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Introduction: Blood samples were collected to explore potential serum biomarkers associated with neurocognitive function in small-cell lung cancer (SCLC) patients who received prophylactic cranial irradiation (PCI)., Methods: This pre-specified study included patients with blood samples available, who participated in a phase III trial (NCT01780675). Blood samples were collected before PCI and 3-days post-initiating PCI. Neurocognitive decline was defined as a decrease of ≥ 5 points on total recall in the Hopkins Verbal Learning Test-Revised (HVLT-R) assessed from pre-PCI to 4-months post-PCI. Biomarkers were screened using univariate logistic regression analysis. P < .1 was considered statistically significant., Results: Forty-eight enrolled patients who had blood samples at baseline were included and 27 were available for analysis as the other 21 did not assess neurocognitive function at 4-months. Lower levels of Tie-2 (OR = 0.999, 90% CI 0.998-1.000, P = .062), and higher levels of MIP-1b (OR = 1.022, 90% CI 1.000-1.044, P = .093), CCL-17 (OR = 1.004, 90% CI 1.001-1.006, P = .029), and IL-1α (OR = 1.597, 90% CI 1.077-2.367, P = .05) before PCI were correlated with neurocognitive decline at 4-months. Decrease of VEGF-C (OR = 0.972, 90% CI 0.949-0.996, P = .055), CCL-17 (OR = 0.993, 90% CI 0.988-0.999, P = .036), IL-1α (OR = 0.788, 90% CI 0.635-0.979, P = .071), and VEGF (OR = 0.981, 90% CI 0.965-0.997, P = .051) 3-days post-initiating PCI were also associated with neurocognitive decline at 4-months., Conclusions: Biomarker levels before PCI and changes in their levels 3-days post-initiating PCI may be linked to subsequent neurocognitive decline at 4-months. If validated, these biomarkers could be used to predict the risk of neurocognitive decline and act as a decision aid for personalized PCI in SCLC., Competing Interests: Disclosure The authors have stated that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Noradrenaline (norepinephrine) is known to modulate many physiological functions and behaviors. In this study, we tested to what extent astrocytes, a type of glial cell, participate in noradrenergic signaling in mouse primary visual cortex (V1). Astrocytes are essential partners of neurons in the central nervous system. They are central to brain homeostasis, but also dynamically regulate neuronal activity, notably by relaying and regulating neuromodulator signaling. Indeed, astrocytes express receptors for multiple neuromodulators, including noradrenaline, but the extent to which astrocytes are involved in noradrenergic signaling remains unclear. To test whether astrocytes are involved in noradrenergic neuromodulation in mice, we employed both short hairpin RNA mediated knockdown as well as pharmacological manipulation of the major noradrenaline receptor in astrocytes, the α1A-adrenoreceptor. Using acute brain slices, we found that the astrocytic α1A-adrenoreceptor subtype contributes to the generation of large intracellular Ca 2+ signals in visual cortex astrocytes, which are generally thought to underlie astrocyte function. To test if reduced α1A-adrenoreceptor signaling in astrocytes affected the function of neuronal circuits in V1, we used both patch-clamp and field potential recordings. These revealed that noradrenergic signaling through the astrocyte α1A-adrenoreceptor is important to not only modulate synaptic activity but also to regulate plasticity in V1, through the potentiation of synaptic responses in circuits involved in visual information processing., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

No single teaching strategy supports all learning styles in veterinary science students. To facilitate more convenient and flexible teaching, learning, and revision, an innovative online digital learning platform-VetCloud-was developed to provide access to modularized programme content across courses to promote active, integrated learning. This study aimed to understand student perceptions regarding the enhancement of the student learning experience in a foundational course in gastrointestinal anatomy and physiology at The University of Queensland across two learning cycles, via applying iterative student feedback in transitioning a flipped classroom approach using VetCloud for the delivery of lecture content in 2022 to a flexible learning approach in 2023. By 2023, the use of VetCloud in the flexible learning approach improved students' work/study/life balance, reduced their stress levels, and enabled a more efficient use of their time when studying, compared to the flipped classroom approach in 2022. Surveying student perceptions was integral to maximizing their learning experience. Data clearly demonstrates that students will mix-and-match how they interact with available options provided via flexible delivery on an individualized basis. This teaching method offers veterinary educators an innovative and efficient approach to veterinary student education in anatomy and physiology while enhancing student well-being.

