Characteristics Of Astrocytes Research Articles

Region-Specific Neuroprotective Features of Astrocytes against Oxidative Stress Induced by 6-Hydroxydopamine.

In previous studies, we found regional differences in the induction of antioxidative molecules in astrocytes against oxidative stress, postulating that region-specific features of astrocytes lead region-specific vulnerability of neurons. We examined region-specific astrocytic features against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) as an oxidative stress using co-culture of mesencephalic neurons and mesencephalic or striatal astrocytes in the present study. The 6-OHDA-induced reduction of mesencephalic dopamine neurons was inhibited by co-culturing with astrocytes. The co-culture of midbrain neurons with striatal astrocytes was more resistant to 6-OHDA than that with mesencephalic astrocytes. Furthermore, glia conditioned medium from 6-OHDA-treated striatal astrocytes showed a greater protective effect on the 6-OHDA-induced neurotoxicity and oxidative stress than that from mesencephalic astrocytes. The cDNA microarray analysis showed that the number of altered genes in both mesencephalic and striatal astrocytes was fewer than that changed in either astrocyte. The 6-OHDA treatment, apparently up-regulated expressions of Nrf2 and some anti-oxidative or Nrf2-regulating phase II, III detoxifying molecules related to glutathione synthesis and export in the striatal astrocytes but not mesencephalic astrocytes. There is a profound regional difference of gene expression in astrocytes induced by 6-OHDA. These results suggest that protective features of astrocytes against oxidative stress are more prominent in striatal astrocytes, possibly by secreting humoral factors in striatal astrocytes.

A Specialized Method to Resolve Fine 3D Features of Astrocytes in Nonhuman Primate (Marmoset, Callithrix jacchus) and Human Fixed Brain Samples.

Astrocytes are among the most numerous cells in the brain and fulfill diverse functions in homeostasis and regulation of neuronal activity. Astrocytes also dramatically change their properties in response to brain injury or disease, a process called reactive gliosis. Precisely how astrocytes contribute to healthy brain function and play differential roles in brain pathology and regeneration remain important areas of investigation. To better understand the properties of astrocytes, more sophisticated approaches for probing their rich and complex anatomical and molecular features are needed to fully determine their contribution to brain physiology. Here we present an efficient and straightforward immunolabeling protocol to obtain high-resolution fluorescence-based images from fixed nonhuman primate (common marmoset Callithrix jacchus) and human brain samples. Importantly, the protocol is useful for obtaining images from samples that have been stored in fixative solutions (such as formalin) for years. This approach is especially useful for three-dimensional, multichannel confocal microscopy and can be optimized for super-resolution techniques such as stimulated emission depletion (STED) microscopy. We also present a strategy for using specific combinations of markers to define the phenotypic variations and cellular/subcellular properties of astrocytes to better predict the function of these cells on their surrounding brain microenvironment.

Single-Cell Analysis of Regional Differences in Adult V-SVZ Neural Stem Cell Lineages

SUMMARYThe ventricular-subventricular zone (V-SVZ) harbors adult neural stem cells. V-SVZ neural stem cells exhibit features of astrocytes, have a regional identity, and depending on their location in the lateral or septal wall of the lateral ventricle, generate different types of neuronal and glial progeny. We performed large-scale single-cell RNA sequencing to provide a molecular atlas of cells from the lateral and septal adult V-SVZ of male and female mice. This revealed regional and sex differences among adult V-SVZ cells. We uncovered lineage potency bias at the single-cell level among lateral and septal wall astrocytes toward neurogenesis and oligodendrogenesis, respectively. Finally, we identified transcription factor co-expression modules marking key temporal steps in neurogenic and oligodendrocyte lineage progression. Our data suggest functionally important spatial diversity in neurogenesis and oligodendrogenesis in the adult brain and reveal molecular correlates of adult NSC dormancy and lineage specialization.

Astrocyte-Neuron Networks: A Multilane Highway of Signaling for Homeostatic Brain Function.

