Microglial morphology in the somatosensory cortex across lifespan. A quantitative study.

BACKGROUND: Microglia are long-lived cells that constantly monitor their microenvironment. To accomplish this task, they constantly change their morphology both in the short and long term under physiological conditions. This makes the process of quantifying physiological microglial morphology difficult. RESULTS: By using a semi-manual and a semi-automatic method to assess fine changes in cortical microglia morphology, we were able to quantify microglia changes in number, surveillance and branch tree starting from the fifth postnatal day to 2 years of life. We were able to identify a fluctuating behavior of most analyzed parameters characterized by a rapid cellular maturation, followed by a long period of relative stable morphology during the adult life with a final convergence to an aged phenotype. Detailed cellular arborization analysis revealed age-induced differences in microglia morphology, with mean branch length and the number of terminal processes changing constantly over time. CONCLUSIONS: Our study provides insight into microglia morphology changes across lifespan under physiological conditions. We were able to highlight, that due to the dynamic nature of microglia several morphological parameters are needed to establish the physiological state of these cells.

A subset of OPCs do not express Olig2 during development which can be increased in the adult by brain injuries and complex motor learning.

Oligodendrocyte precursor cells (OPCs) are uniformly distributed in the mammalian brain; however, their function is rather heterogeneous in respect to their origin, location, receptor/channel expression and age. The basic helix-loop-helix transcription factor Olig2 is expressed in all OPCs as a pivotal determinant of their differentiation. Here, we identified a subset (2%-26%) of OPCs lacking Olig2 in various brain regions including cortex, corpus callosum, CA1 and dentate gyrus. These Olig2 negative (Olig2neg ) OPCs were enriched in the juvenile brain and decreased subsequently with age, being rarely detectable in the adult brain. However, the loss of this population was not due to apoptosis or microglia-dependent phagocytosis. Unlike Olig2pos OPCs, these subset cells were rarely labeled for the mitotic marker Ki67. And, accordingly, BrdU was incorporated only by a three-day long-term labeling but not by a 2-hour short pulse, suggesting these cells do not proliferate any more but were derived from proliferating OPCs. The Olig2neg OPCs exhibited a less complex morphology than Olig2pos ones. Olig2neg OPCs preferentially remain in a precursor stage rather than differentiating into highly branched oligodendrocytes. Changing the adjacent brain environment, for example, by acute injuries or by complex motor learning tasks, stimulated the transition of Olig2pos OPCs to Olig2neg cells in the adult. Taken together, our results demonstrate that OPCs transiently suppress Olig2 upon changes of the brain activity.

Novel algorithms for improved detection and analysis of fluorescent signal fluctuations.

Fluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra- as well as intercellular communication. Using advanced imaging technologies, fluorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and analysis of fluorescence signals require to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection, and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation and automated detection and segmentation of fluorescence fluctuations.

Specific detection and deletion of the sigma-1 receptor widely expressed in neurons and glial cells in vivo.

The chaperon protein sigma-1 receptor (S1R) has been discovered over 40 years ago. Recent pharmacological studies using S1R exogenous ligands demonstrated a promising therapeutical potential of targeting the S1R in several neurological disorders. Although intensive in vitro studies have revealed S1Rs are mainly residing at the membrane of the endoplasmic reticulum (ER), the cell-specific in vivo expression pattern of S1Rs is still unclear, mainly because of the lack of a reliable detection method which also prevented a comprehensive functional analysis. Here, first, we identified a highly specific antibody using S1R knockout (KO) mice and established an immunohistochemical protocol involving a 1% sodium dodecyl sulphate (SDS) antigen retrieval step. Second, we characterized the S1R expression in the mouse brain and can demonstrate that the S1R is widely expressed: in principal neurons, interneurons and all glial cell types. In addition, unlike reported in previous studies, we showed that the S1R expression in astrocytes is not colocalized with the astrocytic cytoskeleton protein GFAP. Thus, our results raise concerns over previously reported S1R properties. Finally, we generated a Cre-dependent S1R conditional KO mouse (S1R flox) to study cell-type-specific functions of the S1R. As a proof of concept, we successfully ablated S1R expressions in neurons or microglia employing neuronal and microglial Cre-expressing mice, respectively. In summary, we provide powerful tools to cell-specifically detect, delete and functionally characterize S1R in vivo.

Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures.

