Glia

Astrocytes are a type of glial cell in the central nervous system responsible for modulating synaptic transmissions, tissue repair, maintaining homeostasis, and are therefore implicated in many neurological diseases. Human cortical astrocytes are more structurally complex, larger, and have unique subtypes in comparison to the commonly studied rodent cortical astrocytes. As access to human cortical tissue is sparse, cerebral organoids (COs) derived from human pluripotent stem cells have emerged as a promising in vitro model for studying the human cortex. Astrocyte subtypes unique to humans are recapitulated in COs but have not been quantitatively assessed (1, 2). In this study, we characterized human astrocytes in situ in sliced COs cultured at the air-liquid interface (ALI-COs). By 4 months of age, ALI-COs express many mature astrocyte markers and showed increasing levels of GFAP with longer culture durations. By employing immunostaining, tissue clearing, morphological reconstruction, and unsupervised clustering analysis, three major GFAP+ astrocyte subtypes were identified in ALI-COs. All subtypes exhibited greater morphological complexity than their mouse counterparts, as revealed by increased branching and longer branch extensions. However, consistent with the mid-gestation fetal stage of the ALI-COs, astrocytes did not fully recapitulate the complexity observed in adult human astrocytes, which are known to continue maturation postnatally. Significance StatementThe limited availability of human brain tissue has long constrained our ability to investigate human-specific neurobiology. While rodent models provide valuable insights, they cannot capture features unique to the human brain, such as distinct cortical astrocyte subtypes. Given the critical role of astrocytes in numerous neurological disorders, elucidating their human-specific properties is essential. Here, we compare the morphology of astrocytes in human cerebral organoids to those in human and rodent brain tissue, demonstrating that cerebral organoids reproduce some key aspects of human astrocyte complexity, and can be further applied to study astrocytes in a human-specific 3D model.

Astrocytes are key players in CNS neuroinflammation and neuroregeneration that may help or hinder recovery, depending on the context of the injury. Although pro-inflammatory factors that promote astrocyte-mediated neurotoxicity have been shown to be secreted by reactive microglia, anti-inflammatory factors that suppress astrocyte activation are not well-characterized. Olfactory ensheathing cells (OECs), glial cells that wrap axons of olfactory sensory neurons, have been shown to moderate astrocyte reactivity, creating an environment conducive to regeneration. Similarly, astrocytes cultured in medium conditioned by cultured OECs (OEC-CM) show reduced nuclear translocation of Nuclear Factor kappa-B (NF{kappa}B), a pro-inflammatory protein that induces neurotoxic reactivity in astrocytes. In this study, we screened primary and immortalized OEC lines to identify these factors and discovered that Alpha B-crystallin (CryAB), an antiinflammatory protein, is secreted by OECs via exosomes, coordinating an intercellular immune response. Our results showed: 1) OEC exosomes block nuclear NF{kappa}B translocation in astrocytes while exosomes from CryAB-null OECs could not; 2) OEC exosomes could be taken up by astrocytes and 3) CryAB treatment suppressed multiple neurotoxicity-associated astrocyte transcripts. Our results indicate that OEC-secreted factors are potential agents that can ameliorate, or even reverse, the growth-inhibitory environment created by neurotoxic reactive astrocytes following CNS injuries. Main PointsO_LIAstrocytes uptake OEC-secreted exosomes. C_LIO_LIWT OEC-exosomes, but not CryAB-null OEC-exosomes, block nuclear NF{kappa}B translocation in astrocytes. C_LIO_LICryAB, and other factors secreted by OECs, suppresses multiple neurotoxicity-associated astrocyte transcripts. C_LI