Research on glial cells over the past 30 years has confirmed the critical role of astrocytes in pathophysiological brain states. However, most of our knowledge about astrocyte physiology and of the interactions between astrocytes and neurons is based on the premises that astrocytes constitute a homogeneous cell type, without considering the particular properties of the circuits or brain nuclei in which the astrocytes are located. Therefore, we argue that more-sophisticated experiments are required to elucidate the specific features of astrocytes in different brain regions, and even within different layers of a particular circuit. Thus, in addition to considering the diverse mechanisms used by astrocytes to communicate with neurons and synaptic partners, it is necessary to take into account the cellular heterogeneity that likely contributes to the outcomes of astrocyte–neuron signaling. In this review article, we briefly summarize the current data regarding the anatomical, molecular and functional properties of astrocyte–neuron communication, as well as the heterogeneity within this communication.

Clonal Glial Response in a Multiple Sclerosis Mouse Model.

Multiple sclerosis (MS) is an autoimmune disease causing central nervous system (CNS) demyelination and axonal injury. In the last years the importance of astrocytes in MS is rapidly increasing, recognizing astrocytes as highly active players in MS pathogenesis. Usually the role assigned to astrocytes in MS lesions has been the formation of the glial scar, but now their implication during lesion formation and the immune response increasingly recognized. Since astrocytes are a heterogeneous cell population with diverse roles in the CNS, the aim of this study was to analyze the putative clonal response of astrocytes in a demyelinating scenario. To undertake this aim, we used the induced experimental autoimmune encephalomyelitis (EAE) as a murine model for MS in previously electroporated mice with in vivo multicolor lineage tracing system, the StarTrack methodology. Our data revealed a variety of morphological changes that were different among distinct clones. In many cases, cells of the same clone responded equally to the injury, while in other cases clonally-related cells responded differently to the injury. Therefore, whereas some clones exhibited a strong morphological alteration, other clones located at similar distances to the lesion were apparently unresponsive. Thus, at present there is no compelling evidences that clonal relationship influences the position or function of astrocytes in the EAE model. Further, the coexistence of different astroglial clonal responses to the bran injury reveals the significance of development to determine the astrocyte features that respond to brain injuries.

Structural and functional identification of two distinct inspiratory neuronal populations at the level of the phrenic nucleus in the rat cervical spinal cord

The diaphragm is driven by phrenic motoneurons that are located in the cervical spinal cord. Although the anatomical location of the phrenic nucleus and the function of phrenic motoneurons at a single cellular level have been extensively analyzed, the spatiotemporal dynamics of phrenic motoneuron group activity have not been fully elucidated. In the present study, we analyzed the functional and structural characteristics of respiratory neuron population in the cervical spinal cord at the level of the phrenic nucleus by voltage imaging, together with histological analysis of neuronal and astrocytic distribution in the cervical spinal cord. We found spatially distinct two cellular populations that exhibited synchronized inspiratory activity on the transversely cut plane at C4–C5 levels and on the ventral surface of the mid cervical spinal cord in the isolated brainstem–spinal cord preparation of the neonatal rat. Inspiratory activity of one group emerged in the central portion of the ventral horn that corresponded to the central motor column, and the other appeared in the medial portion of the ventral horn that corresponded to the medial motor column. We identified by retrogradely labeling study that the anatomical distributions of phrenic and scalene motoneurons coincided with optically detected central and medial motor regions, respectively. Furthermore, we anatomically demonstrated closely located features of putative motoneurons, interneurons and astrocytes in these regions. Collectively, we report that phrenic and scalene motoneuron populations show synchronized inspiratory activities with distinct anatomical locations in the mid cervical spinal cord.

Neuronal and glial regeneration after focal cerebral ischemia in rat, an immunohistochemical and electron microscopical study

ObjectivesUnilateral middle cerebral artery occlusion (MCAO) is an established rat model for stoke studies. It induces focal cerebral ischemia, prior to necrotic and apoptotic loss of tissue in a circumscribed cortical area, paralleled by temporary motor impairment. MethodsHere we examined tissue samples from the peri-infarct zone of rats that had survived unilateral MCAO for up to 90 min. With immunohistochemistry we stained sections for proliferation markers Ki 67 and PCNA and for intermediate filament protein nestin. Electron microscopy was employed to assess ultrastructural changes. ResultsAll MCAO animals developed pronounced lesions in the motor cortex. Numerous cells in the immediate peri-infarct area and scattered cells which seem to have migrated into the infarcted lesion stained positively for Ki 67 and PCNA. Electron microscopy revealed that cells in the lesion site proliferate along the blood vessels. Most of these cells had the ultrastructural features of fibrillary astrocytes while some of the cells were clearly neurons. Endothelia were in part fenestrated. Some of the surrounding cells showed immunostaining for PCNA, indicating proliferation. Oligodendroglia and myelination could not be seen in the lesion site. Single neuronal contacts exhibited the ultrastructural features of synapses. Reformation of cortical layers could not be observed. ConclusionsWe concluded that in spite of extensive proliferation; neuronal and glial regeneration occurs after MCAO only to a small extent. Revascularization seems to be an important initial step. The observed functional recovery of experimental animals may be due to neuronal plasticity in young rats rather than structural regeneration.