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell-neuron interactions. To induce inflammation, we applied T cell conditioned media, which contains a variety of cytokines, during CD8+ T cell polyclonal activation. Release of IFNγ and TNFα from co-cultures was verified by ELISA, confirming an inflammatory response. We also visualized the physical interactions between CD8+ T cells and cortical neurons using live-cell confocal imaging. The imaging revealed that T cells reduced their migration velocity and changed their migratory patterns under inflammatory conditions. CD8+ T cells increased their dwell time at neuronal soma and dendrites in response to added cytokines. These changes were seen in both the in vitro and ex vivo models. The results confirm that these in vitro and ex vivo models provide promising platforms for the study of the molecular details of neuron-immune cell interactions under inflammatory conditions, which allow high-resolution live microscopy and are readily amenable to experimental manipulation.

Cannabidiol Exerts a Neuroprotective and Glia-Balancing Effect in the Subacute Phase of Stroke.

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca2+ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca2+ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca2+ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies.

Epigenetic control of region-specific transcriptional programs in mouse cerebellar and cortical astrocytes.

Astrocytes from the cerebral cortex (CTX) and cerebellum (CB) share basic molecular programs, but also form distinct spatial and functional subtypes. The regulatory epigenetic layers controlling such regional diversity have not been comprehensively investigated so far. Here, we present an integrated epigenome analysis of methylomes, open chromatin, and transcriptomes of astroglia populations isolated from the cortex or cerebellum of young adult mice. Besides a basic overall similarity in their epigenomic programs, cortical astrocytes and cerebellar astrocytes exhibit substantial differences in their overall open chromatin structure and in gene-specific DNA methylation. Regional epigenetic differences are linked to differences in transcriptional programs encompassing genes of region-specific transcription factor networks centered around Lhx2/Foxg1 in CTX astrocytes and the Zic/Irx families in CB astrocytes. The distinct epigenetic signatures around these transcription factor networks point to a complex interconnected and combinatorial regulation of region-specific transcriptomes. These findings suggest that key transcription factors, previously linked to temporal, regional, and spatial control of neurogenesis, also form combinatorial networks important for astrocytes. Our study provides a valuable resource for the molecular basis of regional astrocyte identity and physiology.

Role of sodium channel subtype in action potential generation by neocortical pyramidal neurons.

Neocortical pyramidal neurons express several distinct subtypes of voltage-gated Na+ channels. In mature cells, Nav1.6 is the dominant channel subtype in the axon initial segment (AIS) as well as in the nodes of Ranvier. Action potentials (APs) are initiated in the AIS, and it has been proposed that the high excitability of this region is related to the unique characteristics of the Nav1.6 channel. Knockout or loss-of-function mutation of the Scn8a gene is generally lethal early in life because of the importance of this subtype in noncortical regions of the nervous system. Using the Cre/loxP system, we selectively deleted Nav1.6 in excitatory neurons of the forebrain and characterized the excitability of Nav1.6-deficient layer 5 pyramidal neurons by patch-clamp and Na+ and Ca2+ imaging recordings. We now report that, in the absence of Nav1.6 expression, the AIS is occupied by Nav1.2 channels. However, APs are generated in the AIS, and differences in AP propagation to soma and dendrites are minimal. Moreover, the channels that are expressed in the AIS still show a clear hyperpolarizing shift in voltage dependence of activation, compared with somatic channels. The only major difference between Nav1.6-null and wild-type neurons was a strong reduction in persistent sodium current. We propose that the molecular environment of the AIS confers properties on whatever Na channel subtype is present and that some other benefit must be conferred by the selective axonal presence of the Nav1.6 channel.

Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens.

Multiple sclerosis (MS) has been considered to specifically affect the central nervous system (CNS) for a long time. As autonomic dysfunction including dysphagia can occur as accompanying phenomena in patients, the enteric nervous system has been attracting increasing attention over the past years. The aim of this study was to identify glial and myelin markers as potential target structures for autoimmune processes in the esophagus. RT-PCR analysis revealed glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), and myelin basic protein (MBP) expression, but an absence of myelin oligodendrocyte glycoprotein (MOG) in the murine esophagus. Selected immunohistochemistry for GFAP, PLP, and MBP including transgenic mice with cell-type specific expression of PLP and GFAP supported these results by detection of (1) GFAP, PLP, and MBP in Schwann cells in skeletal muscle and esophagus; (2) GFAP, PLP, but no MBP in perisynaptic Schwann cells of skeletal and esophageal motor endplates; (3) GFAP and PLP, but no MBP in glial cells surrounding esophageal myenteric neurons; and (4) PLP, but no GFAP and MBP in enteric glial cells forming a network in the esophagus. Our results pave the way for further investigations regarding the involvement of esophageal glial cells in the pathogenesis of dysphagia in MS.

