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Editorial
e-Neuroforum 2015 · 6:59–62
DOI 10.1007/s13295-015-0012-0
Published online: 31 July 2015
© Springer-Verlag Berlin Heidelberg 2015
Christine R. Rose1 · Frank Kirchhoff2
1 Institute of Neurobiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
2 Molecular Physiology, Center for Integrative Physiology and Molecular
Medicine (CIPMM), University of Saarland, Homburg, Germany
Glial heterogeneity: the
increasing complexity
of the brain
“How does the brain work?” is one of the
most frequently asked questions. By and
large, the human brain consists of approximately 160 billion cells that mainly represent two types, classified as neurons and
glia, each contributing about 50 % to the
total cell number. Modern imaging techniques such as functional magnetic resonance imaging (fMRI) or diffusion-tensor
imaging (DTI) show us many functional
details of our brain’s plastic activities and
have provided exciting new insights of
how and where the brain processes specific information (. Fig. 1a). Interestingly,
neither fMRI nor DTI are mechanistically based on the electrical activity of neurons, classically thought to be exclusively
responsible for information processing in
the brain. Instead, these techniques enable
the visualization of brain activity based on
changes of cerebral blood flow, i.e. the oxygen content supplied by blood capillaries,
or by anisotropic water diffusion. The cellular building blocks of the smallest fMRI
and DTI signals have already been identified as the neurovascular unit and the myelin-axon unit, respectively (. Fig. 1b and
c). A single fMRI voxel integrates the oxygen consumption of several cell types with
vascular, glial and neuronal origin: endothelial cells, neutrophils, pericytes, astrocytes, microglia, NG2 glia, oligodendrocytes and neurons. The myelin-axon unit
that gives rise to the DTI signal appears
much less complicated: neuronal fiber
tracts, the axons, are surrounded by the
insulating and support-providing myelin
sheaths of the oligodendrocytes. Both imaging techniques take thus advantage of
the strategic positions of glial cells which
enable them to power neurons and to
maintain long-range connections, highlighting the prominent role of neuroglia
in neuronal performance.
The two-dimensional space of histological or vital brain sections already
demonstrated that cells were organized in
functional and morphological networks.
Among these are the columnar modules
of the cortex that contribute to sensory integration and cognition, the layers of the
hippocampus involved in learning and
memory, the rhythmic centers of the brain
stem which regulate breathing, or the cerebellar circuits responsible for fine-tuning
motor coordination. While excitatory and
inhibitory neurons are the main relay stations for the input, processing and output
of electrical signals, the macroglial cells
execute quite different tasks. Astrocytes
are polarized cells that represent a bridge
between blood vessels and neurons. They
take up nutrients from the blood, metabolize them and provide them to neurons.
Astrocytes also regulate extracellular ion
and transmitter homeostasis from the socalled “tri-partite” synapse where they are
in direct contact with neuronal synapses.
Furthermore, by secreting transmitters
and peptide hormones, they can directly
modulate synaptic transmission. Oligodendrocytes insulate neuronal axons with
a lipid-rich structure, the myelin sheath,
to accelerate action potential propagation
and to electrically insulate axons. Recent
data has additionally demonstrated that
oligodendrocytes metabolically support
axons, the long-range links of neural circuits. Glial cells expressing the proteoglycan NG2 (NG2 glia) are a relatively novel class of macroglia and were originally
identified as oligodendroglial progenitor
cells, but appear to represent a more versatile cell reservoir in the adult brain.
Present-day research provides compelling evidence that a neuron-centered
picture of the brain is way too simplistic, indicating that each class of glial cells
is much more diverse than commonly thought. Glial cells appear to have distinct physiological properties in different
brain regions, at different developmental stages and at different activity levels
of the organism. These observations suggest that functional specializations of glia
might have developed to meet the specific requirements of distinct networks
which might as such be critical determinants of brain activity. This new concept
will change the way we think about brain
function and put glial cells into an even
more prominent focus of attention.