Astrocytes in primary cultures express serine racemase, synthesize d-serine and acquire A1 reactive astrocyte features

d-Serine is a co-agonist at forebrain N-methyl-d-aspartate receptors (NMDAR) and is synthesized by serine racemase (SR). Although d-serine and SR were originally reported to be localized to glia, recent studies have provided compelling evidence that under healthy physiologic conditions both are localized primarily in neurons. However, in pathologic conditions, reactive astrocytes can also express SR and synthesize d-serine. Since cultured astrocytes exhibit features of reactive astrocytes, we have characterized d-serine synthesis and the expression of enzymes involved in its disposition in primary glial cultures. The levels of SR were quite low early in culture and increased markedly in all astrocytes with the duration in vitro. The concentration of d-serine in the culture medium increased in parallel with SR expression in the astrocytes. Microglia, identified by robust expression of Iba1, did not express SR. While the levels of glial fibrillary acidic protein (GFAP), glycine decarboxylase (GLDC) and phosphoglycerate dehydrogenase (PHGDH), the initial enzyme in the pathway converting glycine to l-serine, remained constant in culture, the expression of lipocalin-2, a marker for pan-reactive astrocytes, increased several-fold. The cultured astrocytes also expressed Complement-3a, a marker for a subpopulation of reactive astrocytes (A1). Astrocytes grown from mice with a copy number variant associated with psychosis, which have four copies of the GLDC gene, showed a more rapid production of d-serine and a reduction in glycine in the culture medium. These results substantiate the conclusion that A1 reactive astrocytes express SR and release d-serine under pathologic conditions, which may contribute to their neurotoxic effects by activating extra-synaptic NMDARs.

Brain metabolic sensing and metabolic signaling at the level of an astrocyte.

Astrocytes support neuronal function by providing essential structural and nutritional support, neurotransmitter trafficking and recycling and may also contribute to brain information processing. In this article we review published results and report new data suggesting that astrocytes function as versatile metabolic sensors of central nervous system (CNS) milieu and play an important role in the maintenance of brain metabolic homeostasis. We discuss anatomical and functional features of astrocytes that allow them to detect and respond to changes in the brain parenchymal levels of metabolic substrates (oxygen and glucose), and metabolic waste products (carbon dioxide). We report data suggesting that astrocytes are also sensitive to circulating endocrine signals—hormones like ghrelin, glucagon‐like peptide‐1 and leptin, that have a major impact on the CNS mechanisms controlling food intake and energy balance. We discuss signaling mechanisms that mediate communication between astrocytes and neurons and consider how these mechanisms are recruited by astrocytes activated in response to various metabolic challenges. We review experimental data suggesting that astrocytes modulate the activities of the respiratory and autonomic neuronal networks that ensure adaptive changes in breathing and sympathetic drive in order to support the physiological and behavioral demands of the organism in ever‐changing environmental conditions. Finally, we discuss evidence suggesting that altered astroglial function may contribute to the pathogenesis of disparate neurological, respiratory and cardiovascular disorders such as Rett syndrome and systemic arterial hypertension.

Postnatal reduction of tuberous sclerosis complex 1 expression in astrocytes and neurons causes seizures in an age-dependent manner.

