Developmental origin of oligodendrocytes determines their function in the adult brain.

In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function.

Transient Receptor Potential (TRP) Channels in Cochlear Function: Looking Beyond Mechanotransduction.

Transient receptor potential (TRP) channels play key roles in sensory biology as transducers of various stimuli. Although these ion channels are expressed in the cochlea, their functions remain poorly understood. Recent studies by Vélez-Ortega and colleagues indicate that their expression by non-sensory supporting cells helps limit damage from acoustic trauma.

Priming central sound processing circuits through induction of spontaneous activity in the cochlea before hearing onset.

Sensory systems experience a period of intrinsically generated neural activity before maturation is complete and sensory transduction occurs. Here we review evidence describing the mechanisms and functions of this 'spontaneous' activity in the auditory system. Both ex vivo and in vivo studies indicate that this correlated activity is initiated by non-sensory supporting cells within the developing cochlea, which induce depolarization and burst firing of groups of nearby hair cells in the sensory epithelium, activity that is conveyed to auditory neurons that will later process similar sound features. This stereotyped neural burst firing promotes cellular maturation, synaptic refinement, acoustic sensitivity, and establishment of sound-responsive domains in the brain. While sensitive to perturbation, the developing auditory system exhibits remarkable homeostatic mechanisms to preserve periodic burst firing in deaf mice. Preservation of this early spontaneous activity in the context of deafness may enhance the efficacy of later interventions to restore hearing.

Multicore fiber optic imaging reveals that astrocyte calcium activity in the mouse cerebral cortex is modulated by internal motivational state.

Astrocytes are a direct target of neuromodulators and can influence neuronal activity on broad spatial and temporal scales in response to a rise in cytosolic calcium. However, our knowledge about how astrocytes are recruited during different animal behaviors remains limited. To measure astrocyte activity calcium in vivo during normative behaviors, we utilize a high-resolution, long working distance multicore fiber optic imaging system that allows visualization of individual astrocyte calcium transients in the cerebral cortex of freely moving mice. We define the spatiotemporal dynamics of astrocyte calcium changes during diverse behaviors, ranging from sleep-wake cycles to the exploration of novel objects, showing that their activity is more variable and less synchronous than apparent in head-immobilized imaging conditions. In accordance with their molecular diversity, individual astrocytes often exhibit distinct thresholds and activity patterns during explorative behaviors, allowing temporal encoding across the astrocyte network. Astrocyte calcium events were induced by noradrenergic and cholinergic systems and modulated by internal state. The distinct activity patterns exhibited by astrocytes provides a means to vary their neuromodulatory influence in different behavioral contexts and internal states.

Conservation of neuron-astrocyte coordinated activity among sensory processing centers of the developing brain.

Afferent neurons in developing sensory organs exhibit a prolonged period of burst firing prior to the onset of sensory experience. This intrinsically generated activity propagates from the periphery through central processing centers to promote the survival and physiological maturation of neurons and refine their synaptic connectivity. Recent studies in the auditory system indicate that these bursts of action potentials also trigger metabotropic glutamate receptor-mediated calcium increases within astrocytes that are spatially and temporally correlated with neuronal events; however, it is not known if this phenomenon occurs in other sensory modalities. Here we show using in vivo simultaneous imaging of neuronal and astrocyte calcium activity in awake mouse pups that waves of retinal ganglion cell activity induce spatially and temporally correlated waves of astrocyte activity in the superior colliculus that depend on metabotropic glutamate receptors mGluR5 and mGluR3. Astrocyte calcium transients reliably occurred with each neuronal wave, but peaked more than one second after neuronal events. Despite differences in the temporal features of spontaneous activity in auditory and visual processing regions, individual astrocytes exhibited similar overall calcium activity patterns, providing a conserved mechanism to synchronize neuronal and astrocyte maturation within discrete sensory domains.

Oligodendrocyte-axon metabolic coupling is mediated by extracellular K+ and maintains axonal health.