Astrocytes are probably the most versatile class of neuroglia. Functionally positioned between the pia mater, blood
vessels and neuronal synapses, they display a plethora of properties. Astrocytes
contribute to the blood-brain barrier [3],
take up nutrients from the blood, metabolize them and provide energy substrates
to neurons [26]. They link neuronal activity to blood circulation [2, 15], promote
synapse formation [5, 8], and determine
the properties of the extracellular matrix [9, 16, 27]. Furthermore, they regulate extracellular ion and transmitter levels thereby regulating synaptic transmission. Last but not least, astrocytes secrete
compounds which modulate neurotransmission [1, 22]. This impressive list demonstrates via how many routes astrocytes
interact with neurons and influence brain
activity. Astrocytes must be particulare-Neuroforum 3 · 2015 | 59
Editorial
Fig. 1 8 Modern brain imaging techniques are mechanistically based on activity of macroglial cells. a Functional magnetic resonance imaging (fMRI) is able to visualize different functional units (1–3) of the brain in its idle mode (default mode network). A variant of fMRI, diffusion tensor imaging (DTI) can selectively visualize connecting fibre tracts between these centres
(4–6). b The cellular basis of the fMRI signal is the change in oxygen levels in the neurovascular unit composed of capillaries,
astrocytes and neurones. c The axon-myelin unit of fibre tracts causes anisotropic water diffusion that gives rise to DTI signals.
Figure A modified from [13]
ly tailored to fulfil these functions and be
able to adapt to the developmental stage,
the brain region and the activity phases.
Indeed, numerous examples of glial heterogeneity exist. This is especially conspicuous in the morphological specialization of astrocytes (. Fig. 2). In some regions such as the cerebellar cortex and the
retina, they exhibit a radial orientation. In
contrast, astrocytes in the cortex or hippocampus extend processes in all directions displaying a star-like appearance,
while those of the white matter are less
frequently branched and largely lack thin
membrane protrusions. Not surprisingly, first profiling studies of astrocytes isolated from different brain regions display
substantial differences in gene expression
[4, 7, 23]. These include cell surface glycoproteins and components of the extracellular matrix, ion channels, neurotransmitter receptors and transporters, connexins, Eph receptors, and many more [10,
20, 28]. More recent studies show that astroglia sense and compute neuronal activity to feed back to neurones, thereby even
modulating the most visible brain output,
the behaviour. Astroglial cannabinoid receptors in the hippocampus, for example,
are involved in the acquisition of spatial
working memory [14], and in the cerebellum, Bergmann glial AMPA receptors are
important determinants of fine motor coordination [25].
The second class of macroglia, the oligodendrocytes, are the myelin-forming
cells of the CNS. A single oligodendro-
60 | e-Neuroforum 3 · 2015
cyte enwraps up to 50 axons, and myelinating segments can vary in length from 50
to 400 µm. Their morphological heterogeneity has already been described by RioHortega, who distinguished four types. In
white matter fiber tracts such as the optic
nerve or the corpus callosum, axons are
mainly oriented in parallel, and so are the
processes of the oligodendrocytes. In contrast, in grey matter regions where axons
traverse the parenchyma irregularly, oligodendroglial processes point into all directions. The functional characterization
of oligodendroglial heterogeneity is still in
its infancy. They are equipped with a variety of receptors to sense the extracellular
level of transmitters released by neurones
[17]. As a consequence, myelination is regulated by neuronal activity, but is also determined by axon diameter. In addition
to their role in myelination, recent studies
highlight the important role of oligodendrocytes in supporting axons also metabolically [11, 19].