Astrocytes are abundant star-shaped glial cells in the mammalian brain, with essential roles in metabolism, development, homeostasis, response to injury, behavior, and learning. Surprisingly, most regions of the teleost brain are thought to lack astrocytes, based primarily on the use of GFAP (glial fibrillary acidic protein) as a marker1. Here, drawing on recent evidence that astrocytes are molecularly heterogeneous, we propose that astrocytes exist in the teleost brain, albeit of the olig2 subtype2. Highly branched cells are present throughout the zebrafish brain, as shown here in Tg(sox10:EGFP) fish and previously in Tg(olig2:GFP) fish. Transcriptome data indicates the presence of brain cells that are olig2 and sox10 positive, which also express the astrocyte markers sox9b, sparcl1 and slc1a2b but lack gfap and the oligodendrocyte marker mbp. In situ hybridization confirms that stellate sox10:EGFP cells express olig2 and sox9b, while immunofluorescence indicates that they lack HuC/D and GFAP. We suggest that these cells be classified as astrocytes as this may more accurately reflect their functions.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/587565v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1899db6org.highwire.dtl.DTLVardef@1d152fcorg.highwire.dtl.DTLVardef@19f4754org.highwire.dtl.DTLVardef@3f853_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Brain damage triggers diverse cellular and molecular events, with astrocytes playing a crucial role in activating local neuroprotective and reparative signaling within damaged neuronal circuits. Here, we investigated reactive astrocytes using a multidimensional approach to categorize their responses into different subtypes based on morphology using the StarTrack lineage tracer, single-cell imaging reconstruction and multivariate data analysis. Our findings revealed three profiles of reactive astrocyte responses affecting cell size- and shape-related morphological parameters: "moderate," "strong," and "very strong". We also explored the heterogeneity in astrocyte reactivity, with a particular emphasis in the spatial and clonal distribution. Our research highlights the importance of the relationships between the different astrocyte subpopulations with their reactive responses, showing an enrichment of protoplasmic and fibrous astrocytes within the "strong" and "very strong" subtypes. Overall, our study contributes to a better understanding of astrocyte heterogeneity in response to an injury. By elucidating the diverse reactive responses among astrocyte subpopulations, we pave the way for future research aimed at uncovering novel therapeutic targets for mitigating the effects of brain damage and promoting neural repair.

AbstractIschemic brain injuries, including stroke, are highly prevalent neurological diseases. Acute, low-grade inflammation, often resulting from peripheral infection, is a recognised risk factor. White matter axons, and their supporting glia, are susceptible to both ischemic and neuroinflammatory injury. The mechanisms behind ischemic and severe inflammatory injury in white matter are well established. However, the white matter response to acute, low-grade inflammation, and how this can interact with co-morbidities such as cerebral ischemia are yet to be examined. Here, we examine the response of white matter to acute, low-grade inflammation and the downstream effects this had on sensitivity of ischemia. Ex vivo mouse optic nerve and corpus callosum, central white matter tracts, tolerated acute, low-grade inflammation (110-minutes, 0.1g/ml LPS), with preserved action potential conduction, limited microglial activation and no axo-myelinic damage. Exposure to these conditions elevated indicators of stress in oligodendrocytes which, when exposed to ischemic conditions (30 minutes oxygen-glucose deprivation), impaired their ability to maintain healthy myelin. There was a resulting increase in axo-myelinic damage and functional decline associated with ischemic conditions compared to white matter that had not been pre-exposed to LPS. Microglial depletion or dampening microglial responses with the clinically available drug minocycline eliminated the elevated sensitivity of white matter to ischemic injury associated with acute, low-grade inflammation. We demonstrate that acute, low-grade neuroinflammation primes white matter for heightened vulnerability to ischemic injury through microglial-mediated mechanisms. These findings provide a mechanistic explanation for the increased risk of ischemic brain injury observed during systemic inflammatory states and highlights microglial modulation as a clinical strategy to mitigate ischemic damage in susceptible patients.