Sublamina-specific organization of the blood brain barrier in the mouse olfactory nerve layer.

Astrocytes constitute the main glial component of the mammalian blood brain barrier (BBB). However, in the olfactory bulb (OB), the olfactory nerve layer (ONL) is almost devoid of astrocytes, raising the question which glial cells are part of the BBB. We used mice expressing EGFP in astrocytes and tdTomato in olfactory ensheathing cells (OECs), a specialized type of glial cells in the ONL, to unequivocally identify both glial cell types and investigate their contribution to the BBB in the olfactory bulb. OECs were located exclusively in the ONL, while somata of astrocytes were located in deeper layers and extended processes in the inner sublamina of the ONL. These processes surrounded blood vessels and contained aquaporin-4, an astrocytic protein enriched at the BBB. In the outer sublamina of the ONL, in contrast, blood vessels were surrounded by aquaporin-4-negative processes of OECs. Transcardial perfusion of blood vessels with lanthanum and subsequent visualization by electron microscopy showed that blood vessels enwrapped by OECs possessed intact tight junctions. In acute olfactory bulb preparations, injection of fluorescent glucose 6-NBDG into blood vessels resulted in labeling of OECs, indicating glucose transport from the perivascular space into OECs. In addition, Ca2+ transients in OECs in the outer sublamina evoked vasoconstriction, whereas Ca2+ signaling in OECs of the inner sublamina had no effect on adjacent blood vessels. Our results demonstrate that the BBB in the inner sublamina of the ONL contains astrocytes, while in the outer ONL OECs are part of the BBB.

Nerve/glial antigen (NG) 2 is a crucial regulator of intercellular adhesion molecule (ICAM)-1 expression.

The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses.

Panglial gap junctions between astrocytes and olfactory ensheathing cells mediate transmission of Ca2+ transients and neurovascular coupling.

Astrocytes are arranged in highly organized gap junction-coupled networks, communicating via the propagation of Ca2+ waves. Astrocytes are gap junction-coupled not only to neighboring astrocytes, but also to oligodendrocytes, forming so-called panglial syncytia. It is not known, however, whether glial cells in panglial syncytia transmit information using Ca2+ signaling. We used confocal Ca2+ imaging to study intercellular communication between astrocytes and olfactory ensheathing glial cells (OECs) in in-toto preparations of the mouse olfactory bulb. Our results demonstrate that Ca2+ transients in juxtaglomerular astrocytes, evoked by local photolysis of "caged" ATP and "caged" tACPD, led to subsequent Ca2+ responses in OECs. This transmission of Ca2+ responses from astrocytes to OECs persisted in the presence of neuronal inhibition, but was absent when gap junctional coupling was suppressed with carbenoxolone. When Ca2+ transients were directly evoked in OECs by puff application of DHPG, they resulted in delayed Ca2+ responses in juxtaglomerular astrocytes, indicating that panglial transmission of Ca2+ signals occurred in a bidirectional manner. In addition, panglial transmission of Ca2+ signals from astrocytes to OECs resulted in vasoconstriction of OEC-associated blood vessels in the olfactory nerve layer. Our results demonstrate functional transmission of Ca2+ signals between different classes of glial cells within gap junction-coupled panglial networks and the resulting regulation of blood vessel diameter in the olfactory bulb.

Early embryonic NG2 glia are exclusively gliogenic and do not generate neurons in the brain.