NG2 glial cells constitute less than ten
percent of glial cells in the developing and
adult CNS [6]. They have originally been
identified by the expression of the chondroitinsulfate proteoglycan NG2, and
functionally been characterized as oligodendrocyte progenitor cells. Recent studies demonstrate that outside of neurogenic niches, NG2 cells are the most proliferative cells of the adult CNS. NG2 glial
cells are the only glial cells directly innervated by neurons. While in the hippocampus and cerebellum NG2 cells receive both
glutamatergic and GABAergic input, NG2
cells in the medial nucleus of the trapezoid body only receive excitatory glutamatergic synaptic input in parallel to the Calyx of Held [21]. The neuron-glia synapses
even persist during cell division. Although
all NG2 cells can develop into oligodendrocytes, there exist strong differences between white and grey matter. While in the
corpus callosum, almost two third of NG2
glia become oligodendrocytes, in the cortex 90 % remain NG2 glia. Cell proliferation was suggested to be regulated by voltage-gated sodium and potassium channels heterogeneously expressed on NG2
glia during differentiation [12, 18].
The Deutsche Forschungsgemeinschaft acknowledges the demand for further research on the heterogeneity and
funds the special priority program SPP
1757 “Functional specializations of neuroglia ascritical determinants of brain activity”. The Priority Program will pave the
way for a better understanding of the molecular and cellular role of glia in brain pathologies that is urgently needed to develop novel, more customized and targeted
strategies for the treatment of brain injury and disease.
In this special issue of Neuroforum
members of the SPP 1757 will present recent highlights of glia research addressing
the heterogeneity of astrocytes (Christian
Henneberger), NG2 glia (Dirk Dietrich
and Christian Steinhäuser) and oligodendrocytes (Leda Dimou and Michael Wegner). These reviews will be complement-
Fig. 2 8 Heterogeneity of human astroglia. Semi-schematic survey of the
main types of astroglia and related glial cells, and their localization in different layers/specialized regions of the human brain. I: tanicyte (a: pial; b: vascular); II: radial astrocyte (Bergmann glial cell); III: marginal astrocyte; IV: protoplasmic astrocyte; V: velate astrocyte; VI: fibrous astrocyte; VII: perivascular astrocyte; VIII: inter-laminar astrocyte; IX: immature astrocyte/glioblast; X:
ependymocyte; XI: choroid plexus cell. From: Reichenbach and Wolburg [24]
ed by an article of Daniela Dieterich and
Moritz Rossner who describe novel highthroughput approaches with significant
impact for the identification of cellular
specializations in the brain and among glial cells. The SPP 1757 moreover will serve
as a platform to enhance inter-lab communication not only at the national, but
also at the international level. Especially
strong ties have been established with glial research colleagues in Japan. Kazuhiro Ikenaka will describe the Japanese program to foster glial research.
Corresponding addresses
C.R. Rose
Institute of Neurobiology
Heinrich Heine University
Düsseldorf
Universitätsstr. 1; Building
26.02.00, 40225 Düsseldorf
[email protected]
F. Kirchhoff
Molecular Physiology, Center
for Integrative Physiology and
Molecular Medicine (CIPMM)
University of Saarland
Building 48, 66421 Homburg
[email protected]
Christine R. Rose Diploma in Biology (University of
Konstanz); PhD 1990–1993 University of Kaiserslautern
(Dept. of General Zoology, Prof. J. Deitmer);
Postdoctoral Fellow (DFG Fellowship) at the Dept. of
Neurology, Yale University School of Medicine (Bruce
Ransom and Steve Waxman). 1997–1999 Research
Assistant at the Institute of Physiology (University of
Saarland, Homburg; Prof. A. Konnerth), 1999–2003:
Research Assistant at the Institute of Physiology TU/
LMU Munich (A. Konnerth); 2001: Habilitation in
Physiology (LMU Munich), 2003–2005 Heisenberg
Fellow of the DFG DFG. Since 2005: Director of the
Institute of Neurobiology (University Düsseldorf).
Since 2012 Subject Reviewer of the DFG.