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), causing human coronavirus disease 2019 (COVID-19), not only affects the respiratory tract, but also impacts other organs including the brain. A considerable number of COVID-19 patients develop neuropsychiatric symptoms that may linger for weeks and months and contribute to "long-COVID". While the neurological symptoms of COVID-19 are well described, the cellular mechanisms of neurologic disorders attributed to the infection are still enigmatic. Here, we studied the effect of an infection with SARS-CoV-2 on the structure and expression of marker proteins of astrocytes and microglial cells in the frontal cortex of patients who died from COVID-19 in comparison to non-COVID-19 controls. Most of COVID-19 patients had microglial cells with retracted processes and rounded and enlarged cell bodies in both gray and white matter, as visualized by anti-Iba1 staining and confocal fluorescence microscopy. In addition, gray matter astrocytes in COVID-19 patients were frequently labeled by intense anti-GFAP staining, whereas in non-COVID-19 controls, most gray matter astrocytes expressed little GFAP. The most striking difference between astrocytes in COVID-19 patients and controls was found by anti-aquaporin-4 (AQP4) staining. In COVID-19 patients, a large number of gray matter astrocytes showed an increase in AQP4. In addition, AQP4 polarity was lost and AQP4 covered the entire cell, including the cell body and all cell processes, while in controls, AQP4 immunostaining was mainly detected in endfeet around blood vessels and did not visualize the cell body. In summary, our data suggest neuroinflammation upon SARS-CoV-2 infection including microgliosis and astrogliosis, including loss of AQP4 polarity.

Reactive astrogliosis, a hallmark of central nervous system pathologies, involves cellular responses, including morphological remodelling and upregulation of intermediate filaments such as vimentin. These changes are driven by cytoskeletal dynamics and are mediated by focal adhesions (FAs). Our study identifies plectin, a versatile cytoskeletal linker protein, as a critical modulator of FA-associated processes in mouse astrocytes. We demonstrate that plectin localizes to astrocyte FAs, where it regulates their number, maturation, turnover, and the mobility of FA components. Plectin also polarizes within FAs, depending on their maturation state, and controls the recruitment of key cytoskeletal elements particularly vimentin. In plectin-deficient astrocytes, the vimentin network exhibits impaired connectivity, accompanied by altered viscoelastic properties of the cells. In a model of reactive astrocytes FA number and size were elevated along with the expression of plectin, highlighting involvement of plectin in pathological conditions.

Astrocytes have long been considered to be a largely homogeneous cell population. Recent studies however suggest that astrocytes are highly adapted to the local neuronal circuitry. Glucose utilization in the retinorecipient superior colliculus (SC) is one of the highest in the brain. Since metabolic support to neurons is a major function of astrocytes, they could be of particular relevance in this region and display specific features. However, little is known about astrocytes and their interactions with neurons in this multisensory brain area. We thus here investigated region-specific cellular and structural properties of astrocytes in the visual layer of the SC. Using morphological reconstructions, fluorescent recovery after photobleaching (FRAP) and superresolution imaging, we found that astrocytes from the visual layers of the SC are highly distinct with a higher cellular density, a more complex morphology and a stronger proximity to synapses compared to astrocytes from the primary visual cortex and the hippocampus. These data point to astroglial diversity and specialization within neural circuits integrating sensory information in the adult brain.

In the central nervous system (CNS), myelin formation by oligodendrocytes (OLs) relies on actin dynamics. Actin polymerization supports the ensheathment step, when the OL process contacts the axon, while a drastic shift to actin depolymerization is required to enable the following step of wrapping and expansion of myelin membranes. The molecular mechanisms triggering this switch, essential for proper myelination, have yet to be elucidated. Here, we identify P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. We show that PAK1 accumulates in OLs in a kinase inhibited form, triggering actin disassembly and, consequently, myelin expansion. Remarkably, we identify NF2/Merlin as an endogenous inhibitor of PAK1 by proteomics analysis of its binding partners. We found that Nf2 knockdown in OLs results in PAK1 activation and impairs myelin formation, and that pharmacological inhibition of PAK1 in Nf2-knockdown OLs rescues these defects. Moreover, we demonstrate that modulating PAK1 activity in OLs controls myelin expansion and provide compelling evidence indicating that specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo. Overall, our data indicate that PAK1-NF2/Merlin duo plays a key role in actin cytoskeleton remodeling in OLs, required for proper myelin formation. These findings have broad mechanistic and therapeutic implications for demyelinating diseases and neurodevelopmental disorders. SignificanceRemodeling actin cytoskeleton plays a crucial role in myelin formation by oligodendrocytes (OLs). Recent studies have shown that expansion and wrapping of myelin membranes around axons depends on actin depolymerization. However, the molecular mechanisms triggering this key step in myelination are not fully elucidated. Using genetic and pharmacological tools as well as proteomics analyses, we found that PAK1 (P21 Activated Kinase 1) kinase activity is maintained inhibited by NF2/Merlin in OLs to allow actin depolymerization and, consequently, myelin membrane expansion. Pak1 loss-of-function in OLs leads to an increase in myelin thickness in the white matter of adult mice, confirming the role of PAK1 inactivation in myelin membrane expansion.