Traumatic brain injury (TBI) can result in long-lasting changes in hippocampal function. The changes induced by TBI on the hippocampus contribute to cognitive deficits. The adult hippocampus harbors neural stem cells (NSCs) that generate neurons (neurogenesis), and astrocytes (astrogliogenesis). While deregulation of hippocampal NSCs and neurogenesis have been observed after TBI, it is not known how TBI may affect hippocampal astrogliogenesis. Using a controlled cortical impact model of TBI in male mice, single cell RNA sequencing and spatial transcriptomics, we assessed how TBI affected hippocampal NSCs and the neuronal and astroglial lineages derived from them. We observe an increase in NSC-derived neuronal cells and a concomitant decrease in NSC-derived astrocytic cells, together with changes in gene expression and cell dysplasia within the dentate gyrus. Here, we show that TBI modifies NSC fate to promote neurogenesis at the cost of astrogliogenesis and identify specific cell populations as possible targets to counteract TBI-induced cellular changes in the adult hippocampus., (© 2024. The Author(s).)

Parkinson's Disease (PD) is the second most common neurodegenerative disorder. The pathological hallmark of PD is loss of dopaminergic neurons and the presence of aggregated α-synuclein, primarily in the substantia nigra pars compacta (SNpc) of the midbrain. However, the molecular mechanisms that underlie the pathology in different cell types is not currently understood. Here, we present a single nucleus transcriptome analysis of human post-mortem SNpc obtained from 15 sporadic Parkinson's Disease (PD) cases and 14 Controls. Our dataset comprises ∼84K nuclei, representing all major cell types of the brain, allowing us to obtain a transcriptome-level characterization of these cell types. Importantly, we identify multiple subpopulations for each cell type and describe specific gene sets that provide insights into the differing roles of these subpopulations. Our findings reveal a significant decrease in neuronal cells in PD samples, accompanied by an increase in glial cells and T cells. Subpopulation analyses demonstrate a significant depletion of tyrosine hydroxylase (TH) enriched astrocyte, microglia and oligodendrocyte populations in PD samples, as well as TH enriched neurons, which are also depleted. Moreover, marker gene analysis of the depleted subpopulations identified 28 overlapping genes, including those associated with dopamine metabolism (e.g., ALDH1A1, SLC6A3 & SLC18A2). Overall, our study provides a valuable resource for understanding the molecular mechanisms involved in dopaminergic neuron degeneration and glial responses in PD, highlighting the existence of novel subpopulations and cell type-specific gene sets., (© 2024. The Author(s).)

Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pereira, Ayana, Holt and Arckens.)

Astrocytes are integral components of brain circuits, where they sense, process, and respond to surrounding activity, maintaining homeostasis and regulating synaptic transmission, the sum of which results in behavior modulation. These interactions are possible due to their complex morphology, composed of a tree-like structure of processes to cover defined territories ramifying in a mesh-like system of fine leaflets unresolved by conventional optic microscopy. While recent reports devoted more attention to leaflets and their dynamic interactions with synapses, our knowledge about the tree-like "backbone" structure in physiological conditions is incomplete. Recent transcriptomic studies described astrocyte molecular diversity, suggesting structural heterogeneity in regions such as the hippocampus, which is crucial for cognitive and emotional behaviors. In this study, we carried out the structural analysis of astrocytes across the hippocampal subfields of Cornu Ammonis area 1 (CA1) and dentate gyrus in the dorsoventral axis. We found that astrocytes display heterogeneity across the hippocampal subfields, which is conserved along the dorsoventral axis. We further found that astrocytes appear to contribute in an exocytosis-dependent manner to a signaling loop that maintains the backbone structure. These findings reveal astrocyte heterogeneity in the hippocampus, which appears to follow layer-specific cues and depend on the neuro-glial environment., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Cognitive decline is a common pathological outcome during aging, with an ill-defined molecular and cellular basis. In recent years, the concept of inflammaging, defined as a low-grade inflammation increasing with age, has emerged. Infiltrating T cells accumulate in the brain with age and may contribute to the amplification of inflammatory cascades and disruptions to the neurogenic niche observed with age. Recently, a small resident population of regulatory T cells has been identified in the brain, and the capacity of IL2-mediated expansion of this population to counter neuroinflammatory disease has been demonstrated. Here, we test a brain-specific IL2 delivery system for the prevention of neurological decline in aging mice. We identify the molecular hallmarks of aging in the brain glial compartments and identify partial restoration of this signature through IL2 treatment. At a behavioral level, brain IL2 delivery prevented the age-induced defect in spatial learning, without improving the general decline in motor skill or arousal. These results identify immune modulation as a potential path to preserving cognitive function for healthy aging., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Astrocytes are ubiquitous within the central nervous system (CNS). These cells possess many individual processes which extend out into the neuropil, where they interact with a variety of other cell types, including neurons at synapses. Astrocytes are now known to be active players in all aspects of the synaptic life cycle, including synapse formation and elimination, synapse maturation, maintenance of synaptic homeostasis and modulation of synaptic transmission. Traditionally, astrocytes have been studied as a homogeneous group of cells. However, recent studies have uncovered a surprising degree of heterogeneity in their development and function, suggesting that astrocytes may be matched to neurons to support local circuits. Hence, a better understanding of astrocyte heterogeneity and its implications are needed to understand brain function., (© 2023 The Author(s).)