Frank Kirchhoff 1981–1985: Diploma in Biochemistry
at the University of Hannover; 1986–1990 PhD
University Heidelberg (Institute of Neurobiology,
Prof. M. Schachner); 1991–1994 Postdoktoral Fellow
(Institute of Neurobiology, Prof. Helmut Kettenmann);
1995–1999 Max Delbrück Center for Molecular
Medicine Berlin (Cellular Neurosciences, Prof. H.
Kettenmann), 1997 Habilitation in Biochemistry at
the Free University of Berlin; 2000–2008 research
group leader at the Max Planck Institute of
Experimental Medicine (Neurogenetics, Prof. K.-A.
Nave). Since 2009 University Professor (Molecular
Physiology, University of Saarland, Homburg).
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62 | e-Neuroforum 3 · 2015
Review article
e-Neuroforum 2015 · 6:63–68
DOI 10.1007/s13295-015-0009-8
Published online: 11 August 2015
© Springer-Verlag Berlin Heidelberg 2015
Daniela C. Dieterich1,2,3 · Moritz J. Rossner4
1 Institute for Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
2 Leibniz Institute for Neurobiology, Magdeburg, Germany
3 Center for Behavioral Brain Sciences, Magdeburg, Germany
4 Laboratory of Molecular and Behavioral Neurobiology, Department of Psychiatry, Munich, Germany
Dissecting the regional
diversity of glial cells by
applying -omic technologies
Background
Increasing evidence suggest that both
neuronal and glial heterogeneity contribute to and are essential for higher order
brain functions. A plethora of observations have been made showing that neurons and glial cells are not only physically
highly intermingled but are physiologically tightly connected and mutually depend
at various levels on each other. For example, astroglial cells are essential for maintaining the metabolic integrity of neurons
and participate in synaptic transmission
(“tripartite synapse”) [1, 4]. Therefore, the
view emerges that disturbed neuron–glia
interactions may contribute to the initiation and progression of neurodegenerative and psychiatric diseases. Intriguingly and similarly to neurons, macroglia
classes like astrocytes, NG2 cells, and oligodendrocytes are not at all homogenous
cell populations but do possess a markedly heterogeneity in morphology, functionality, and cellular activity as well, that only recently has been started to be acknowledged and integrated into a concept of
brain function that pictures a neural world
rather than a puristical neuronal world. In
the light of these findings, it is not too surprising that glial cells are also increasingly
considered as attractive targets for clinical
intervention strategies aiming at re-balancing disturbed brain function.
So far, the application of conventional approaches to characterize neurons and
more recently also glial cells at global levels by applying so-called -omic technologies has been limited for the brain [8]. This
has several reasons: (1) the cellular com-
plexity of the nervous system is extreme,
with a high degree of regional variability
of the different cell types and, hence, susceptibility in different diseases. (2) Under
physiological and pathological conditions,
the proper function of neurons cannot be
dissociated from supporting glial cells—
and vice versa. (3) Most neurodegenerative and psychiatric disorders are late-onset chronic “systems-diseases” that affect
different neuron–glia networks, which
complicates comparison between different studies (4). The analysis of brain tissue is still state of the art for molecular and
biochemical studies, although cell-type
relevant processes are likely to be masked
due to the fact that all cells share a rather large pool of identical proteins ranging from housekeeping proteins, protein
signaling cascades, adhesion molecules,
and receptors and ion channels. Because
of these facts, several methods needed to
be developed and are still in progress of
refined development that allow to specifically label and isolate either different glial and neuronal cells or cell-specific subcellular compartments to high purity and
at conditions that allow for the application
of state-of-the art -omic approaches. The
appendix “omics” added to the class of biological molecules under investigation
circumscribes respective globally scaled
technologies, such as Genomics, Transcriptomics, Proteomics, Lipidomics, and
Metabolomics. By definition, omics technologies claim to cover the majority of different molecules of a given subclass within
one experiment providing a systems-level
view onto a biological sample. Thus, unbiased and exploratory (“hypothesis-gen-
erating”) approaches toward an enhanced
understanding of physiological or pathological cellular states are possible. Subsequently, we will provide an overview on
current technical approaches used to perform transcriptomics and proteomics to
dissect glial heterogeneity of the brain.
