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Myelin is essential for rapid nerve signaling and is increasingly found to play important roles in learning and in diverse diseases of the CNS. Morphological parameters of myelin such as sheath length are thought to precisely tune conduction velocity, but the mechanisms controlling sheath morphology are poorly understood. Local calcium signaling has been observed in nascent myelin sheaths and can be modulated by neuronal activity. However, the role of calcium signaling in sheath formation remains incompletely understood. Here, we use genetic tools to attenuate oligodendrocyte calcium signaling during myelination in the developing mouse CNS. Surprisingly, genetic calcium attenuation does not grossly affect the number of myelinated axons or myelin thickness. Instead, calcium attenuation causes myelination defects resulting in shorter, dysmorphic sheaths. Mechanistically, calcium attenuation reduces actin filaments in oligodendrocytes, and an intact actin cytoskeleton is necessary and sufficient to achieve accurate myelin morphology. Together, our work reveals a cellular mechanism required for accurate CNS myelin formation and may provide mechanistic insight into how oligodendrocytes respond to neuronal activity to sculpt and refine myelin sheaths.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.

Astrocytes respond to neurotransmitters and neuromodulators using G-protein-coupled receptors (GPCRs) to mediate physiological responses. Despite their importance, there has been no method to genetically, specifically, and effectively attenuate astrocyte Gq GPCR pathways to explore consequences of this prevalent signaling mechanism in vivo. We report a 122-residue inhibitory peptide from β-adrenergic receptor kinase 1 (iβARK; and inactive D110A control) to attenuate astrocyte Gq GPCR signaling. iβARK significantly attenuated Gq GPCR Ca2+ signaling in brain slices and, in vivo, altered behavioral responses, spared other GPCR responses, and did not alter astrocyte spontaneous Ca2+ signals, morphology, electrophysiological properties, or gene expression in the striatum. Furthermore, brain-wide attenuation of astrocyte Gq GPCR signaling with iβARK using PHP.eB adeno-associated viruses (AAVs), when combined with c-Fos mapping, suggested nuclei-specific contributions to behavioral adaptation and spatial memory. iβARK extends the toolkit needed to explore functions of astrocyte Gq GPCR signaling within neural circuits in vivo.

Astrocytes exist throughout the CNS and affect neural circuits and behavior through intracellular Ca2+ signaling. Studying the function(s) of astrocyte Ca2+ signaling has proven difficult because of the paucity of tools to achieve selective attenuation. Based on recent studies, we generated and used male and female knock-in mice for Cre-dependent expression of mCherry-tagged hPMCA2w/b to attenuate astrocyte Ca2+ signaling in genetically defined cells in vivo (CalExflox mice for Calcium Extrusion). We characterized CalExflox mice following local AAV-Cre microinjections into the striatum and found reduced astrocyte Ca2+ signaling (∼90%) accompanied with repetitive self-grooming behavior. We also crossed CalExflox mice to astrocyte-specific Aldh1l1-Cre/ERT2 mice to achieve inducible global CNS-wide Ca2+ signaling attenuation. Within 6 d of induction in the bigenic mice, we observed significantly altered ambulation in the open field, disrupted motor coordination and gait, and premature lethality. Furthermore, with histologic, imaging, and transcriptomic analyses, we identified cellular and molecular alterations in the cerebellum following mCherry-tagged hPMCA2w/b expression. Our data show that expression of mCherry-tagged hPMCA2w/b with CalExflox mice throughout the CNS resulted in substantial attenuation of astrocyte Ca2+ signaling and significant behavioral alterations in adult mice. We interpreted these findings candidly in relation to the ability of CalEx to attenuate astrocyte Ca2+ signaling, with regards to additional mechanistic interpretations of the data, and their relation to past studies that reduced astrocyte Ca2+ signaling throughout the CNS. The data and resources provide complementary ways to interrogate the function(s) of astrocytes in multiple experimental scenarios.SIGNIFICANCE STATEMENT Astrocytes represent a significant fraction of all brain cells and tile the entire central nervous system. Unlike neurons, astrocytes lack propagated electrical signals. Instead, astrocytes are proposed to use diverse and dynamic intracellular Ca2+ signals to communicate with other cells. An open question concerns if and how astrocyte Ca2+ signaling regulates behavior in adult mice. We approached this problem by generating a new transgenic mouse line to achieve inducible astrocyte Ca2+ signaling attenuation in vivo We report our data with this mouse line and we interpret the findings candidly in relation to past studies and within the framework of different mechanistic interpretations.

