the secondary progression of autoimmune encephalomyelitis.

Secondary progressive multiple sclerosis (SPMS) is an autoimmune disease of the central nervous system (CNS) characterized by progressive motor dysfunction, sensory deficits, and visual problems. The pathological mechanism of SPMS remains poorly understood. In this study, we investigated the role of microglia, immune cells in the CNS, in a secondary progressive form of experimental autoimmune encephalomyelitis (EAE), the mouse model of SPMS. We induced EAE in nonobese diabetic mice and treated the EAE mice with PLX3397, an antagonist of colony stimulating factor-1 receptor, during secondary progression in order to deplete microglia. The results showed that PLX3397 treatment significantly exacerbated secondary progression of EAE and increased mortality rates. Additionally, histological analysis showed that PLX3397 treatment significantly promoted inflammation, demyelination, and axonal degeneration. Moreover, the number of CD4+ T cells in the spinal cord of EAE mice was expanded due to PLX3397-mediated proliferation. These results suggest that microglia suppressed secondary progression of EAE by inhibiting the proliferation of CD4+ T cells in the CNS.

Streamlined sensory motor communication through cortical reciprocal connectivity in a visually guided eye movement task.

Cortical computation is distributed across multiple areas of the cortex by networks of reciprocal connectivity. However, how such connectivity contributes to the communication between the connected areas is not clear. In this study, we examine the communication between sensory and motor cortices. We develop an eye movement task in mice and combine it with optogenetic suppression and two-photon calcium imaging techniques. We identify a small region in the secondary motor cortex (MOs) that controls eye movements and reciprocally connects with a rostrolateral part of the higher visual areas (VRL/A/AL). These two regions encode both motor signals and visual information; however, the information flow between the regions depends on the direction of the connectivity: motor information is conveyed preferentially from the MOs to the VRL/A/AL, and sensory information is transferred primarily in the opposite direction. We propose that reciprocal connectivity streamlines information flow, enhancing the computational capacity of a distributed network.