In the central nervous system, the type I transmembrane glycoprotein NG2 (nerve-glia antigen 2) is only expressed by pericytes and oligodendrocyte precursor cells (OPCs). Therefore, OPCs are also termed NG2 glia. Their fate during development has been investigated systematically in several genetically modified mouse models. Consensus exists that postnatal NG2 glia are restricted to the oligodendrocyte (OL) lineage, while, at least in the forebrain, embryonic NG2 glia could also generate astrocytes. In addition, experimental evidence for a neurogenic potential of NG2 glia in the early embryonic brain (before E16.5) has been provided. However, this observation is still controversial. Here, we took advantage of reliable transgene expression in NG2-EYFP and NG2-CreERT2 knock-in mice to study the fate of early embryonic NG2 glia. While pericytes were the main cells with robust NG2 gene activity at E12.5, only a few OPCs expressed NG2 at this early stage of embryogenesis. Subsequently, this proportion of OPCs increased from 3% (E12.5) to 11% and 25% at E14.5 and E17.5, respectively. When Cre DNA recombinase activity was induced at E12.5 and E14.5 and pups were analyzed at postnatal day 0 (P0) and P10, the vast majority of recombined cells, besides pericytes, belonged to the OL lineage cells, with few astrocytes in the ventral forebrain. In other brain regions such as brain stem, cerebellum, and olfactory bulb only OL lineage cells were detected. Therefore, we conclude that NG2 glia from early embryonic brain are restricted to a gliogenic fate and do not differentiate into neurons after birth.

Nerve/glial antigen 2 is crucially involved in the revascularization of freely transplanted pancreatic islets.

Pancreatic islets are highly vascularized endocrine units. Accordingly, their adequate revascularization is of major importance for successful islet transplantation. The proteoglycan, nerve/glial antigen 2 (NG2) expressed in pericytes is a crucial regulator of angiogenesis. Therefore, we herein analyze whether this surface protein contributes to the revascularization of grafted islets. Islets were isolated from NG2+/+ (wild-type) and NG2-/- mice and their cellular composition was analyzed by immunohistochemical detection of insulin, glucagon, somatostatin and CD31. Moreover, insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). In addition, isolated islets were transplanted into dorsal skinfold chambers of wild-type mice and their revascularization was determined by intravital fluorescence microscopy and immunohistochemistry. NG2+/+ and NG2-/- islets did not differ in their cellular composition and insulin secretion. However, transplanted NG2-/- islets exhibited a significantly lower functional capillary density and a reduced number of CD31-positive microvessels. These findings demonstrate that the loss of NG2 impairs the revascularization of transplanted islets, underlining the importance of this pericytic proteoglycan for islet engraftment.

Tanycytes and a differential fatty acid metabolism in the hypothalamus.

Although the brain controls all main metabolic pathways in the whole organism, its lipid metabolism is partially separated from the rest of the body. Circulating lipids and other metabolites are taken up into brain areas like the hypothalamus and are locally metabolized and sensed involving several hypothalamic cell types. In this study we show that saturated and unsaturated fatty acids are differentially processed in the murine hypothalamus. The observed differences involve both lipid distribution and metabolism. Key findings were: (i) hypothalamic astrocytes are targeted by unsaturated, but not saturated lipids in lean mice; (ii) in obese mice labeling of these astrocytes by unsaturated oleic acid cannot be detected unless ß-oxidation or ketogenesis is inhibited; (iii) the hypothalamus of obese animals increases ketone body and neutral lipid synthesis while tanycytes, hypothalamic cells facing the ventricle, increase their lipid droplet content; and (iv) tanycytes show different labeling for saturated or unsaturated lipids. Our data support a metabolic connection between tanycytes and astrocytes likely to impact hypothalamic lipid sensing. GLIA 2017;65:231-249.

TRPC1- and TRPC3-dependent Ca2+ signaling in mouse cortical astrocytes affects injury-evoked astrogliosis in vivo.

Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.

The Role of the Oligodendrocyte Lineage in Acute Brain Trauma.

An acute brain injury is commonly characterized by an extended cellular damage. The post-injury process of scar formation is largely determined by responses of various local glial cells and blood-derived immune cells. The role of astrocytes and microglia have been frequently reviewed in the traumatic sequelae. Here, we summarize the diverse contributions of oligodendrocytes (OLs) and their precursor cells (OPCs) in acute injuries. OLs at the lesion site are highly sensitive to a damaging insult, provoked by Ca2+ overload after hyperexcitation originating from increased levels of transmitters. At the lesion site, differentiating OPCs can replace injured oligodendrocytes to guarantee proper myelination that is instrumental for healthy brain function. In contrast to finally differentiated and non-dividing OLs, OPCs are the most proliferative cells of the brain and their proliferation rate even increases after injury. There exist even evidence that OPCs might also generate some type of astrocyte beside OLs. Thereby, OPCs can contribute to the generation and maintenance of the glial scar. In the future, detailed knowledge of the molecular cues that help to prevent injury-evoked glial cell death and that control differentiation and myelination of the oligodendroglial lineage will be pivotal in developing novel therapeutic approaches.

