GABA-Glycine Cotransmitting Neurons in the Ventrolateral Medulla: Development and Functional Relevance for Breathing.

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.

Novel Mutations in the Asparagine Synthetase Gene (ASNS) Associated With Microcephaly.

Microcephaly is a devastating condition defined by a small head and small brain compared to the age- and sex-matched population. Mutations in a number of different genes causative for microcephaly have been identified, e.g., MCPH1, WDR62, and ASPM. Recently, mutations in the gene encoding the enzyme asparagine synthetase (ASNS) were associated to microcephaly and so far 24 different mutations in ASNS causing microcephaly have been described. In a family with two affected girls, we identified novel compound heterozygous variants in ASNS (c.1165G > C, p.E389Q and c.601delA, p.M201Wfs∗28). The first mutation (E389Q) is a missense mutation resulting in the replacement of a glutamate residue evolutionary conserved from Escherichia coli to Homo sapiens by glutamine. Protein modeling based on the known crystal structure of ASNS of E. coli predicted a destabilization of the protein by E389Q. The second mutation (p.M201Wfs∗28) results in a premature stop codon after amino acid 227, thereby truncating more than half of the protein. The novel variants expand the growing list of microcephaly causing mutations in ASNS.

Open housing drives the expression of immune response genes in the nasal mucosa, but not the olfactory bulb.

Nasal mucosa and olfactory bulb are separated by the cribriform plate which is perforated by olfactory nerves. We have previously demonstrated that the cribriform plate is permissive for T cells and monocytes and that viruses can enter the bulb upon intranasal injection by axonal transportation. Therefore, we hypothesized that nasal mucosa and olfactory bulb are equipped to deal with constant infectious threats. To detect genes involved in this process, we compared gene expression in nasal mucosa and bulb of mice kept under specific pathogen free (SPF) conditions to gene expression of mice kept on non-SPF conditions using RNA deep sequencing. We found massive alterations in the expression of immune-related genes of the nasal mucosa, while the bulb did not respond immunologically. The absence of induction of immune-related genes in the olfactory bulb suggests effective defence mechanisms hindering entrance of environmental pathogens beyond the outer arachnoid layer. The genes detected in this study may include candidates conferring susceptibility to meningitis.

Versatile and simple approach to determine astrocyte territories in mouse neocortex and hippocampus.

BACKGROUND: Besides their neuronal support functions, astrocytes are active partners in neuronal information processing. The typical territorial structure of astrocytes (the volume of neuropil occupied by a single astrocyte) is pivotal for many aspects of glia-neuron interactions. METHODS: Individual astrocyte territorial volumes are measured by Golgi impregnation, and astrocyte densities are determined by S100ß immunolabeling. These data are compared with results from conventionally applied methods such as dye filling and determination of the density of astrocyte networks by biocytin loading. Finally, we implemented our new approach to investigate age-related changes in astrocyte territories in the cortex and hippocampus of 5- and 21-month-old mice. RESULTS: The data obtained by our simplified approach based on Golgi impregnation were compared to previously published dye filling experiments, and yielded remarkably comparable results regarding astrocyte territorial volumes. Moreover, we found that almost all coupled astrocytes (as indicated by biocytin loading) were immunopositive for S100ß. A first application of this new experimental approach gives insight in age-dependent changes in astrocyte territorial volumes. They increased with age, while cell densities remained stable. In 5-month-old mice, the overlap factor was close to 1, revealing little or no interdigitation of astrocyte territories. However, in 21-month-old mice, the overlap factor was more than 2, suggesting that processes of adjacent astrocytes interdigitate. CONCLUSION: Here we verified the usability of a simple, versatile method for assessing astrocyte territories and the overlap factor between adjacent territories. Second, we found that there is an age-related increase in territorial volumes of astrocytes that leads to loss of the strict organization in non-overlapping territories. Future studies should elucidate the physiological relevance of this adaptive reaction of astrocytes in the aging brain and the methods presented in this study might be a powerful tool to do so.

Deletion of the cell adhesion adaptor protein vinculin disturbs the localization of GFAP in Bergmann glial cells.