Epilepsy is one of the most prominent symptoms of tuberous sclerosis complex (TSC), a genetic disorder, and may be related to developmental defects resulting from impaired TSC1 or TSC2 gene function in astrocytes and neurons. Inactivation of the Tsc1 gene driven by a glial-fibrillary acidic protein (GFAP) promoter during embryonic brain development leads to widespread pathologic effects on astrocytes and neurons, culminating in severe, progressive epilepsy in mice (Tsc1GFAP-Cre mice). However, the developmental timing and cellular specificity relevant to epileptogenesis in this model has not been well defined. The present study evaluates the effect of postnatal Tsc1 gene inactivation on pathologic features of astrocytes and neurons and development of epilepsy. An inducible Tsc1 knock-out mouse was created utilizing a tamoxifen-driven GFAP-CreER line (Tsc1GFAP-CreER mice) with TSC1 reduction induced postnatally at 2 and 6 weeks of age, and compared to conventional Tsc1GFAP-Cre mice with prenatal TSC1 reduction. Western blotting, immunohistochemistry, histology, and video-electroencephalography (EEG) assessed mechanistic target of rapamycin (mTOR) pathway activation, astrogliosis, neuronal organization, and spontaneous seizures, respectively. Tsc1 gene inactivation at 2 weeks of age was sufficient to cause astrogliosis and mild epilepsy in Tsc1GFAP-CreER mice, but the phenotype was much less severe than that observed with prenatal Tsc1 gene inactivation in Tsc1GFAP-Cre mice. Both astrocytes and neurons were affected by prenatal and postnatal Tsc1 gene activation to a degree similar to the severity of epilepsy, suggesting that both cellular types may contribute to epileptogenesis. These findings support a model in which the developmental timing of TSC1 loss dictates the severity of neuronal and glial abnormalities and resulting epilepsy.

Alterations in the Interplay between Neurons, Astrocytes and Microglia in the Rat Dentate Gyrus in Experimental Models of Neurodegeneration.

The hippocampus is negatively affected by aging and neurodegenerative diseases leading to impaired learning and memory abilities. A diverse series of progressive modifications in the intercellular communication among neurons, astrocytes and microglia occur in the hippocampus during aging or inflammation. A detailed understanding of the neurobiological modifications that contribute to hippocampal dysfunction may reveal new targets for therapeutic intervention. The current study focussed on the interplay between neurons and astroglia in the Granule Layer (GL) and the Polymorphic Layer (PL) of the Dentate Gyrus (DG) of adult, aged and LPS-treated rats. In GL and PL of aged and LPS-treated rats, astrocytes were less numerous than in adult rats. In GL of LPS-treated rats, astrocytes acquired morphological features of reactive astrocytes, such as longer branches than was observed in adult rats. Total and activated microglia increased in the aged and LPS-treated rats, as compared to adult rats. In the GL of aged and LPS-treated rats many neurons were apoptotic. Neurons decreased significantly in GL and PL of aged but not in rats treated with LPS. In PL of aged and LPS-treated rats many damaged neurons were embraced by microglia cells and were infiltrated by branches of astrocyte, which appeared to be bisecting the cell body, forming triads. Reactive microglia had a scavenging activity of dying neurons, as shown by the presence of neuronal debris within their cytoplasm. The levels of the chemokine fractalkine (CX3CL1) increased in hippocampal homogenates of aged rats and rats treated with LPS, and CX3CL1 immunoreactivity colocalized with activated microglia cells. Here we demonstrated that in the DG of aged and LPS-treated rats, astrocytes and microglia cooperate and participate in phagocytosis/phagoptosis of apoptotic granular neurons. The differential expression/activation of astroglia and the alteration of their intercommunication may be responsible for the different susceptibility of the DG in comparison to the CA1 and CA3 hippocampal areas to neurodegeneration during aging and inflammation.

Contribution of Astroglial Cx43 Hemichannels to the Modulation of Glutamatergic Currents by D-Serine in the Mouse Prefrontal Cortex.