The integrity of myelinated axons relies on homeostatic support from oligodendrocytes (OLs). To determine how OLs detect axonal spiking and how rapid axon-OL metabolic coupling is regulated in the white matter, we studied activity-dependent calcium (Ca2+) and metabolite fluxes in the mouse optic nerve. We show that fast axonal spiking triggers Ca2+ signaling and glycolysis in OLs. OLs detect axonal activity through increases in extracellular potassium (K+) concentrations and activation of Kir4.1 channels, thereby regulating metabolite supply to axons. Both pharmacological inhibition and OL-specific inactivation of Kir4.1 reduce the activity-induced axonal lactate surge. Mice lacking oligodendroglial Kir4.1 exhibit lower resting lactate levels and altered glucose metabolism in axons. These early deficits in axonal energy metabolism are associated with late-onset axonopathy. Our findings reveal that OLs detect fast axonal spiking through K+ signaling, making acute metabolic coupling possible and adjusting the axon-OL metabolic unit to promote axonal health.

Norepinephrine modulates calcium dynamics in cortical oligodendrocyte precursor cells promoting proliferation during arousal in mice.

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated from oligodendrocyte precursor cells (OPCs) that express neurotransmitter receptors. However, the mechanisms that affect OPC activity in vivo and the physiological roles of neurotransmitter signaling in OPCs are unclear. In this study, we generated a transgenic mouse line that expresses membrane-anchored GCaMP6s in OPCs and used longitudinal two-photon microscopy to monitor OPC calcium (Ca2+) dynamics in the cerebral cortex. OPCs exhibit focal and transient Ca2+ increases within their processes that are enhanced during locomotion-induced increases in arousal. The Ca2+ transients occur independently of excitatory neuron activity, rapidly decline when OPCs differentiate and are inhibited by anesthesia, sedative agents or noradrenergic receptor antagonists. Conditional knockout of α1A adrenergic receptors in OPCs suppresses spontaneous and locomotion-induced Ca2+ increases and reduces OPC proliferation. Our results demonstrate that OPCs are directly modulated by norepinephrine in vivo to enhance Ca2+ dynamics and promote population homeostasis.

Loss of Astrocytic µ Opioid Receptors Exacerbates Aversion Associated with Morphine Withdrawal in Mice: Role of Mitochondrial Respiration.

Astrocytes express mu/µ opioid receptors, but the function of these receptors remains poorly understood. We evaluated the effects of astrocyte-restricted knockout of µ opioid receptors on reward- and aversion-associated behaviors in mice chronically exposed to morphine. Specifically, one of the floxed alleles of the Oprm1 gene encoding µ opioid receptor 1 was selectively deleted from brain astrocytes in Oprm1 inducible conditional knockout (icKO) mice. These mice did not exhibit changes in locomotor activity, anxiety, or novel object recognition, or in their responses to the acute analgesic effects of morphine. Oprm1 icKO mice displayed increased locomotor activity in response to acute morphine administration but unaltered locomotor sensitization. Oprm1 icKO mice showed normal morphine-induced conditioned place preference but exhibited stronger conditioned place aversion associated with naloxone-precipitated morphine withdrawal. Notably, elevated conditioned place aversion lasted up to 6 weeks in Oprm1 icKO mice. Astrocytes isolated from the brains of Oprm1 icKO mice had unchanged levels of glycolysis but had elevated oxidative phosphorylation. The basal augmentation of oxidative phosphorylation in Oprm1 icKO mice was further exacerbated by naloxone-precipitated withdrawal from morphine and, similar to that for conditioned place aversion, was still present 6 weeks later. Our findings suggest that µ opioid receptors in astrocytes are linked to oxidative phosphorylation and they contribute to long-term changes associated with opioid withdrawal.

Preservation of developmental spontaneous activity enables early auditory system maturation in deaf mice.