Astrocytes are a large and diverse population of morphologically complex cells that exist throughout nervous systems of multiple species. Progress over the last two decades has shown that astrocytes mediate developmental, physiological, and pathological processes. However, a long-standing open question is how astrocytes regulate neural circuits in ways that are behaviorally consequential. In this regard, we summarize recent studies using Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, and Mus musculus. The data reveal diverse astrocyte mechanisms operating in seconds or much longer timescales within neural circuits and shaping multiple behavioral outputs. We also refer to human diseases that have a known primary astrocytic basis. We suggest that including astrocytes in mechanistic, theoretical, and computational studies of neural circuits provides new perspectives to understand behavior, its regulation, and its disease-related manifestations.

Astrocytes tile the central nervous system and are widely implicated in brain diseases, but the molecular mechanisms by which astrocytes contribute to brain disorders remain incompletely explored. By performing astrocyte gene expression analyses following 14 experimental perturbations of relevance to the striatum, we discovered that striatal astrocytes mount context-specific molecular responses at the level of genes, pathways, and upstream regulators. Through data mining, we also identified astrocyte pathways in Huntington's disease (HD) that were reciprocally altered with respect to the activation of striatal astrocyte G protein-coupled receptor (GPCR) signaling. Furthermore, selective striatal astrocyte stimulation of the Gi-GPCR pathway in vivo corrected several HD-associated astrocytic, synaptic, and behavioral phenotypes, with accompanying improvement of HD-associated astrocyte signaling pathways, including those related to synaptogenesis and neuroimmune functions. Overall, our data show that astrocytes are malleable, using context-specific responses that can be dissected molecularly and used for phenotypic benefit in brain disorders.

Astrocytes tile the central nervous system, but their functions in neural microcircuits in vivo and their roles in mammalian behavior remain incompletely defined. We used two-photon laser scanning microscopy, electrophysiology, MINIscopes, RNA-seq, and a genetic approach to explore the effects of reduced striatal astrocyte Ca2+ signaling in vivo. In wild-type mice, reducing striatal astrocyte Ca2+-dependent signaling increased repetitive self-grooming behaviors by altering medium spiny neuron (MSN) activity. The mechanism involved astrocyte-mediated neuromodulation facilitated by ambient GABA and was corrected by blocking astrocyte GABA transporter 3 (GAT-3). Furthermore, in a mouse model of Huntington's disease, dysregulation of GABA and astrocyte Ca2+ signaling accompanied excessive self-grooming, which was relieved by blocking GAT-3. Assessments with RNA-seq revealed astrocyte genes and pathways regulated by Ca2+ signaling in a cell-autonomous and non-cell-autonomous manner, including Rab11a, a regulator of GAT-3 functional expression. Thus, striatal astrocytes contribute to neuromodulation controlling mouse obsessive-compulsive-like behavior.

Astrocytes are ubiquitous in the brain and are widely held to be largely identical. However, this view has not been fully tested, and the possibility that astrocytes are neural circuit specialized remains largely unexplored. Here, we used multiple integrated approaches, including RNA sequencing (RNA-seq), mass spectrometry, electrophysiology, immunohistochemistry, serial block-face-scanning electron microscopy, morphological reconstructions, pharmacogenetics, and diffusible dye, calcium, and glutamate imaging, to directly compare adult striatal and hippocampal astrocytes under identical conditions. We found significant differences in electrophysiological properties, Ca2+ signaling, morphology, and astrocyte-synapse proximity between striatal and hippocampal astrocytes. Unbiased evaluation of actively translated RNA and proteomic data confirmed significant astrocyte diversity between hippocampal and striatal circuits. We thus report core astrocyte properties, reveal evidence for specialized astrocytes within neural circuits, and provide new, integrated database resources and approaches to explore astrocyte diversity and function throughout the adult brain. VIDEO ABSTRACT.