Advances in single cell sequencing have enabled the identification of a large number of genes, expressed in many different cell types, and across a variety of model organisms. In particular, the nervous system harbors an immense number of interacting cell types, which are poorly characterized. Future loss- and gain-of-function experiments will be essential in determining how novel genes play critical roles in diverse cellular, as well as evolutionarily adapted, contexts. However, functional analysis across species is often hampered by technical limitations, in non-genetic animal systems. Here, we describe a new single plasmid system, misPiggy. The system is based around the hyperactive piggyBac transposon system, which combines stable genomic integration of transgenes (for long-term expression) with large cargo capacity. Taking full advantage of these characteristics, we engineered novel expression modules into misPiggy that allow for cell-type specific loss- and gain-of-gene function. These modules work widely across species from frog to ferret. As a proof of principle, we present a loss-of-function analysis of the neuronal receptor Deleted in Colorectal Cancer (DCC) in retinal ganglion cells (RGCs) of Xenopus tropicalis tadpoles. Single axon tracings of mosaic knock-out cells reveal a specific cell-intrinsic requirement of DCC, specifically in axonal arborization within the frog tectum, rather than retina-to-brain axon guidance. Furthermore, we report additional technical advances that enable temporal control of knock-down or gain-of-function analysis. We applied this to visualize and manipulate labeled neurons, astrocytes and other glial cells in the central nervous system (CNS) of mouse, rat and ferret. We propose that misPiggy will be a valuable tool for rapid, flexible and cost-effective screening of gene function across a variety of animal models., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

Prion infections cause conformational changes of the cellular prion protein (PrP C ) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and β2-α2 loops of PrP C . Expression of a PrP 2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrP R207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrP C , but not PrP 2Cys . These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity., (© 2022. The Author(s).)

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (T reg ) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident T reg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients., (© 2022. The Author(s).)

Systemic exposure to nanoparticles (NPs) adversely affects different organs, including the nervous system. We systematically extracted data from publication on PubMed and Embase database up to the year 2020, and analyzed in vitro and in vivo neurotoxicity of 4 of the most well studied NPs (silver NPs, carbon-based NPs, iron NPs and silica NPs). A relatively good correlation was observed between in vitro and in vivo effects, including genotoxicity, oxidative stress, apoptosis and pro-inflammatory effects. However, crucial knowledge gap exists in current understanding of the underlying mechanisms. Some of the critical knowledge gaps and research needs identified in relation to neurotoxicity of nanoparticles include (1) lack of physio-chemical characteristics of NPs used, (2) cellular/tissue uptake of NP, (3) NP translocation across the blood-brain barrier (BBB), (4) Effect of exposure routes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In the last four decades, monoclonal antibodies and their derivatives have emerged as a powerful class of therapeutics, largely due to their exquisite targeting specificity. Several clinical areas, most notably oncology and autoimmune disorders, have seen the successful introduction of monoclonal-based therapeutics. However, their adoption for treatment of Central Nervous System diseases has been comparatively slow, largely due to issues of efficient delivery resulting from limited permeability of the Blood Brain Barrier. Nevertheless, CNS diseases are becoming increasingly prevalent as societies age, accounting for ~6.5 million fatalities worldwide per year. Therefore, harnessing the full therapeutic potential of monoclonal antibodies (and their derivatives) in this clinical area has become a priority. Adeno-associated virus-based vectors (AAVs) are a potential solution to this problem. Preclinical studies have shown that AAV vector-mediated antibody delivery provides protection against a broad range of peripheral diseases, such as the human immunodeficiency virus (HIV), influenza and malaria. The parallel identification and optimization of AAV vector platforms which cross the Blood Brain Barrier with high efficiency, widely transducing the Central Nervous System and allowing high levels of local transgene production, has now opened a number of interesting scenarios for the development of AAV vector-mediated antibody delivery strategies to target Central Nervous System proteinopathies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marino and Holt.)