Strategies to isolate neuronal
and glial cell types of the brain
Defined ex vivo culture conditions have
been developed for all major cell types of
the brain, such as astrocytes, oligodendrocyte precursor cells (OPCs or NG2 glia),
mature oligodendrocytes, and different
neuronal subtypes as well as microglia.
Thus, it is possible to culture large quantities of defined cell types for extended periods in vitro. Although the basic electrophysiological and molecular characteristics of cultured neuronal and glial cells
appear to be sustained in vitro, it is widely accepted that ex vivo cultures will only partially reflect the in vivo situation
even when different culture conditions
are employed and compared. The molecular identity of a given cell type depends not only on the availability of nutrients and soluble survival factors (“media and supplements”) but also critically
on regional cues depending for example
on the nature and activity of surrounding
cells, composition of the local extracellular matrix as well as many other factors.
Thus, the local microenvironment determines the molecular and structural features of cells in vivo, which cannot be exactly mirrored in vitro. For globally scaled
proteomics and also the analysis of none-Neuroforum 3 · 2015 | 63
Review article
Fig. 1 8 Identification of transporter genes enriched in forebrain astrocytes by transcriptome analysis. a Experimental strategy: Cortex (Cx), Hippocampus (Hi), and Brainstem (Bs) tissues from brains of mice expressing enhanced green fluorescent
protein (EGFP) under the control of the glial-specific promoter glial fibrillary acidic protein (hGFAP-EGFP) were triturated and
50k EGFP positive cells were purified by fluorescence-activated cell sorting (FACS), followed by RNA isolation and transcriptome profiling with RNAseq. SR101 candidate uptake transporter genes (of the solute carrier, Slc family) were hypothesized to
display a higher expression in Cx and Hi versus Bs astrocytes (Cx/Hi > Bs). b Scatter plots of FACS analysis with green fluorescent protein intensities plotted versus forward scatter (FSC) given as arbitrary units. Blue dots represent Hoechst 33342 positive and GFP negative cells, green dots represent Hoechst 33342 and GFP double positive cells representing different astrocyte populations. c Venn diagram of all forebrain-enriched transporter genes. Seven mRNAs were significantly elevated both
in Cx and Hi. d Differentially expressed mRNAs coding for solute carriers (Slc and Slco gene families). Depicted are the gene
symbols, accession, and average read numbers in Cx, Hi, and Bs samples. The seven detected forebrain-enriched mRNAs correspond to five genes (Slc1a2 and Slco1c1 annotated by two mRNAs each). Detection cut-off was determined with an adjusted p value cut-off < 0.05. Cx (n = 2), Hi (n = 3), Bs (n = 4) biological replicates. e Scatter plot analysis shows high degree of reproducibility of technical replicates, biological replicates, and astrocyte from different region as well as comparison of astrocytes and neurons display as expected increasing levels of divergence. f Inspection of ISH data (from Allen Brain Atlas) for top
candidates being differentially expressed between Hi and Bs validates regional NGS profiles (a–d). (modified from (Schnell et
al. 2013)
coding RNAs (ncRNAs), however, large
homogenous pools of intact cells including all compartments and membranous
extrusions (filopodia, neurites, etc.) may
be the preferred substrate for analysis. Under these circumstances, ex vivo cultures
for example of different glial cells—both
primary and cell lines—at high purity are
still the substrate of choice to date.