Astrocytes are the most common glial cell type in the brain, yet, it is unclear how their activation affects the transcriptome of neighboring cells. Engineered G protein-coupled receptors (GPCRs) called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) enable selective activation of specific cell types, such as astrocytes. Here, we combine activation of astrocytes in the hippocampus and cortex of healthy mice with single-cell RNA sequencing. Our data show that long-term activation of astrocytes dramatically alters the transcriptome of astrocytes and microglia. Genes that were differentially expressed in Gq-DREADD-activated astrocytes are involved in neurogenesis and low-density lipoprotein particle biology, while those in the microglia were involved in lipoprotein handling, purinergic receptor activity, and immune cell migration and chemotaxis. Furthermore, network analysis showed that Gq-DREADD-mediated activation in astrocytes resulted in an upregulation of genes involved in the GPCR signaling pathways and calcium ion homeostasis, confirming astrocyte activation. This dataset will serve as a resource for the broader neuroscience community, and our findings highlight the importance of studying transcriptomic alterations in microglia after astrocyte activation in vivo.

The injury responses of tissue-resident macrophages in the CNS (microglia) and blood-derived macrophages (BDMs) play key roles in successful regeneration of the zebrafish spinal cord, but the origins and dynamic behaviours of these immune cells are not well characterized. Here, we find that microglia, labelled by the p2ry12:GFP reporter gene, migrate long-distance through neural tissue from the brain to the spinal lesion site, while BDMs, labelled by the mpeg1:mCherry reporter gene, migrate mainly from the caudal hematopoietic tissue to the lesion and back. Half of p2ry12:GFP-positive microglia co-express mpeg1:mCherry, while mpeg1:mCherry-positive BDMs are mostly p2ry12:GFP-negative. However, a BDM sub-population starts to express p2ry12:GFP in the lesion. This indicates heterogeneous and dynamic gene expression in macrophage populations. Gene expression profiling reveals several microglia-like and BDM-like clusters in the lesion with gene expression profiles related to proliferation, phagocytosis, pro- and anti-inflammatory phenotypes and distinct expression of regeneration-relevant genes. The most abundant cell cluster are densely-packed microglia-like cells in the lesion core, which express the novel marker g0s2, as well as phagocytosis-related genes. Hence, regenerative success of the zebrafish spinal cord is linked to a heterogeneous and dynamic response of microglia and BDM subpopulations.

The cerebellum is involved in the coordination of movement. Its cellular composition is dominated by GABAergic neuronal types, and glial cells are known to express functional receptors. GABAergic signaling regulates cell proliferation, differentiation, and migration during neurodevelopment. However, little is known about the functional expression of GABA receptors in the cerebellar white matter (WM). Thus, the aim of this study was to test whether glial cells express functional GABA receptors during postnatal development (P7-P9) of cerebellar WM. Immunofluorescence studies showed that half of the astrocytes express GAD67, suggesting that GABA is synthetized by glial cells. Calcium imaging in cerebellar slices revealed that GABA and the GABAA agonist muscimol evoked calcium transients in sulforhodamine B (SRB) negative cells, whereas the GABAB agonist baclofen failed to evoke responses in cerebellar WM. Whole-cell patch-clamp recordings of GFAP+ cells showed dye coupling and a passive current-voltage relation typical of astrocytes. Surprisingly, these cells did not respond to muscimol. Two additional populations were identified as GFAP- cells. The first population showed dye coupling, slow decaying inward and outward currents with no voltage dependence and did not respond to GABAA agonists. The second population showed an outward-rectifying current-voltage relationship and responded to muscimol, but dye coupling was absent. These cells received synaptic input and were NG2+, but evoked calcium waves failed to modulate the frequency of sPSCs or signal directly to NG2 glia. We conclude that GABAA receptor-mediated signaling is selective for NG2 glia in the WM of the cerebellum.