Astrocytes interact dynamically with neurons by modifying synaptic activity and plasticity. This interplay occurs through a process named gliotransmission, meaning that neuroactive molecules are released by astrocytes. Acting as a gliotransmitter, D-serine, a co-agonist of the NMDA receptor at the glycine-binding site, can be released by astrocytes in a calcium [Ca2+]i-dependent manner. A typical feature of astrocytes is their high expression level of connexin43 (Cx43), a protein forming gap junction channels and hemichannels associated with dynamic neuroglial interactions. Pharmacological and genetic inhibition of Cx43 hemichannel activity reduced the amplitude of NMDA EPSCs in mouse layer 5 prefrontal cortex pyramidal neurons without affecting AMPA EPSC currents. This reduction of NMDA EPSCs was rescued by addition of D-serine in the extracellular medium. LTP of NMDA and AMPA EPSCs after high-frequency stimulation was reduced by prior inhibition of Cx43 hemichannel activity. Inactivation of D-serine synthesis within the astroglial network resulted in the reduction of NMDA EPSCs, which was rescued by adding extracellular D-serine. We showed that the activity of Cx43 hemichannels recorded in cultured astrocytes was [Ca2+]I dependent. Accordingly, in acute cortical slices, clamping [Ca2+]i at a low level in astroglial network resulted in an inhibition of NMDA EPSC potentiation that was rescued by adding extracellular D-serine. This work demonstrates that astroglial Cx43 hemichannel activity is associated with D-serine release. This process, occurring by direct permeation of D-serine through hemichannels or indirectly by Ca2+ entry and activation of other [Ca2+]i-dependent mechanisms results in the modulation of synaptic activity and plasticity.SIGNIFICANCE STATEMENT We recorded neuronal glutamatergic (NMDA and AMPA) responses in prefrontal cortex (PFC) neurons and used pharmacological and genetic interventions to block connexin-mediated hemichannel activity specifically in a glial cell population. For the first time in astrocytes, we demonstrated that hemichannel activity depends on the intracellular calcium concentration and is associated with D-serine release. Blocking hemichannel activity reduced the LTP of these excitatory synaptic currents triggered by high-frequency stimulation. These observations may be particularly relevant in the PFC, where D-serine and its converting enzyme are highly expressed.

Astrocytes in the Optic Nerve Head of Glaucomatous Mice Display a Characteristic Reactive Phenotype.

PurposeOptic nerve head astrocytes, a subtype of white-matter astrocytes, become reactive early in the course of glaucoma. It was shown recently that in the DBA/2J mouse model of inherited glaucoma optic nerve astrocytes extend new longitudinal processes into the axon bundles before ganglion cell loss becomes apparent. The present study aims at testing whether this behavior of astrocytes is typical of early glaucomatous damage.MethodsMice expressing green fluorescent protein in individual astrocytes were used to evaluate the early response of astrocytes in the glial lamina of the optic nerve head after increasing the IOP using the microbead occlusion method. Tissue sections from the glial lamina were imaged consecutively by confocal and electron microscopy.ResultsConfocal and electron microscope images show that astrocytes close to the myelination transition zone in the hypertensive nerve heads extend new processes that follow the longitudinal axis of the optic nerve and invade axon bundles in the nerve head. Ultrastructurally, the longitudinal processes were largely devoid of subcellular organelles except for degenerating mitochondria.ConclusionsThe longitudinal processes are a common feature of glaucomatous optic nerve astrocytes, whereas they are not observed after traumatic nerve injury. Thus, astrocytes appear to fine-tune their responses to the nature and/or timing of the injury to the neurons that they surround.

In Vivo Two-Photon Imaging of Astrocytes in GFAP-GFP Transgenic Mice.

Astrocytes play important roles in normal brain function and neurological diseases. In vivo two-photon excitation laser scanning microscopy has the potential to reveal rapid, dynamic structural changes in cells in a variety of physiological and pathological conditions. The type of in vivo imaging method has been shown to affect the plasticity of dendritic spines of neurons, but the optimal in vivo imaging methods of astrocytes have not been established. We compared open-skull and thinned-skull imaging methods for two-photon laser microscopy of live astrocytes in neocortex of GFAP-GFP transgenic mice. The thinned-skull method provided stable image intensity and morphological features of astrocytes in vivo over at least one week, with no evidence of astrogliosis. In contrast, the open-skull method resulted in significant changes in image intensity and induced astrogliosis. The thinned-skull method is the preferred approach for in vivo imaging of astrocytes under most conditions involving gross astrocyte modulation or causing astrogliosis.

Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

Employing an open-source tool to assess astrocyte tridimensional structure

Astrocytes display important features that allow them to maintain a close dialog with neurons, ultimately impacting brain function. The complex morphological structure of astrocytes is crucial to the role of astrocytes in brain networks. Therefore, assessing morphologic features of astrocytes will help provide insights into their physiological relevance in healthy and pathological conditions. Currently available tools that allow the tridimensional reconstruction of astrocytes present a number of disadvantages, including the need for advanced computational skills and powerful hardware, and are either time-consuming or costly. In this study, we optimized and validated the FIJI-ImageJ, Simple Neurite Tracer (SNT) plugin, an open-source software that aids in the reconstruction of GFAP-stained structure of astrocytes. We describe (1) the loading of confocal microscopy Z-stacks, (2) the selection criteria, (3) the reconstruction process, and (4) the post-reconstruction analysis of morphological features (process length, number, thickness, and arbor complexity). SNT allows the quantification of astrocyte morphometric parameters in a simple, efficient, and semi-automated manner. While SNT is simple to learn, and does not require advanced computational skills, it provides reproducible results, in different brain regions or pathophysiological states.

Human astrocytes are distinct contributors to the complexity of synaptic function.

Cellular components of synaptic circuits have been adjusted for increased human brain size, neural cell density, energy consumption and developmental duration. How does the human brain make these accommodations? There is evidence that astrocytes are one of the most divergent neural cell types in primate brain evolution and it is now becoming clear that they have critical roles in controlling synaptic development, function and plasticity. Yet, we still do not know how the precise developmental appearance of these cells and subsequent astrocyte-derived signals modulate diverse neuronal circuit subtypes. Here, we discuss what is currently known about the influence of glial factors on synaptic maturation and focus on unique features of human astrocytes including their potential roles in regenerative and translational medicine. Human astrocyte distinctiveness may be a major contributor to high level neuronal processing of the human brain and act in novel ways during various neuropathies ranging from autism spectrum disorders, viral infection, injury and neurodegenerative conditions.

Spatial organization of astrocytes in ferret visual cortex.

ABSTRACTAstrocytes form an intricate partnership with neural circuits to influence numerous cellular and synaptic processes. One prominent organizational feature of astrocytes is the “tiling” of the brain with non‐overlapping territories. There are some documented species and brain region–specific astrocyte specializations, but the extent of astrocyte diversity and circuit specificity are still unknown. We quantitatively defined the rules that govern the spatial arrangement of astrocyte somata and territory overlap in ferret visual cortex using a combination of in vivo two‐photon imaging, morphological reconstruction, immunostaining, and model simulations. We found that ferret astrocytes share, on average, half of their territory with other astrocytes. However, a specific class of astrocytes, abundant in thalamo‐recipient cortical layers (“kissing” astrocytes), overlap markedly less. Together, these results demonstrate novel features of astrocyte organization indicating that different classes of astrocytes are arranged in a circuit‐specific manner and that tiling does not apply universally across brain regions and species. J. Comp. Neurol. 524:3561–3576, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

High Resolution Dissection of Reactive Glial Nets in Alzheimer's Disease.

Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

Effects of Salt Loading on the Morphology of Astrocytes in the Ventral Glia Limitans of the Rat Supraoptic Nucleus.

In the ventral glial limitans (VGL) of the supraoptic nucleus (SON) of the rat, a unique astrocyte type is found with an ability to undergo striking morphological plasticity in response to a wide range of physiological stimulations such as chronic hypernatraemia. This includes a thinning of the VGL, which contains the somata and proximal processes of these astrocytes, as well as an almost complete withdrawal of their vertically-oriented distal processes. Currently, there is little information available on the types of astrocytes that reside in the SON-VGL and which of these exhibit state-dependent structural plasticity. To address this, we enabled the visualisation of single SON-VGL glia using two novel cell labelling techniques with fluorescence microscopy. First, we used an inducible genetic reporter mouse line that allowed the specific labelling of a low density of astrocytes expressing glutamate and aspartate transporter (GLAST)/excitatory amino acid transporter 1. This approach revealed a high degree of variability in the morphology of mouse SON-VGL astrocytes, in contrast to what has been reported for cortical astrocytes. Next, we used the DiOlistlic labelling approach to label single glial cells with DiI in the SON-VGL of rats. Astrocytes observed using this approach shared the morphological features of GLAST-expressing astrocytes in the mouse SON-VGL. Specific structural aspects of these cells were modified by chronic hypernatraemia achieved by 7-day salt loading. Notably, the average area of cells exhibiting protoplasmic features was significantly reduced in the horizontal plane, and the size of varicosities present on fibrous projections was significantly enlarged. These observations indicate that novel cell labelling methods can significantly advance our understanding of SON-VGL cells and reveal specific forms of morphological plasticity that can be driven by chronic hypernatraemia.