Astrocytes operate in close spatial relationship to other cells including neurons. Structural interaction is controlled by a dynamic interplay between actin-based cell motility and contact formation via cell-cell and cell-extracellular matrix adhesions. A central player in the control of cell adhesion is the cytoskeletal adaptor protein Vinculin. Incorporation of Vinculin affects mechanical properties and turnover of cell adhesion sites. To study the in vivo function of Vinculin in astrocytes, a mouse line with astrocyte specific and inducible deletion of vinculin was generated. Deletion of vinculin decreased the expression of the glial acidic fibrillary protein (GFAP) in Bergmann glial cells in the cerebellum. In addition, localization of GFAP to Bergmann glial endfeet was disturbed, indicating a role for vinculin in controlling its expression and localization. In contrast, vimentin expression, morphology, activation state and polarity of the targeted cells as well as the localization of the extracellular matrix protein laminin was not compromised. Furthermore, stab wound lesions were performed in the cerebellar cortex. In both wildtype and vinculin knockout mice GFAP expression was upregulated in Bergmann glial cells of the lesioned area with no differences observed between genotypes in expression and localization of GFAP. These results propose a selective requirement for vinculin in cellular events related to cell adhesion in vivo. As in vitro data suggested a major role for vinculin in the control of the cytoskeletal connection affecting mechanical stability and cell motility, our data add a note of caution to the extrapolation of in vitro data to in vivo function.

Ca²âº signals of astrocytes are modulated by the NAD⁺/NADH redox state.

Astrocytes are important glial cells in the brain providing metabolic support to neurons as well as contributing to brain signaling. These different functional levels have to be highly coordinated to allow for proper cell and brain function. In this study, we show that in astrocytes the NAD(+) /NADH redox state modulates dopamine-induced Ca(2+) signals thereby connecting metabolism and Ca(2+) signaling. Application of dopamine induced a dose-dependent increase in Ca(2+) signal frequency in these cells, which was dependent on D(1) -receptor signaling, glycolytic activity, an increase in cytosolic NADH and inositol 1,4,5-triphosphate receptor operated intracellular Ca(2+) stores. Application of dopamine at a low concentration (1 µM) did not induce an increase in Ca(2+) signal frequency by itself. However, simultaneously increasing cytosolic NADH content either by direct application of NADH or by application of lactate resulted in a pronounced increase in Ca(2+) signal frequency. This increase could be blocked by co-application of pyruvate, suggesting that indeed the NAD(+) /NADH redox state is regulating Ca(2+) signals. We conclude that at the NAD(+) /NADH redox state metabolic and signaling information is integrated in astrocytes, thereby most likely contributing to precisely coordinate these different tasks of astrocytes.

The human ubiquitin C promoter drives selective expression in principal neurons in the brain of a transgenic mouse line.

The specificity of promoters used to drive the expression of proteins of interest is a crucial determinant of transgenesis. Numerous strategies have been developed to restrict expression on a certain cell population. On the other hand it has also remained challenging to obtain ubiquitous expression of transgenes which is needed for example to generate recombination reporter mice or to induce expression by recombination mediated excision of STOP-cassettes. We have generated transgenic mice with the expression of nuclear ß-galactosidase driven by the human ubiquitin C promoter thought to mediate ubiquitous expression. However, in the brains of these transgenic mice the expression of the transgene was strikingly limited to principal neurons, while no expression was detected in interneurons or glial cells. These results indicate that the human ubiquitin C promoter might be useful to selectively target projections neurons of the brain.

A role for glia in the progression of Rett's syndrome.

Rett's syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed in most tissues, loss of MeCP2 expression results primarily in neurological symptoms. Earlier studies suggested the idea that RTT is due exclusively to loss of MeCP2 function in neurons. Although defective neurons clearly underlie the aberrant behaviours, we and others showed recently that the loss of MECP2 from glia negatively influences neurons in a non-cell-autonomous fashion. Here we show that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter VGLUT1. Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports the targeting of glia as a strategy for improving the associated symptoms.

Genetic deletion of laminin isoforms ß2 and γ3 induces a reduction in Kir4.1 and aquaporin-4 expression and function in the retina.