Phenotype and distribution pattern of nestin-GFP-expressing cells in murine myenteric plexus.

The enteric nervous system has to adapt to altering dietary or environmental conditions and presents an enormous plasticity that is conserved over the whole lifespan. It harbours neural-crest-derived neurons, glial cells and their precursors. Based on a nestin-green fluorescent protein (NGFP) transgenic model, a histological inventory has been performed to deliver an overview of neuronal and glial markers for the various parts of the gastrointestinal tract in newborn (postnatal day 7) and adult mice under homeostatic conditions. Whereas NGFP-positive glial cells can be found in all parts of the gut at any individual age, a specific NGFP population is present with both neuronal morphology and marker expression in the myenteric plexus (nNGFP). These cells appear in variable quantities, depending on age and location. Their overall abundance decreases from newborn to adults and their spatial distribution is also age-dependent. In newborn gut, nNGFP cells are found in similar quantities throughout the gut, with a significantly lower presence in the duodenum. Their expression increases in the adult mouse from the stomach to the colon. All of these nNGFP cells expressed either (but not both) of the glia markers S100 or glial fibrillary acidic protein (GFAP). In the S100-positive glia population, a subset of cells also shows a neuronal morphology (nS100), without expressing nestin. Thus, the presence of premature neurons that express NGFP demonstrates that neurogenesis takes place far beyond birth. In enteric neurons, NGFP acts as a marker for neuronal plasticity showing the differentiation and change in the phenotype of neuronal precursor cells.

Evaluation of TgH(CX3CR1-EGFP) mice implanted with mCherry-GL261 cells as an in vivo model for morphometrical analysis of glioma-microglia interaction.

BACKGROUND: Glioblastoma multiforme is the most aggressive brain tumor. Microglia are prominent cells within glioma tissue and play important roles in tumor biology. This work presents an animal model designed for the study of microglial cell morphology in situ during gliomagenesis. It also allows a quantitative morphometrical analysis of microglial cells during their activation by glioma cells. METHODS: The animal model associates the following cell types: 1- mCherry red fluorescent GL261 glioma cells and; 2- EGFP fluorescent microglia, present in the TgH(CX3CR1-EGFP) mouse line. First, mCherry-GL261 glioma cells were implanted in the brain cortex of TgH(CX3CR1-EGFP) mice. Epifluorescence - and confocal laser-scanning microscopy were employed for analysis of fixed tissue sections, whereas two-photon laser-scanning microscopy (2P-LSM) was used to track tumor cells and microglia in the brain of living animals. RESULTS: Implanted mCherry-GL261 cells successfully developed brain tumors. They mimic the aggressive behavior found in human disease, with a rapid increase in size and the presence of secondary tumors apart from the injection site. As tumor grows, mCherry-GL261 cells progressively lost their original shape, adopting a heterogeneous and diffuse morphology at 14-18 d. Soma size increased from 10-52 µm. At this point, we focused on the kinetics of microglial access to glioma tissues. 2P-LSM revealed an intense microgliosis in brain areas already shortly after tumor implantation, i.e. at 30 min. By confocal microscopy, we found clusters of microglial cells around the tumor mass in the first 3 days. Then cells infiltrated the tumor area, where they remained during all the time points studied, from 6-18 days. Microglia in contact with glioma cells also present changes in cell morphology, from a ramified to an amoeboid shape. Cell bodies enlarged from 366 ± 0.0 µm(2), in quiescent microglia, to 1310 ± 146.0 µm(2), and the cell processes became shortened. CONCLUSIONS: The GL261/CX3CR1 mouse model reported here is a valuable tool for imaging of microglial cells during glioma growth, either in fixed tissue sections or living animals. Remarkable advantages are the use of immunocompetent animals and the simplified imaging method without the need of immunohistochemical procedures.

Novel NG2-CreERT2 knock-in mice demonstrate heterogeneous differentiation potential of NG2 glia during development.

NG2 (nerve/glia antigen-2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2-expressing (NG2(+) ) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2(+) cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter(+) astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen-sensitive and NG2 gene locus-dependent gene recombination could be detected in a small, but persistent population of cortical NeuN(+) neurons starting from the second postnatal week.