Glial cell adhesion molecule (GlialCAM) is an astrocyte- and oligodendrocyte-expressed transmembrane protein with two extracellular IgG-like domains and a cytoplasmic tail with putative signaling functions. While numerous studies have explored functions for the GlialCAM IgG-like domains in brain development and physiology, functions for its cytoplasmic signaling tail remain largely unknown. Therefore, we developed a mutant mouse model that expresses a truncated GlialCAM construct (GlialCAM {Delta}CT) that contains intact extracellular and transmembrane domains but lacks the cytoplasmic tail. Deletion of the GlialCAM cytoplasmic domain in glial cells of the brain results in vacuolization within white matter regions without disrupting neurovascular barrier integrity. Consequently, mutant mice exhibited selective deficits in motor coordination, muscular strength, and memory. Single cell transcriptome sequencing identifies GlialCAM-dependent defects in ECM remodeling pathways in white matter tracts. In situ spatial profiling revealed robust activation of astrocytes and microglia in the mutant brain. Proteomic analysis identified GlialCAM cytoplasmic tail interactors with links to MAPK signaling and cytoskeletal regulatory networks. These data reveal important functions for the GlialCAM cytoplasmic tail in homeostasis of white matter tracts in the adult murine brain. The GlialCAM {Delta}CT model may also be useful for studying the pathogenesis and possible treatment of neurological diseases linked to white matter degeneration.

Astrocytes are an active element of brain signalling, capable of release of small molecule gliotransmitters by vesicular and channel-mediated mechanisms. However, specific physiological roles of astroglial exocytosis of glutamate and D-Serine remain controversial. Our data demonstrate that cortical astrocytes can release glutamate and D-Serine by combination of SNARE-dependent exocytosis and non-vesicular mechanisms dependent on TREK-1 and Best1 channels. Astrocyte-derived glutamate and D-serine elicited complex multicomponent phasic response in neocortical pyramidal neurons, which is mediated by extra-synaptic GluN2B receptors. Impairment of either pathway of gliotransmission (in the TREK1 KO, Best-1 KO or dnSNARE mice) strongly affected the NMDAR-dependent long-term synaptic plasticity in the hippocampus and neocortex. Moreover, impairment of astroglial exocytosis in dnSNARE mice led to the deficit in the spatial working memory which was rescued by environmental enrichment. We conclude that synergism between vesicular and non-vesicular gliotransmission is crucial for astrocyte-neuron communication and astroglia-driven regulation of synaptic plasticity and memory. HighlightsO_LIAstrocytes in situ release glutamate via exocytosis and channel-mediated release. C_LIO_LIAstroglia-derived glutamate and D-Serine activate phasic NMDAR currents in neurons C_LIO_LIBoth vesicular and non-vesicular gliotransmission are required for synaptic plasticity C_LIO_LIImpaired exocytosis of gliotransmitters causes deficit in working memory C_LI