To overcome the limitations of ex vivo culturing and enrichment, several isolation techniques have been developed. A
powerful method for isolating small defined brain regions and individual celltypes is “Laser-capture microdissection”
(LCM), which combines high-resolution
64 | e-Neuroforum 3 · 2015
microscopy with precise microlaser cutting in one single device. Simple Nisslstaining can be applied to precisely guide
regional selection and to identify a few
neuronal cell types such as large-sized
Purkinje cells of the cerebellum and motor neurons of the spinal cord [13, 19]. We
have adapted this technique by using a nuclear targeted fluorescent protein to genetically label projection neurons of the adult
cortex and could show that samples with
cellular resolution revealed transcriptomic signatures, which were masked even in
layer-specific cortical microregions [19].
Nonetheless, LCM is considered rather
an enrichment than purification strate-
gy given the fact that for example minor
proportions of glial transcripts originating
most likely from cellular processes will be
co-isolated from semithin cryosections.
Small-sized glial cells are at the limit of
the resolution of the technique. Moreover,
due to the low amount of isolated sample
globally scaled proteomics has not been
combined with LCM purification.
A more widely applied and less labor
intensive isolation technique, particularly
for small-sized glial cells, relies on tissue
trituration followed by fluorescence-activated cell sorting (FACS). This strategy
has been successfully used by us and others in combination with quantitative real-
Abstract
time polymerase chain reaction, microarray analysis, and more recently RNAseq to
profile mRNA expression of glial cell populations [3, 12, 14, 15]. Both, transgenic labels introducing for example green fluorescent protein in astrocytes and antibodies directed against extracellular epitopes have been applied [14]. The caveats
of FACS mediated approaches are ex vivo incubation steps that need to be strictly standardized and which could potentially cause artifacts. The induction of immediate-early or stress-associated genes
has, however, not been observed [14].
The advantage of this method is that all
RNA species, mRNAs as well as noncoding transcripts, can be harvested simultaneously (see below). Of particular importance for regulatory mechanisms in nuclear organization and transcriptional, posttranscriptional as well as epigenetic processes in the brain appear to be small and
long ncRNAs [18]. Comprehensive characterizations of these molecules have not
been described in different glia populations so far. Given the enormous progress
in the sensitivity and coverage of mass
spectometry based technologies, globally
scaled proteomic analyses for example of
different FACS enriched cell populations
are already on the way to deliver the full
complement of all expressed proteins in
neuronal and glial cell types.
A partially complementary approach
relies on ribosome pull-down protocols
that allow, by using a transgenically tagged
ribosome component, to study selectively the translated transcriptome of a pool
of defined cell types from different brain
regions, however, ncRNAs cannot be analyzed [6].
Transcriptomic approaches to
unravel glial heterogeneity
Transcriptomic analyses with DNA microarrays were among the most widely
applied globally scaled genomic technologies and have been successfully used to
profile neural gene expression in the context of normal brain physiology and disease, however, mostly not at the cell-typespecific level [8]. More recently, second
or next generation sequencing technologies (“Deep-Sequencing”) principally allow obtaining a much deeper insight in-
to the “whole transcriptome” in an unbiased matter [22]. With RNA-Seq, the detection and quantification of all expressed
mRNA splice variants and ncRNAs is possible without the a priori knowledge of
their exact nature. It has been shown that
RNAseq provides a more accurate, quantitative, and sensitive analysis of global
transcriptomes as compared with hybridization based methods such as microarrays [22]. The maximally meaningful application of these globally scaled technologies most likely requires a coordinated
approach since particularly ncRNAs are
essential players in the control of mRNA
abundance and initiation of protein translation.
As a first step toward the characterization of region-specific molecular signatures of astrocytes, we have combined
FACS with 3’ digital gene expression [20].
In this RNAseq approach, we selectively
focused on mRNAs expression profiles
of astrocytes FACS isolated from the cortex (Cx), hippocampus (Hi), and brainstem (Bs). We hypothesized that differential expression levels of genes encoding for
transporter proteins correlating with the
Cx- and Hi-specific uptake of sulforhodamine (SR101) into astrocytes would allow us to identify the SR101 transporter.