Intrinsically generated neural activity propagates through the developing auditory system to promote maturation and refinement of sound processing circuits prior to hearing onset. This early patterned activity is induced by non-sensory supporting cells in the organ of Corti, which are highly interconnected through gap junctions containing connexin 26 (Gjb2). Although loss of function mutations in Gjb2 impair cochlear development and are the most common cause of congenital deafness, it is not known if these variants disrupt spontaneous activity and the developmental trajectory of sound processing circuits in the brain. Here, we show in a new mouse model of Gjb2-mediated congenital deafness that cochlear supporting cells adjacent to inner hair cells (IHCs) unexpectedly retain intercellular coupling and the capacity to generate spontaneous activity, exhibiting only modest deficits prior to hearing onset. Supporting cells lacking Gjb2 elicited coordinated activation of IHCs, leading to coincident bursts of activity in central auditory neurons that will later process similar frequencies of sound. Despite alterations in the structure of the sensory epithelium, hair cells within the cochlea of Gjb2-deficient mice were intact and central auditory neurons could be activated within appropriate tonotopic domains by loud sounds at hearing onset, indicating that early maturation and refinement of auditory circuits was preserved. Only after cessation of spontaneous activity following hearing onset did progressive hair cell degeneration and enhanced auditory neuron excitability manifest. This preservation of cochlear spontaneous neural activity in the absence of connexin 26 may increase the effectiveness of early therapeutic interventions to restore hearing.

Multicore fiber optic imaging reveals that astrocyte calcium activity in the cerebral cortex is modulated by internal motivational state.

Astrocytes are a direct target of neuromodulators and can influence neuronal activity on broad spatial and temporal scales through their close proximity to synapses. However, our knowledge about how astrocytes are functionally recruited during different animal behaviors and their diverse effects on the CNS remains limited. To enable measurement of astrocyte activity patterns in vivo during normative behaviors, we developed a high-resolution, long working distance, multi-core fiber optic imaging platform that allows visualization of cortical astrocyte calcium transients through a cranial window in freely moving mice. Using this platform, we defined the spatiotemporal dynamics of astrocytes during diverse behaviors, ranging from circadian fluctuations to novelty exploration, showing that astrocyte activity patterns are more variable and less synchronous than apparent in head-immobilized imaging conditions. Although the activity of astrocytes in visual cortex was highly synchronized during quiescence to arousal transitions, individual astrocytes often exhibited distinct thresholds and activity patterns during explorative behaviors, in accordance with their molecular diversity, allowing temporal sequencing across the astrocyte network. Imaging astrocyte activity during self-initiated behaviors revealed that noradrenergic and cholinergic systems act synergistically to recruit astrocytes during state transitions associated with arousal and attention, which was profoundly modulated by internal state. The distinct activity patterns exhibited by astrocytes in the cerebral cortex may provide a means to vary their neuromodulatory influence in response to different behaviors and internal states.

Cross-modality supervised image restoration enables nanoscale tracking of synaptic plasticity in living mice.

Learning is thought to involve changes in glutamate receptors at synapses, submicron structures that mediate communication between neurons in the central nervous system. Due to their small size and high density, synapses are difficult to resolve in vivo, limiting our ability to directly relate receptor dynamics to animal behavior. Here we developed a combination of computational and biological methods to overcome these challenges. First, we trained a deep-learning image-restoration algorithm that combines the advantages of ex vivo super-resolution and in vivo imaging modalities to overcome limitations specific to each optical system. When applied to in vivo images from transgenic mice expressing fluorescently labeled glutamate receptors, this restoration algorithm super-resolved synapses, enabling the tracking of behavior-associated synaptic plasticity with high spatial resolution. This method demonstrates the capabilities of image enhancement to learn from ex vivo data and imaging techniques to improve in vivo imaging resolution.

MHC class I and MHC class II reporter mice enable analysis of immune oligodendroglia in mouse models of multiple sclerosis.

Oligodendrocytes and their progenitors upregulate MHC pathways in response to inflammation, but the frequency of this phenotypic change is unknown and the features of these immune oligodendroglia are poorly defined. We generated MHC class I and II transgenic reporter mice to define their dynamics in response to inflammatory demyelination, providing a means to monitor MHC activation in diverse cell types in living mice and define their roles in aging, injury, and disease.