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the App NL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

The development and evolution of mammalian higher cognition are represented by gyrification of the laminar cerebral cortex and astrocyte development, but their mechanisms and interrelationships remain unknown. Here, we show that localized astrogenesis plays an important role in gyri formation in the gyrencephalic cerebral cortex. In functional genetic experiments, we show that reducing astrocyte number prevents gyri formation in the ferret cortex, while increasing astrocyte number in mice, which do not have cortical folds, can induce gyrus-like protrusions. Morphometric analyses demonstrate that the vertical expansion of deep pallial regions achieved by localized astrogenesis is crucial for gyri formation. Furthermore, our findings suggest that localized astrogenesis by a positive feedback loop of FGF signaling is an important mechanism underlying cortical folding in gyrencephalic mammalian brains. Our findings reveal both the cellular mechanisms and the mechanical principle of gyrification in the mammalian brain.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics ' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions., (© 2022 The Authors.)

Plasticity is a fundamental property of neuronal circuits, allowing them to adapt to alterations in activation. Generally speaking, plasticity has been viewed from a 'neuron-centric' perspective, with changes in circuit function attributed to alterations in neuronal excitability, synaptic strength or neuronal connectivity. However, it is now clear that glial cells, in particular astrocytes, are key regulators of neuronal plasticity. This article reviews recent progress made in understanding astrocyte function and attempts to summarize these functions into a coherent framework that positions astrocytes as central players in the plasticity process., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.

Noradrenaline is a major neuromodulator in the central nervous system (CNS). It is released from varicosities on neuronal efferents, which originate principally from the main noradrenergic nuclei of the brain - the locus coeruleus - and spread throughout the parenchyma. Noradrenaline is released in response to various stimuli and has complex physiological effects, in large part due to the wide diversity of noradrenergic receptors expressed in the brain, which trigger diverse signaling pathways. In general, however, its main effect on CNS function appears to be to increase arousal state. Although the effects of noradrenaline have been researched extensively, the majority of studies have assumed that noradrenaline exerts its effects by acting directly on neurons. However, neurons are not the only cells in the CNS expressing noradrenaline receptors. Astrocytes are responsive to a range of neuromodulators - including noradrenaline. In fact, noradrenaline evokes robust calcium transients in astrocytes across brain regions, through activation of α1-adrenoreceptors. Crucially, astrocytes ensheath neurons at synapses and are known to modulate synaptic activity. Hence, astrocytes are in a key position to relay, or amplify, the effects of noradrenaline on neurons, most notably by modulating inhibitory transmission. Based on a critical appraisal of the current literature, we use this review to argue that a better understanding of astrocyte-mediated noradrenaline signaling is therefore essential, if we are ever to fully understand CNS function. We discuss the emerging concept of astrocyte heterogeneity and speculate on how this might impact the noradrenergic modulation of neuronal circuits. Finally, we outline possible experimental strategies to clearly delineate the role(s) of astrocytes in noradrenergic signaling, and neuromodulation in general, highlighting the urgent need for more specific and flexible experimental tools., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wahis and Holt.)