From the identified candidates, Slco1c1
was validated with corresponding biochemical assays and mouse mutants as
the Cx/Hi-specific astrocyte SR101 transporter (. Fig. 1a–d) [20]. This study was
purely hypothesis driven and utilized only a small fraction of the obtained data.
Nonetheless, it clearly showed that transcriptomic datasets from different astrocytes population can help to better understand functional consequences of expression differences. By performing quality controls at all steps of the protocol (average r2 values of > 0.99 for technical replicates indicate a high level of reproducibility, see . Fig. 1e) and by applying stringent statistical criteria, we are convinced
that an unbiased bioinformatic analysis of
the complete data set will reveal a deep insight into the regional differences of the
transcriptomes of astrocytes isolated at
P10 from Cx, Hi, and Bs. We can conclude
from a preliminary analysis that astrocytes from these regions are highly heterogenous with respect to differential gene
e-Neuroforum 2015 · 6:63–68
DOI 10.1007/s13295-015-0009-8
© Springer-Verlag Berlin Heidelberg 2015
D.C. Dieterich · M.J. Rossner
Dissecting the regional
diversity of glial cells by
applying -omic technologies
Abstract
Neuronal as well as glial cells contribute to
higher order brain functions. Many observations show that neurons and glial cells
are not only physically highly intermingled but are physiologically tightly connected and mutually depend at various
levels on each other. Moreover, macroglia classes like astrocytes, NG2 cells and oligodendrocytes are not at all homogenous
cell populations but do possess a markedly heterogeneity in various aspects similar to neurons. The diversity of differences in morphology, functionality and, cellular activity has been acknowledged recently and will be integrated into a concept of
brain function that pictures a neural rather than a puristical neuronal world. With
the recent progress in “omic” technologies,
an unbiased and exploratory approach toward an enhanced understanding of glial
heterogeneity has become possible. Here,
we provide an overview on current technical transcriptomic and proteomic approaches used to dissect glial heterogeneity of the brain.
Keywords
Glial cell types · Brain · Transcriptomics ·
Proteomics · Cell isolation
expression beyond transporter. The number of differentially expressed genes at a
stringent cutoff (p value > 10−5) is high (Hi
vs. Bs = 277, Hi vs. Cx = 1105, and Cx vs.
Bs = 724), which most likely reflects several additional subregion-specific mechanisms operating in these astrocyte populations. A preliminary pathway analysis
shows, for example, that—among many
others—Ca2+ signaling-related gene sets
are enriched in forebrain astrocytes (Kannayian and Rossner, unpublished).
Proteomic approaches to
unravel glial heterogeneity
With an estimated number of approximately 10,000 different proteins in each
single mammalian cell [16], in-depth
identification of a cell’s entire proteome
e-Neuroforum 3 · 2015 | 65
Review article
Fig. 2 8 Marcoglial proteomics approaches. Schematic strategy for the investigation of marcoglial proteomes. a In vitro cultivation of purified primary marcoglia cells or respective cell lines enables the analysis of the intracellular protein entity as well
as secretomes, whereas fractionation and extraction procedures of brain homogenates b allows the identification of subcellular proteomes such as astroglial gliosomes, myelin, and others, however, at the expense of true cell-type specificity. Metabolic
labeling of newly synthesized proteins using the noncanonical amino acid azidonorleucine (ANL) and a mutated methionine
tRNA synthetase expressed in selected cell types may facility cell-type-specific proteome analysis in combination with “click
chemistry” under in situ conditions prior biochemical and mass spectrometrical analyses
is still a major challenge for modern proteomic technologies. In addition, the total
number of different/unique proteins per
synapse is estimated to be at approximately 2000–2500 [17], which are often under
control of different activity-dependent
turnover rates. While today’s state-of-theart MS instruments routinely sequence
single purified proteins with subfemtomolar sensitivity, the effective identification
of low-abundance proteins is orders of
magnitude lower in complex mixtures due
to limited dynamic range and sequencing
speed and due to the common, strong bias toward acquiring MS/MS data on higher abundance molecules. Hence, to tackle
activity-dependent proteome alterations
entire, functionally heterogeneous brain
regions with different subtypes of neurons and macroglial cells are usually being sampled at the loss of cell-type specificity and spatial resolution. The charac-
66 | e-Neuroforum 3 · 2015
terization of a proteome is an even more
difficult challenge if temporal and spatial
aspects of a proteome or a subpopulation
of the proteome have to be taken into consideration. Thus, the separation and enrichment of the subproteome in question
is key for its successful characterization.