BackgroundFragile X syndrome (FXS) is the most common type of mental retardation attributable to a single-gene mutation. It is caused by FMR1 gene silencing and the consequent loss of its protein product, fragile X mental retardation protein. Fmr1 global knockout (KO) mice recapitulate many behavioral and synaptic phenotypes associated with FXS. Abundant evidence suggests that astrocytes are important contributors to neurological diseases. This study investigates astrocytic contributions to the progression of synaptic abnormalities and learning impairments associated with FXS.MethodsTaking advantage of the Cre-lox system, we generated and characterized mice in which fragile X mental retardation protein is selectively deleted or exclusively expressed in astrocytes. We performed in vivo two-photon imaging to track spine dynamics/morphology along dendrites of neurons in the motor cortex and examined associated behavioral defects.ResultsWe found that adult astrocyte-specific Fmr1 KO mice displayed increased spine density in the motor cortex and impaired motor-skill learning. The learning defect coincided with a lack of enhanced spine dynamics in the motor cortex that normally occurs in response to motor skill acquisition. Although spine density was normal at 1 month of age in astrocyte-specific Fmr1 KO mice, new spines formed at an elevated rate. Furthermore, fragile X mental retardation protein expression in only astrocytes was insufficient to rescue most spine or behavioral defects.ConclusionsOur work suggests a joint astrocytic-neuronal contribution to FXS pathogenesis and reveals that heightened spine formation during adolescence precedes the overabundance of spines and behavioral defects found in adult Fmr1 KO mice.

The mammalian cerebral cortex is typically organized in six layers containing multiple types of neurons, with pyramidal neurons (PNs) being the most abundant. PNs in different cortical layers have distinct morphology, physiology and functional roles in neural circuits. Therefore, their development and synaptic plasticity may also differ. Using in vivo transcranial two-photon microscopy, we followed the structural dynamics of dendritic spines on apical dendrites of layer (L) 2/3 and L5 PNs at different developmental stages. We show that the density and dynamics of spines are significantly higher in L2/3 PNs than L5 PNs in both adolescent (1 month old) and adult (4 months old) mice. While spine density of L5 PNs decreases during adolescent development due to a higher rate of spine elimination than formation, there is no net change in the spine density along apical dendrites of L2/3 PNs over this period. In addition, experiences exert differential impact on the dynamics of apical dendritic spines of PNs resided in different cortical layers. While motor skill learning promotes spine turnover on L5 PNs in the motor cortex, it does not change the spine dynamics on L2/3 PNs. In addition, neonatal sensory deprivation decreases the spine density of both L2/3 and L5 PNs, but leads to opposite changes in spine dynamics among these two populations of neurons in adolescence. In summary, our data reveal distinct dynamics and plasticity of apical dendritic spines on PNs in different layers in the living mouse cortex, which may arise from their distinct functional roles in cortical circuits.

The membrane protein Nogo-A is known as an inhibitor of axonal outgrowth and regeneration in the CNS. However, its physiological functions in the normal adult CNS remain incompletely understood. Here, we investigated the role of Nogo-A in cortical synaptic plasticity and motor learning in the uninjured adult rodent motor cortex. Nogo-A and its receptor NgR1 are present at cortical synapses. Acute treatment of slices with function-blocking antibodies (Abs) against Nogo-A or against NgR1 increased long-term potentiation (LTP) induced by stimulation of layer 2/3 horizontal fibers. Furthermore, anti-Nogo-A Ab treatment increased LTP saturation levels, whereas long-term depression remained unchanged, thus leading to an enlarged synaptic modification range. In vivo, intrathecal application of Nogo-A-blocking Abs resulted in a higher dendritic spine density at cortical pyramidal neurons due to an increase in spine formation as revealed by in vivo two-photon microscopy. To investigate whether these changes in synaptic plasticity correlate with motor learning, we trained rats to learn a skilled forelimb-reaching task while receiving anti-Nogo-A Abs. Learning of this cortically controlled precision movement was improved upon anti-Nogo-A Ab treatment. Our results identify Nogo-A as an influential molecular modulator of synaptic plasticity and as a regulator for learning of skilled movements in the motor cortex.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Refinement of mammalian neural circuits involves substantial experience-dependent synapse elimination. Using in vivo two-photon imaging, we found that experience-dependent elimination of postsynaptic dendritic spines in the cortex was accelerated in ephrin-A2 knockout (KO) mice, resulting in fewer adolescent spines integrated into adult circuits. Such increased spine removal in ephrin-A2 KOs depended on activation of glutamate receptors, as blockade of the N-methyl-D-aspartate (NMDA) receptors eliminated the difference in spine loss between wild-type and KO mice. We also showed that ephrin-A2 in the cortex colocalized with glial glutamate transporters, which were significantly downregulated in ephrin-A2 KOs. Consistently, glial glutamate transport was reduced in ephrin-A2 KOs, resulting in an accumulation of synaptic glutamate. Finally, inhibition of glial glutamate uptake promoted spine elimination in wild-type mice, resembling the phenotype of ephrin-A2 KOs. Together, our results suggest that ephrin-A2 regulates experience-dependent, NMDA receptor-mediated synaptic pruning through glial glutamate transport during maturation of the mouse cortex.