Oligodendrocyte dysfunction has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron loss. The failure of trophic support provided by oligodendrocytes is associated with a concomitant reduction in oligodendroglial monocarboxylate transporter 1 (MCT1) expression and is detrimental for the long-term survival of motor neuron axons. Therefore, we established an adeno-associated virus 9 (AAV9)-based platform by which MCT1 was targeted mostly to white matter oligodendrocytes to investigate whether this approach could provide a therapeutic benefit in the SOD1 G93A mouse model of ALS. Despite good oligodendrocyte transduction and AAV-mediated MCT1 transgene expression, the disease outcome of SOD1 G93A mice was not altered. Our study further increases our current understanding about the complex nature of oligodendrocyte pathology in ALS and provides valuable insights into the future development of therapeutic strategies to efficiently modulate these cells., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Background: Three amino acid differences between rodent and human APP affect medically important features, including β-secretase cleavage of APP and Aβ peptide aggregation (De Strooper et al., EMBO J 14:4932-38, 1995; Ueno et al., Biochemistry 53:7523-30, 2014; Bush, 2003, Trends Neurosci 26:207-14). Most rodent models for Alzheimer's disease (AD) are, therefore, based on the human APP sequence, expressed from artificial mini-genes randomly inserted in the rodent genome. While these models mimic rather well various biochemical aspects of the disease, such as Aβ-aggregation, they are also prone to overexpression artifacts and to complex phenotypical alterations, due to genes affected in or close to the insertion site(s) of the mini-genes (Sasaguri et al., EMBO J 36:2473-87, 2017; Goodwin et al., Genome Res 29:494-505, 2019). Knock-in strategies which introduce clinical mutants in a humanized endogenous rodent APP sequence (Saito et al., Nat Neurosci 17:661-3, 2014) represent useful improvements, but need to be compared with appropriate humanized wildtype (WT) mice., Methods: Computational modelling of the human β-CTF bound to BACE1 was used to study the differential processing of rodent and human APP. We humanized the three pivotal residues we identified G676R, F681Y and R684H (labeled according to the human APP770 isoform) in the mouse and rat genomes using a CRISPR-Cas9 approach. These new models, termed mouse and rat App hu/hu , express APP from the endogenous promotor. We also introduced the early-onset familial Alzheimer's disease (FAD) mutation M139T into the endogenous Rat Psen1 gene., Results: We show that introducing these three amino acid substitutions into the rodent sequence lowers the affinity of the APP substrate for BACE1 cleavage. The effect on β-secretase processing was confirmed as both humanized rodent models produce three times more (human) Aβ compared to the original WT strain. These models represent suitable controls, or starting points, for studying the effect of transgenes or knock-in mutations on APP processing (Saito et al., Nat Neurosci 17:661-3, 2014). We introduced the early-onset familial Alzheimer's disease (FAD) mutation M139T into the endogenous Rat Psen1 gene and provide an initial characterization of Aβ processing in this novel rat AD model., Conclusion: The different humanized APP models (rat and mouse) expressing human Aβ and PSEN1 M139T are valuable controls to study APP processing in vivo allowing the use of a human Aβ ELISA which is more sensitive than the equivalent system for rodents. These animals will be made available to the research community.

Astrocytes are vital for preserving correct brain functioning by continuously sustaining neuronal activity and survival. They are in contact with multiple synapses at once allowing the expansion of local synaptic events into activity changes in neuronal networks. Furthermore, cortical astrocytes integrate local sensory inputs and behavioral state. From an anatomical, molecular, and functional perspective, astrocytes are thus ideal candidates to influence complex large-scale brain mechanisms such as plasticity. We collected evidence for the astrocytic potential for plasticity modulation, using the monocular enucleation (ME) mouse model of visual cortex plasticity. The impact of one-eyed vision involves the functional recruitment of the deprived visual cortex by the spared senses within a 7-week time frame, reflecting a substantial change in sensory information processing. In visually deprived cortex, a swift upregulation in Aldh1l1-positive astrocyte density lasts until maximal functional recovery is reached. Transient metabolic silencing of visual cortex astrocytes at the time of ME induction, through intracranial fluorocitrate injections, reveals that astrocytes are required on site to achieve adequate long-term neuronal reactivation. In addition, chronic stimulation by G i but not G q G-protein coupled receptor activation of local astrocytes boosts the cortical plasticity phenomenon. Hence, functional manipulation of protoplasmic astrocytes has long-lasting effects on the functional recovery of cortical neurons upon sensory loss, possibly by influencing the neuronal threshold to reactivate. Together, our results highlight an integral role for astrocytes in mediating adult cortical plasticity and unmask astrocyte specific G i signaling as an interesting therapeutic pathway for brain plasticity regulation., (© 2020 Wiley Periodicals, Inc.)

Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.