Nerve cells in the brain and spinal cord are surrounded by a layer of insulation called myelin that allows cells to transmit messages to each other more quickly and efficiently. This protective sheath is produced by cells called oligodendrocytes which together with their immature counterparts can also repair damage caused to myelin. In the inflammatory disease multiple sclerosis (MS), this insulation is disrupted and oligodendroglia fail to repair breaks in the myelin sheath, leaving nerves vulnerable to further damage. Recently it was discovered that mature and immature oligodendrocytes (which are collectively known as oligodendroglia) sometimes express proteins normally restricted to the immune system called major histocompatibility complexes (or MHCs for short). Researchers believe that MHC expression may allow oligodendroglia to interact with immune cells, potentially leading to the removal of oligodendroglia by the immune system as well as inflammation that exacerbates damage to nerves and hinders myelin repair. Knowing when oligodendroglia start producing MHCs and where these MHC-expressing cells are located is therefore important for understanding their role in MS. However, it is difficult to identify the location of MHC-expressing oligodendroglia using methods that are currently available. To address this, Harrington, Catenacci et al. created a genetically engineered mouse model in which the MHC-expressing oligodendroglia also generated a red fluorescent protein that could be detected under a microscope. This revealed that only a small number of oligodendroglia in the nervous system had MHCs, but these cells were located in areas of the brain and spinal cord with the highest inflammatory activity. Further microscopy studies in mice that developed MS-like symptoms revealed that MHC production in oligodendroglia increased compared with healthy animals, and that the proportion of oligodendroglia that produced MHC was highest in mice with the most severe symptoms. MHC-expressing oligodendroglia also congregated in the most damaged areas of the brain and spinal cord. These results suggest that MHC expression may contribute to inflammation and impact the function of oligodendroglia that have these molecules. In the future, Harrington et al. hope that their new mouse model will help researchers study the role of MHC expression in different diseases, and in the case of MS, aid the development of new treatments.

Oligodendrocyte precursor cells ingest axons in the mouse neocortex.

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

Developmental spontaneous activity promotes formation of sensory domains, frequency tuning and proper gain in central auditory circuits.

Neurons that process sensory information exhibit bursts of electrical activity during development, providing early training to circuits that will later encode similar features of the external world. In the mammalian auditory system, this intrinsically generated activity emerges from the cochlea prior to hearing onset, but its role in maturation of auditory circuitry remains poorly understood. We show that selective suppression of cochlear supporting cell spontaneous activity disrupts patterned burst firing of central auditory neurons without affecting cell survival or acoustic thresholds. However, neurons in the inferior colliculus of these mice exhibit enhanced acoustic sensitivity and broader frequency tuning, resulting in wider isofrequency laminae. Despite this enhanced neural responsiveness, total tone-responsive regions in the auditory cortex are substantially smaller. Thus, disruption of pre-hearing cochlear activity causes profound changes in neural encoding of sound, with important implications for restoration of hearing in individuals who experience reduced activity during this critical developmental period.

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.

Olig2 Ablation in Immature Oligodendrocytes Does Not Enhance CNS Myelination and Remyelination.