Many lines of evidence suggest that memory in the mammalian brain is stored with distinct spatiotemporal patterns. Despite recent progresses in identifying neuronal populations involved in memory coding, the synapse-level mechanism is still poorly understood. Computational models and electrophysiological data have shown that functional clustering of synapses along dendritic branches leads to nonlinear summation of synaptic inputs and greatly expands the computing power of a neural network. However, whether neighbouring synapses are involved in encoding similar memory and how task-specific cortical networks develop during learning remain elusive. Using transcranial two-photon microscopy, we followed apical dendrites of layer 5 pyramidal neurons in the motor cortex while mice practised novel forelimb skills. Here we show that a third of new dendritic spines (postsynaptic structures of most excitatory synapses) formed during the acquisition phase of learning emerge in clusters, and that most such clusters are neighbouring spine pairs. These clustered new spines are more likely to persist throughout prolonged learning sessions, and even long after training stops, than non-clustered counterparts. Moreover, formation of new spine clusters requires repetition of the same motor task, and the emergence of succedent new spine(s) accompanies the strengthening of the first new spine in the cluster. We also show that under control conditions new spines appear to avoid existing stable spines, rather than being uniformly added along dendrites. However, succedent new spines in clusters overcome such a spatial constraint and form in close vicinity to neighbouring stable spines. Our findings suggest that clustering of new synapses along dendrites is induced by repetitive activation of the cortical circuitry during learning, providing a structural basis for spatial coding of motor memory in the mammalian brain.

As earth system modeling develops ever finer grid resolutions, the inputting and outputting (I/O) of the increasingly large data fields becomes a processing bottleneck. Many models developed in China, as well as the community coupler (C-Coupler), do not fully benefit from existing parallel I/O supports. This paper reports the design and implementation of a common parallel input/output framework (CIOFC1.0) based on C-Coupler2.0. The CIOFC1.0 framework can accelerate the I/O of large data fields by parallelizing data read/write operations among processes. The framework also allows convenient specification by users of the I/O settings, e.g., the data fields for I/O, the time series of the data files for I/O, and the data grids in the files. The framework can also adaptively input data fields from a time series dataset during model integration, automatically interpolate data when necessary, and output fields either periodically or irregularly. CIOFC1.0 demonstrates the cooperative development of an I/O framework and coupler, and thus enables convenient and simultaneous use of a coupler and an I/O framework. [ABSTRACT FROM AUTHOR]

The community coupler (C-Coupler) for Earth system modelling is a coupler family that was developed in China in 2010. C-Coupler3.0, the latest version, is fully compatible with the previous version, C-Coupler2, and is an integrated infrastructure with new features, i.e. a series of parallel-optimization technologies for accelerating coupling initialization and reducing memory usage, a common halo-exchange library for developing a parallel version of a model, a common module-integration framework for integrating a software module (e.g. a flux algorithm, a parameterization scheme, and a data assimilation method), a common framework for conveniently developing a weakly coupled ensemble data assimilation system, and a common framework for flexibly inputting and outputting fields in parallel. Specifically, C-Coupler3.0 is able to handle coupling under much finer resolutions (e.g. more than 100 million horizontal grid cells) with fast coupling initialization and successful generation of remapping-weight files. [ABSTRACT FROM AUTHOR]

Novel motor skills are learned through repetitive practice and, once acquired, persist long after training stops (1,2). Earlier studies have shown that such learning induces an increase in the efficacy of synapses in the primary motor cortex, the persistence of which is associated with retention of the task (3-5). However, how motor learning affects neuronal circuitry at the level of individual synapses and how long-lasting memory is structurally encoded in the intact brain remain unknown. Here we show that synaptic connections in the living mouse brain rapidly respond to motor-skill learning and permanently rewire. Training in a forelimb reaching task leads to rapid (within an hour) formation of postsynaptic dendritic spines on the output pyramidal neurons in the contralateral motor cortex. Although selective elimination of spines that existed before training gradually returns the overall spine density back to the original level, the new spines induced during learning are preferentially stabilized during subsequent training and endure long after training stops. Furthermore, we show that different motor skills are encoded by different sets of synapses. Practice of novel, but not previously learned, tasks further promotes dendritic spine formation in adulthood. Our findings reveal that rapid, but long-lasting, synaptic reorganization is closely associated with motor learning. The data also suggest that stabilized neuronal connections are the foundation of durable motor memory., Fine motor movements require accurate muscle synergies that rely on coordinated recruitment of intracortical synapses onto corticospinal neurons (6,7). Obtaining new motor skills has been shown to strengthen the horizontal [...]