Microglia and astrocytes undertake numerous essential roles in nervous systems but we know little of their anatomical distribution within numerous nuclei. In the principal nuclei of the mammalian auditory midbrain, the inferior colliculi (IC), the cellular density and relative distribution of glutamate synthetase (GS) expressing astrocytes and ionized calcium-binding adapter molecule 1 (Iba1) expressing microglia is unknown. To address this, the IC of young adult, male Wistar rats were immunohistochemically labelled for GS and Iba1, using chromogenic methods. Sub-regions of imaged IC sections were demarked and soma density of both cell types determined. GS labelled somata were twice more densely packed as Iba1 labelled somata throughout IC parenchyma and peri-vascular regions. Furthermore, GS labelled somata density was significantly lower in dorsal cortex than external cortex or central nucleus. Iba1 labelled somata density exhibited the opposite trend, revealing an inverse density of these glial cell types between IC sub-regions. GS labelled neuropil was strongest in the cortices with and a gradual transition of lighter labelling towards central nucleus. These data provide the first detailed descriptions of GS labelling in IC and demonstrate sub-regional differences in IC glial cell density. Taken together, these findings suggest neurochemical specialization of glia in IC sub-regions, likely related to local physiological and metabolic demands, with implications for IC function.

Astrocytes and microglia maintain brain homeostasis and respond to inflammation through functions coordinated by molecular mediators they produce. Growing evidence shows that cellular metabolism is key to how these cells adapt to challenges. However, little is known about what drives glial metabolic reprogramming or whether molecules involved in astrocyte- microglia crosstalk also regulate this process. Here, we explored this question focusing on Secreted Frizzled-Related Protein 1 (SFRP1). SFRP1 is an astrocyte-derived factor induced by inflammatory cues and overexpressed in neurodegeneration, which fosters microglial response to inflammation through NF-{kappa}B/HIF-dependent programs. We combined mitochondrial morphometry (MitoTracker Red and MiNA analysis) with Seahorse extracellular flux assays (Mito Stress Test) to determine whether SFRP1 modulates glial bioenergetics in primary cultures of astrocytes and microglia from wild-type and Sfrp1-/- mice. We report that SFRP1 acts as a driver of astrocytic metabolic activation, preferentially enhancing glycolysis over mitochondrial respiration. This effect is most pronounced during inflammation, when oxidative phosphorylation is restricted and SFRP1 enhances glycolytic flexibility to sustain energy demands. By contrast, microglia showed the expected LPS-driven glycolytic shift with minimal dependence on SFRP1 under monoculture conditions. These findings position SFRP1 as a candidate regulator of astrocyte-centered metabolic tuning during neuroinflammation, with implications for disorders such as Alzheimers disease, in which SFRP1 is elevated.

Myelination of CNS axons requires oligodendrocytes to undergo extensive morphological changes by producing large amounts of myelin membrane with defined protein composition and structure. The formation of myelin sheaths thus involves efficient trafficking and sorting of future myelin constituents via vesicles that fuse with prospective myelin membranes by exocytotic mechanisms. However, the functional relevance of other trafficking steps in oligodendocytes for myelin biogenesis is largely unknown. Here, we followed the hypothesis that developmental myelination involves endocytic mechanisms. In this model, Golgi-derived vesicles fuse with the oligodendroglial plasma membrane, from which myelin constituents are retrieved by endocytosis into endosomal/lysosomal organelles before their final integration into the growing sheath. Considering that adaptor protein complex-2 subunit-{micro} (AP2M) facilitates AP2-dependent endocytosis, we recombined the Ap2m-gene in myelin-forming oligodendrocytes, causing both hypomyelination and specific changes in the myelin proteome. Most strikingly, lysosomal membrane proteins accumulate in the abaxonal (outermost) myelin layer, identifying this membrane as an active site for retrieving constituents from myelin sheaths. These data demonstrate that the AP2 complex serves a critical function in developmental myelination in vivo. Unexpectedly, we also observed pathological myelin outfoldings indicative of focal hypermyelination. Consistent with the hypothesis that this phenotype reflects impaired maintenance rather than biogenesis of myelin sheaths, recombination of the Ap2m-gene in oligodendrocytes of adult mice caused late-onset progressive focal hypermyelination. These results indicate that, in addition to astrocytic and microglial phagocytosis, oligodendrocytes cell-autonomously contribute to maintaining the structure of healthy myelin sheaths via AP2-dependent mechanisms.