Astrocytes are ubiquitous in the central nervous system (CNS). These cells possess thousands of individual processes, which extend out into the neuropil, interacting with neurons, other glia and blood vessels. Paralleling the wide diversity of their interactions, astrocytes have been reported to play key roles in supporting CNS structure, metabolism, blood-brain-barrier formation and control of vascular blood flow, axon guidance, synapse formation and modulation of synaptic transmission. Traditionally, astrocytes have been studied as a homogenous group of cells. However, recent studies have uncovered a surprising degree of heterogeneity in their development and function, in both the healthy and diseased brain. A better understanding of astrocyte heterogeneity is urgently needed to understand normal brain function, as well as the role of astrocytes in response to injury and disease.

Astrocytes, a major cell type found throughout the central nervous system, have general roles in the modulation of synapse formation and synaptic transmission, blood-brain barrier formation, and regulation of blood flow, as well as metabolic support of other brain resident cells. Crucially, emerging evidence shows specific adaptations and astrocyte-encoded functions in regions, such as the spinal cord and cerebellum. To investigate the true extent of astrocyte molecular diversity across forebrain regions, we used single-cell RNA sequencing. Our analysis identifies five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Validation of our data in situ reveals distinct spatial positioning of defined subtypes, reflecting the distribution of morphologically and physiologically distinct astrocyte populations. Our findings are evidence for specialized astrocyte subtypes between and within brain regions. The data are available through an online database (https://holt-sc.glialab.org/), providing a resource on which to base explorations of local astrocyte diversity and function in the brain.

Astrocytes are a major cell type in the mammalian nervous system, are in close proximity to neurons, and show rich Ca 2+ activity thought to mediate cellular outputs. Astrocytes show activity linked to sensory [1, 2] and motor [3, 4] events, reflecting local neural activity and brain-wide neuromodulatory inputs. Sensory responses are highly variable [5-10], which may reflect interactions between distinct input types [6, 7, 9]. However, the diversity of inputs generating astrocyte activity, particularly during sensory stimulation and behavior, is not fully understood [11, 12]. Using a combination of Ca 2+ imaging, a treadmill assay, and visual stimulation, we examined the properties of astrocyte activity in mouse visual cortex associated with motor or sensory events. Consistent with previous work, motor activity activated astrocytes across the cortex with little specificity, reflecting a diffuse neuromodulatory mechanism. In contrast, moving visual stimuli generated specific activity patterns that reflected the stimulus' trajectory within the visual field, precisely as one would predict if astrocytes reported local neural activity. Visual responses depended strongly on behavioral state, with astrocytes showing high amplitude Ca 2+ transients during locomotion and little activity during stillness. Furthermore, the amplitudes of visual responses were highly correlated with pupil size, suggesting a role of arousal. Interestingly, while depletion of cortical noradrenaline abolished locomotor responses, visual responses were only reduced in amplitude and their spatiotemporal organization remained intact, suggesting two distinct types of inputs underlie visual responses. We conclude that cortical astrocytes integrate local sensory information and behavioral state, suggesting a role in information processing., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Until recently, adeno-associated virus 9 (AAV9) was considered the AAV serotype most effective in crossing the blood-brain barrier (BBB) and transducing cells of the central nervous system (CNS), following systemic injection. However, a newly engineered capsid, AAV-PHP.B, is reported to cross the BBB at even higher efficiency. We investigated how much we could boost CNS transgene expression by using AAV-PHP.B carrying a self-complementary (sc) genome. To allow comparison, 6 weeks old C57BL/6 mice received intravenous injections of scAAV2/9-GFP or scAAV2/PHP.B-GFP at equivalent doses. Three weeks postinjection, transgene expression was assessed in brain and spinal cord. We consistently observed more widespread CNS transduction and higher levels of transgene expression when using the scAAV2/PHP.B-GFP vector. In particular, we observed an unprecedented level of astrocyte transduction in the cortex, when using a ubiquitous CBA promoter. In comparison, neuronal transduction was much lower than previously reported. However, strong neuronal expression (including spinal motor neurons) was observed when the human synapsin promoter was used. These findings constitute the first reported use of an AAV-PHP.B capsid, encapsulating a scAAV genome, for gene transfer in adult mice. Our results underscore the potential of this AAV construct as a platform for safer and more efficacious gene therapy vectors for the CNS.

Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Drosophila is widely used as a genetic model in questions of development, cellular function and disease. Genetic screens in flies have proven to be incredibly powerful in identifying crucial components for synapse formation and function, particularly in the case of the presynaptic release machinery. Although modern biochemical methods can identify individual proteins and lipids (and their binding partners), they have typically been excluded from use in Drosophila for technical reasons. To bridge this essential gap between genetics and biochemistry, we developed a fractionation method to isolate various parts of the synaptic machinery from Drosophila, thus allowing it to be studied in unprecedented biochemical detail. This is only possible because our protocol has unique advantages in terms of enriching and preserving endogenous protein complexes. The procedure involves decapitation of adult flies, homogenization and differential centrifugation of fly heads, which allow subsequent purification of presynaptic (and to a limited degree postsynaptic) components. It is designed to require only a rudimentary knowledge of biochemical fractionation, and it takes ∼3.5 h. The yield is typically 4 mg of synaptic membrane protein per gram of Drosophila heads.

CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. In this study, we report that the CIDE-N protein Drep-2 is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation.

Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstitution of vesicle fusion from purified synaptic proteins and lipids has played a major role in elucidating the synaptic exocytotic fusion machinery with ever increasing detail. However, one limitation of most reconstitution approaches has been the relatively slow rate of fusion that can be produced in these systems. In a related study, a notable exception is an approach measuring fusion of single reconstituted vesicles bearing the vesicle fusion protein synaptobrevin with supported planar membranes harboring the presynaptic plasma membrane proteins syntaxin and SNAP-25. Fusion times of ∼20 ms were achieved in this system. Despite this advance, an important question with reconstituted systems is how well they mimic physiological systems they are supposed to reproduce. In this work, we demonstrate that purified synaptic vesicles from rat brain fuse with acceptor-SNARE containing planar bilayers equally fast as equivalent reconstituted vesicles and that their fusion efficiency is increased by divalent cations. Calcium boosts fusion through a combined general electrostatic and synaptotagmin-specific mechanism., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

One hundred fifty multiparous cows were balanced to 1 of 3 treatments (2 pens/trt) according to previous lactation 305-d mature equivalent yield to evaluate supplementation with yeast culture (YC; A-Max, Vi-COR, Mason, IA) and YC plus enzymatically hydrolyzed yeast (YC+EHY; Celmanax, Vi-COR) on production performance in dairy cattle. Cows entered pens at calving and remained through 14 wk postpartum. Treatment assignment to pens was random throughout the barn. Pens were identical in layout and each contained an exit alley to eliminate feed and animal mixing. The 3 treatments were control: nonsupplemented; YC: control diet with YC (56 g/d); and YC+EHY: control diet plus YC and EHY (28 g/d). Mean pen dry matter intake was similar across treatments. Cows supplemented with YC and YC+EHY produced more milk, fat-corrected milk, and energy-corrected milk than control cows (1.4 and 1.6, 1.6 and 1.8, 1.7 and 1.9 kg, respectively). Treatments YC and YC+EHY did not differ. Milk fat and lactose percentages were not affected by treatment. Milk protein percentage was higher for cows supplemented with YC+EHY than for those on YC and control treatments (2.98, 2.93, and 2.91%, respectively) with control and YC-supplemented cows not being different from each other. Differences in fat and protein yields were primarily reflective of milk yield. Treatment had no effect on milk urea nitrogen. No differences in the incidence of metabolic health were observed; however, cases of clinical mastitis for YC+EHY were less than half those for control and YC during wk 8 to 14 on trial. Somatic cell count was higher for cows fed control and YC diets compared with YC+EHY, primarily during wk 8 to 14 on trial. Supplementation of early lactation cows with YC improved milk production performance; furthermore, EHY supplementation improved milk protein percentage and mammary gland health., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In eukaryotes, most intracellular membrane fusion reactions are mediated by the interaction of SNARE proteins that are present in both fusing membranes. However, the minimal number of SNARE complexes needed for membrane fusion is not known. Here we show unambiguously that one SNARE complex is sufficient for membrane fusion. We performed controlled in vitro Förster resonance energy transfer (FRET) experiments and found that liposomes bearing only a single SNARE molecule are still capable of fusion with other liposomes or with purified synaptic vesicles. Furthermore, we demonstrated that multiple SNARE complexes do not act cooperatively, showing that synergy between several SNARE complexes is not needed for membrane fusion. Our findings shed new light on the mechanism of SNARE-mediated membrane fusion and call for a revision of current views of fusion events such as the fast release of neurotransmitters.