The oligodendrocyte (OL) lineage transcription factor Olig2 is expressed throughout oligodendroglial development and is essential for oligodendroglial progenitor specification and differentiation. It was previously reported that deletion of Olig2 enhanced the maturation and myelination of immature OLs and accelerated the remyelination process. However, by analyzing multiple Olig2 conditional KO mouse lines (male and female), we conclude that Olig2 has the opposite effect and is required for OL maturation and remyelination. We found that deletion of Olig2 in immature OLs driven by an immature OL-expressing Plp1 promoter resulted in defects in OL maturation and myelination, and did not enhance remyelination after demyelination. Similarly, Olig2 deletion during premyelinating stages in immature OLs using Mobp or Mog promoter-driven Cre lines also did not enhance OL maturation in the CNS. Further, we found that Olig2 was not required for myelin maintenance in mature OLs but was critical for remyelination after lysolecithin-induced demyelinating injury. Analysis of genomic occupancy in immature and mature OLs revealed that Olig2 targets the enhancers of key myelination-related genes for OL maturation from immature OLs. Together, by leveraging multiple immature OL-expressing Cre lines, these studies indicate that Olig2 is essential for differentiation and myelination of immature OLs and myelin repair. Our findings raise fundamental questions about the previously proposed role of Olig2 in opposing OL myelination and highlight the importance of using Cre-dependent reporter(s) for lineage tracing in studying cell state progression.SIGNIFICANCE STATEMENT Identification of the regulators that promote oligodendrocyte (OL) myelination and remyelination is important for promoting myelin repair in devastating demyelinating diseases. Olig2 is expressed throughout OL lineage development. Ablation of Olig2 was reported to induce maturation, myelination, and remyelination from immature OLs. However, lineage-mapping analysis of Olig2-ablated cells was not conducted. Here, by leveraging multiple immature OL-expressing Cre lines, we observed no evidence that Olig2 ablation promotes maturation or remyelination of immature OLs. Instead, we find that Olig2 is required for immature OL maturation, myelination, and myelin repair. These data raise fundamental questions about the proposed inhibitory role of Olig2 against OL maturation and remyelination. Our findings highlight the importance of validating genetic manipulation with cell lineage tracing in studying myelination.

Deficient mitochondrial respiration in astrocytes impairs trace fear conditioning and increases naloxone-precipitated aversion in morphine-dependent mice.

Mitochondria are abundant in the fine processes of astrocytes, however, potential roles for astrocyte mitochondria remain poorly understood. In the present study, we performed a systematic examination of the effects of abnormal oxidative phosphorylation in astrocytes on several mouse behaviors. Impaired astrocyte oxidative phosphorylation was produced by astrocyte-specific deletion of the nuclear mitochondrial gene, Cox10, that encodes an accessory protein of complex IV, the protoheme:heme-O-farnesyl transferase. As expected, conditional deletion of the Cox10 gene in mice (cKO mice) significantly reduced expression of COX10 and Cytochrome c oxidase subunit I (MTCO1) of Complex IV, resulting in decreased oxidative phosphorylation without significantly affecting glycolysis. No effects of the deletion were observed on locomotor activity, anxiety-like behavior, nociception, or spontaneous alternation. Cox10 cKO female mice exhibited mildly impaired novel object recognition, while Cox10 cKO male mice were moderately deficient in trace fear conditioning. No group-related changes were observed in conditional place preference (CPP) that assessed effects of morphine on reward. In contrast to CPP, Cox10 cKO mice demonstrated significantly increased aversive behaviors produced by naloxone-precipitated withdrawal following chronic exposure to morphine, that is, jumping and avoidance behavior as assessed by conditional place aversion (CPA). Our study suggests that astrocyte oxidative phosphorylation may contribute to behaviors associated with greater cognitive load and/or aversive and stressful conditions.

Deep-learning two-photon fiberscopy for video-rate brain imaging in freely-behaving mice.

Scanning two-photon (2P) fiberscopes (also termed endomicroscopes) have the potential to transform our understanding of how discrete neural activity patterns result in distinct behaviors, as they are capable of high resolution, sub cellular imaging yet small and light enough to allow free movement of mice. However, their acquisition speed is currently suboptimal, due to opto-mechanical size and weight constraints. Here we demonstrate significant advances in 2P fiberscopy that allow high resolution imaging at high speeds (26 fps) in freely-behaving mice. A high-speed scanner and a down-sampling scheme are developed to boost imaging speed, and a deep learning (DL) algorithm is introduced to recover image quality. For the DL algorithm, a two-stage learning transfer strategy is established to generate proper training datasets for enhancing the quality of in vivo images. Implementation enables video-rate imaging at ~26 fps, representing 10-fold improvement in imaging speed over the previous 2P fiberscopy technology while maintaining a high signal-to-noise ratio and imaging resolution. This DL-assisted 2P fiberscope is capable of imaging the arousal-induced activity changes in populations of layer2/3 pyramidal neurons in the primary motor cortex of freely-behaving mice, providing opportunities to define the neural basis of behavior.