BACKGROUND: Glial cells such as retinal Müller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Müller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Müller cells. METHODOLOGY/PRINCIPAL FINDINGS: The aim of the present study was to test the function of the ß2 and γ3 containing laminins in murine Müller cells. We used knockout mice with genetic deletion of both ß2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Müller cells from laminin ß2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice. CONCLUSION: These findings demonstrate a strong impact of laminin ß2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Müller cells.

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression.

Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia-reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild-type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)) in which glial cells are consequently devoid of IFs, and normal Long-Evans rats. In response to ischemia-reperfusion, glial cells stiffened significantly in wild-type mice and rats but were unchanged in GFAP(-/-)Vim(-/-) mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up-regulation in reactive glial cells may facilitate neuroregeneration.

Signalling of sphingosine-1-phosphate in Müller glial cells via the S1P/EDG-family of G-protein-coupled receptors.

Signalling of sphingosine-1-phosphate (S1P) via G-protein-coupled receptors of the Endothelial Differentiation Gene family differentially regulates cellular processes such as migration, proliferation and morphogenesis in a variety of cell types. Proliferation and migration of retinal Müller glial cells are involved in pathological events such as proliferative vitreoretinopathy and proliferative diabetic retinopathy. Investigation of possible functional roles of S1P receptors might thus open new insights into Müller cell pathophysiology. Here we show that cultured Müller cells from the guinea pig retina respond to application of S1P with an increase in the intracellular calcium content in a concentration-dependent manner (EC(50) 11nM). This calcium increase consists of two components; an initial fast peak and a slow plateau component. The initial transient is caused by a release of calcium from intracellular stores and is suppressed by U-73122, a selective phospholipase C inhibitor. The slow plateau component is caused by a calcium influx. These results suggest that the S1P-induced calcium response in Müller cells partially involves signalling via G-protein-coupled receptors. Moreover, S1P slightly induced Müller cell migration but no proliferation. Thus, the data indicate that Müller cells might be involved in S1P signalling in the retina.

Alterations in protein expression and membrane properties during Müller cell gliosis in a murine model of transient retinal ischemia.

Retinal Müller glial cells are involved in K+ ion homeostasis of the tissue. Inwardly rectifying K(+) (Kir) channels play a decisive role in the process of spatial K+ buffering. It has been demonstrated that Kir-mediated currents of Müller cells are downregulated in various cases of retinal neurodegeneration. However, this has not yet been verified for any murine animal model. The aim of the present study was to investigate Müller cells after transient retinal ischemia in mice. High intraocular pressure was applied for 1h; the retina was analysed 1 week later. We studied protein expression in the tissue by immunohistochemistry, and membrane currents of isolated cells by patch-clamp experiments. We found the typical indicators of reactive gliosis such as upregulation of glial fibrillary acidic protein. Moreover, the membrane capacitance of isolated Müller cells was increased and the amplitudes of Kir-mediated currents were slightly, but significantly decreased. This murine high intraocular pressure model of transient retinal ischemia is proposed as a versatile tool for further studies on Müller cell functions in retinal degeneration.

Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation.

BACKGROUND: DNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2. METHODOLOGY/PRINCIPAL FINDINGS: In the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC(50)-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM. CONCLUSIONS/SIGNIFICANCE: These characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters.

Split-cre complementation indicates coincident activity of different genes in vivo.

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive "split-Cre" fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting.

Osmotic swelling characteristics of glial cells in the murine hippocampus, cerebellum, and retina in situ.

