NeuroGLIA Consortium: neuron-astroglia in brain function and dysfunction

Brief description and aims of work

The Department of Molecular Physiology is part of the Institute of Physiology at the Medical Faculty University of Saarland in Homburg, Germany. Our research focuses on the molecular and cellular mechanisms of neuron-glia interaction in the central nervous system.

We are pursuing three main research questions:

• How do neurons and astrocytes interact with each other? Are Ca²⁺ signals mediators or only indicators of coordinated network activities?
• How do glial transmitter receptors sense and modulate synaptic transmission? What is the impact for living organisms?
• How do glial cells respond to injuries within the central nervous system?

For functional analyses we generated (and are still continuing to develop) transgenic mouse models with cell-type specific expression of various fluorescent proteins (FPs) and inducible gene deletion. We are applying a combination of biochemical and molecular biological methods together with imaging techniques such as two-photon laser-scanning microscopy (2P-LSM) or CCD imaging.

Previous findings

Transgenic mouse lines with cell-type specific fluorescent protein expression in the nervous system
Over the last years my group has generated a series of transgenic mouse models which enabled us and others to study various plastic processes of the nervous system in vitro, in situ and in vivo (Hirrlinger et al., 2005; Nimmerjahn et al., 2004; Malatesta et al., 2003; Matthias et al., 2003; Nolte et al., 2001). The individual mouse lines express various spectrally different FPs under the control of promoters specific for neurons, astrocytes and oligodendrocytes. In various brain regions, cells and their respective cell-type can be easily identified by conventional fluorescence light microscopy without prior cumbersome staining techniques. Imaging of these mice allows short- and long-term analyses of individual cells and their compartments in vivo or in situ, e.g., in the forebrain or the spinal cord. In this way, we were able to identify functional NMDA-type glutamate receptors at the distal processes of astrocytes (Schipke et al., 2001; Lalo et al., 2006; Kirchhoff and Verkhratsky, 2007). In addition, we could show that such process endings which are in close contact to active synaptic terminals are highly motile (Hirrlinger et al., 2004). This work is supported by the DFG Research Center Molecular Physiology of the Brain.

Temporal control control of astroglial gene deletion in vivo
Our latest transgenic mouse line sets us in a position to study the functional impact of genes expressed by astrocytes in the living mouse. In the progeny of TgN(hGFAP-CreERT2) mice crossbred to mice with loxP-flanked DNA fragments, the synthetic estrogen receptor ligand tamoxifen induces specific astroglial gene recombination, i.e., gene ablation (Hirrlinger et al., 2006). Currently, we are studying the role of astroglial AMPA-type glutamate receptors.

Analysis of glial cells by in vivo two-photon laser-scanning microscopy
The strength of in vivo 2-photon laser scanning microscopy is best illustrated by the publications resulting from the collaborations with Fritjof Helmchen (University Zurich; Nimmerjahn et al., 2004, 2005). Using transgenic mice with cell-type specific FP expression and/or vital dye labelling we could simultaneously demonstrate the dynamics of neuronal and astroglial Ca²⁺ signals. In addition, by imaging through the thinned skull of mice with green fluorescent microglia, we were the first who observed the highly dynamic motility of microglial processes permanently surveying their environment under so-called resting conditions. After localized, laser-induced micro-lesions to a small brain capillary, microglial cells sent immediately processes toward the lesion site, which then protected the brain parenchyma from further damage by closing the hole in the capillary.

Implications of our research

Transgenic mouse models with cell-type specific fluorescent protein expression and two-photon laser-scanning microscopy are important tools and techniques to study the dynamic interactions of individual cells and cell types — in our case, between neurons and astrocytes, within the nervous system in situ and in vivo. In addition, the generation of transgenic mice with inducible and astroglia-specific gene deletion represent an essential paradigm to evaluate the impact on a living animal of a given molecular component expressed in astrocytes.

Selected references

1. Nolte C, Matyash M, Pivneva T, Schipke CG, Ohlemeyer C, Hanisch UK, Kirchhoff F, Kettenmann H (2001) GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33:72-86.
2. Schipke CG, Ohlemeyer C, Matyash M, Nolte C, Kettenmann H, Kirchhoff F (2001) Astrocytes of the mouse neocortex express functional N-methyl-D-aspartate receptors. FASEB J. 15:1270-2.
3. Hirrlinger J, Hülsmann S, Kirchhoff F (2004) Astroglial processes show spontaneous motility at active synaptic terminals in situ. Eur. J. Neurosci. 20:2235-9.
4. Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1:31-7.
5. Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314-8.
6. Hirrlinger PG, Scheller A, Braun C, Quintela-Schneider M, Fuss B, Hirrlinger J, Kirchhoff F (2005) Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice. Mol. Cell. Neurosci. 30:291-303.
7. Hirrlinger PG, Scheller A, Braun C, Hirrlinger J, Kirchhoff F (2006) Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2. Glia 54:11-20.

Past and present collaborations

• Arthur Butt · Cellular Neurophysiology, University of Portsmouth, UK
• Babette Fuss · Institute of Anatomy and Neurobiology, Virginia Commwealth University, Richmond, VA, USA
• Magdalena Götz · Institute of Stem Cell Research, GSF, Munich, Germany
• Fritjof Helmchen · Institute of Brain Research, University of Zurich, Switzerland
• Johannes Hirrlinger · Interdisciplinary Centre for Clinical Research (IZKF) Leipzig, University of Leipzig, Germany
• Swen Hülsmann · Dept of Sensory and Neurophysiology, University of Göttingen, Göttingen, Germany
• Helmut Kettenmann · Cellular Neurosciences, Max Delbrück Center for Molecular Medicine, Berlin, Germany
• Clemens Neusch · Dept of Neurology, University of Göttingen, Göttingen, Germany
• Axel Nimmerjahn · Stanford University, CA, USA
• Martin Oheim · Neurophysiology & New Microscopies Laboratory, University Déscartes, Paris, France
• Andreas Reichenbach · Paul Flechsig Institute of Brain Research, University of Leipzig, Germany
• Christian Steinhäuser · Institute of Cellular Neurosciences, University of Bonn, Germany
• Jacky Trotter · Molecular Cell Biology, University of Mainz, Germany
• Alexej Verkhratsky · Faculty for Life Sciences, University of Manchester, UK