Astrocytes play key roles in maintaining brain homeostasis, metabolism, and neurovascular integrity, yet their diversity and age-related modulation remain insufficiently understood, particularly across primate lineages. While rodent studies have generated extensive knowledge, notable species differences highlight the need for comparative analyses in non-human primates. The gray mouse lemur (Microcebus murinus), a small primate widely used in aging research, offers a valuable but underexplored model for studying astroglial aging. In this study, we characterized astrocyte distribution, morphology, and reactivity in 17 mouse lemurs aged 1.0-11.5 years using GFAP and vimentin immunohistochemistry. We identified marked regional and morphological heterogeneity, with dense astrocytic labeling in white matter, hippocampus, and sparse but diverse cortical populations. Distinct astrocyte subtypes--including fibrous, protoplasmic, projection, pial and subpial interlaminar, radial glia-like cells, tanycytes--were documented. Varicosity-bearing processes were common across multiple astroglial subtypes and may indicate altered physiological states. Quantitative analyses revealed pronounced age-related increases in astrocytic reactivity, particularly in white matter and interlaminar astrocytes. Cortical and hippocampal changes were comparatively modest. These findings indicate region-specific astrocytic vulnerability during aging and support the translational value of the mouse lemur for investigating glial aging in primates. Main PointsO_LIThe mouse lemur is the smallest primate on earth with a key role to understand primate brain characteristics. C_LIO_LIWe characterized seven different astrocyte subtypes: from fibrous to primate-specific astrocyte as interlaminar astrocytes in different brain regions of this primate. C_LIO_LIVaricosities were reported in different astrocyte subtypes found close to brain borders. C_LIO_LIMain age-related changes concerned fibrous astrocytes in the white matter and interlaminar astrocytes at the cortical border. C_LI

Myelin sheaths comprise compacted layers of oligodendroglial membrane wrapped spirally around axons. Each sheath, if imagined unwrapped, has a cytoplasm-filled space at its perimeter, linking it to the oligodendrocyte soma via a short process. By electron microscopy (EM), this space, which we term the myelinic channel system contains microtubules and membranous organelles, but whether these are remnants of development or serve a function is unknown. Performing live imaging of myelinating oligodendrocytes expressing fluorescent reporters, we found that the myelinic channel system serves microtubule-dependent organelle transport. Further, the intra-myelinic movement of peroxisomes was modulated by neuronal electrical activity in these mixed neural cell cultures. Loss of oligodendroglial Kif21b or CNP in vivo led to apparent stasis of myelin organelles and secondary axon pathology. This suggests that oligodendrocytes require motor transport in myelin to maintain axonal integrity.

Microglia have classically been viewed as the endogenous phagocytes of the brain, however, emerging evidence suggests roles for microglia in the healthy, mature nervous system. We know little of the contribution microglia make to ongoing processing in sensory systems. To explore Iba1+ microglial diversity, we employed the inferior colliculi (IC) as model nuclei, as they are characterized by sub-regions specialized for differing aspects of auditory processing. We conducted fluorescent multi-channel immunohistochemistry and confocal microscopy in guinea pigs of both sexes and discovered that the density and morphology of Iba1+ labelling varied between parenchymal sub-regions of IC, while GFAP+ labelling of astrocytes was confined to the glia limitans externa and peri-vascular regions. The density of Iba1+ microglia somata was similar across sub-regions, however a greater amount of labelling was found in dorsal cortex than ventral central nucleus or lateral cortex. To further understand these differences between sub-regions in IC, Sholl and skeleton analyses of individual microglia revealed a greater number of branching ramifications in dorsal cortex. We also quantified abutments of Iba1+ microglial processes onto GAD67+ (putative GABAergic) somata. Cluster analyses revealed two novel sub-types of GAD67+ neuron, which can be distinguished solely based on the quantity of axo-somatic Iba1+ abutments they receive. These data demonstrate Iba1+ microglia exhibit different morphologies and interactions with GAD67+ neurons in distinct sub-regions of the mature, healthy IC. Taken together, these findings suggest significant heterogeneity amongst microglia in the auditory system, possibly related to the ongoing functional demands of their niche.