Glial cells are proposed to play a major role in the ionic and osmotic homeostasis in the CNS. Swelling of glial cells contributes to the development of edema in neural tissue under pathological conditions such as trauma and ischemia. In this study, we compared the osmotic swelling characteristics of murine hippocampal astrocytes, cerebellar Bergmann glial cells, and retinal Müller glial cells in acutely isolated tissue slices in response to hypoosmotic stress and pharmacological blockade of Kir channels. Hypoosmotic challenge induced an immediate swelling of somata in the majority of Bergmann glial cells and hippocampal astrocytes investigated, whereas Müller cell bodies displayed a substantial delay in the onset of swelling and hippocampal astroglial processes remained unaffected. Blockade of Kir channels under isoosmotic conditions had no swelling-inducing effect in Müller cell somata but caused a swelling in brain astrocytic somata and processes. Blockade of Kir channels under hypoosmotic conditions induced an immediate and strong swelling in Müller cell somata, but had no cumulative effect to brain astroglial somata. No regulatory volume decrease could be observed in all cell types. The data suggest that Kir channels are differently implicated in cell volume homeostasis of retinal Müller cells and brain astrocytes and that Müller cells and brain astrocytes differ in their osmotic swelling properties.

Glycine transporter 1 expression in the ventral respiratory group is restricted to protoplasmic astrocytes.

The lack of the glycine transporter 1 (GlyT1) leads to early postnatal death due to failure of respiratory network activity. Here we demonstrate a segregated expression of GlyT1 on different astroglial cell populations of the ventral respiratory group. In TgN(hGFAP-EGFP) mice a combined immunohistochemical and electrophysiological approach was used to define the cellular expression of GlyT1 in the respiratory network. EGFP-labeled cells with outwardly rectifying current-voltage relationship did not express glycine transporter 1, while GlyT1 was abundantly expressed in mature protoplasmic astrocytes, which are electrophysiologically characterized by a large potassium conductance, a more negative membrane potential and the expression of glutamate transporters. Taken together, the vital capacity for the clearance of extracellular glycine is restricted to a subpopulation of astroglial cells.

Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2.

Inducible gene modification using the Cre/loxP system provides a valuable tool for the analysis of gene function in the active animal. GFAP-Cre transgenic mice have been developed to achieve gene recombination in astrocytes, the most abundant cells of the central nervous system, with pivotal roles during brain function and pathology. Unfortunately, these mice displayed neuronal recombination as well, since the GFAP promoter is also active in embryonic radial glia, which possess a substantial neurogenic potential. To enable the temporal control of gene deletions in astrocytes only, we generated a transgenic mouse with expression of CreERT2, a fusion protein of the DNA recombinase Cre and a mutated ligand-binding domain of the estrogen receptor, under the control of the human GFAP promoter. In offspring originating from crossbreedings of GFAP-CreERT2-transgenic mice with various Cre-sensitive reporter mice, consecutive intraperitoneal injections of tamoxifen induced genomic recombination selectively in astrocytes of almost all brain regions. In Bergmann glia, which represent the main astroglial cell population of the cerebellum, virtually all cells showed successful gene recombination. When adult mice received cortical stab wound lesions, simultaneously given tamoxifen induced substantial recombination in reactive glia adjacent to the site of injury. These transgenic GFAP-CreERT2 mice will allow the functional analysis of loxP-modified genes in astroglia of the postnatal and adult brain.

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing.

Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission "bleed-through" of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies.

Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice.

Reef coral fluorescent proteins (RCFPs) are bright fluorescent proteins (FPs) covering a wide spectral range. We used various RCFP genes to transgenically color different cell populations in the brain. The mouse Thy1.2 promoter was used to target expression of HcRed1 in neurons, the human glial fibrillary acidic protein (GFAP) promoter to label astrocytes with AmCyan1, AsRed2 and mRFP1 as well as the mouse proteolipid protein promoter to mark oligodendrocytes with DsRed1. In brain sections of transgenic mice, RCFP expression was found to be highly specific using immunohistochemistry and fluorescence microscopy. In contrast to transgenic mice with expression of jellyfish FP variants, RCFPs formed numerous fluorescent precipitates. These aggregates were primarily found in cell somata and also in cell processes. Older mice were more affected than younger ones. Despite these fluorescent deposits, physiological properties of RCFP expressing brain cells such as whole-cell membrane currents or glutamate-evoked calcium signaling seemed to be unaffected. While brightness and spectral variation of RCFPs are optimal for expression in transgenic animals used in physiological experiments, the formation of fluorescent precipitates in various cell types limits their use for morphological cell analysis in situ.