To overcome the above mentioned limitations, several cellular and biochemical
(subcellular) enrichment strategies combined with proteomics have been developed to increase sensitivity and selectivity for the analysis of neuronal and glial
proteomes, and to finally dissolve cellular
heterogeneity of neural cells in the brain
(. Fig. 2a-b).
Concerning cellular selectivity, intracellular proteomes and secretomes from
cultured primary astrocytes and astroglial cell lines have been characterized in detail by several labs as proteomic approaches as indicated above require a much larg-
er quantity than transcriptomic approaches. Analysis of primary cortical astrocytes
revealed the astonishingly high number of
proteins in conditioned media including
1247 putative secreted proteins [21]. More
recently 6000 unique protein groups belonging to the secretome and 7265 unique
intracellular protein groups were identified from cultured C8-D1A astrocytes
[10]. Moreover, these experiments demonstrate activity-dependent changes in
intracellular and secreted proteomes even
under ex vitro conditions. Similar to indepth analysis of postsynaptic and presynaptic protein fractions, biochemical fractionation approaches have been recently used to attain detailed pictures of defined subcellular glial proteomes, such as
myelin membranes of oligodendrocytes
or astroglial membrane fractions. Analysis of freshly purified human and murine
myelin fractions revealed more than 1000
proteins and about 700 different lipids [9].
In a very recent study, Carney et al. characterized the proteome of gliosomes purified from murine brain tissue. Gliosomes
are enriched for proteins of the astrocytic exocytosis machinery (marker protein
VAMP3), perisynaptic astrocyte processes (marker protein Ezrin), and astrocyte
plasma membrane proteins (e.g.astrocyte
membrane glycoprotein Basigin44).
While common synaptosomal proteins
(such as the presynaptic SNARE proteins
and postsynaptic proteins NR1 and PSD95) were depleted in the gliosome fraction, the fraction contained glutamate receptors and a plethora of heteromeric Gprotein subunits and small GTPases related to perisynaptic function. In combination with fluorescence-activated sorting, such as recently done for VGLUT1Venus-labeled synaptosomes [2], specialized astrocytic gliosomes could be investigated in unprecedented detail in the future. Another approach to decipher glial
proteomic heterogeneity might employ
bioorthogonal metabolic protein labeling
(. Fig. 2c) as recently shown by us for different cell types including glial and neuronal cells in Drosophila melanogaster [7].
This technique relies on a mutated methionine tRNA synthetase (MetRSLtoG),
which incorporates, instead of methionine, the noncanonical amino acid azidonorleucine (ANL) into nascent proteins.
By transgenically expressing the MetRSLtoG cell-specificity can be achieved,
whereas restricting feeding ANL to transgenic flies within a desired time enables
temporal specificity. Thus, only in the
cells expressing the mutated MetRSLtoG
and only during the ANL feeding, the synthesized proteins incorporate ANL. Subsequently and depending of the type of
analysis, ANL is clicked either to a fluorescent tag for visualization of ANL-harboring proteins (FUNCAT; [5]) or clicked
to an affinity tag with subsequent analysis
of tagged proteins using immunoblot or
bulk identification via mass spectrometry
(BONCAT [11]). Notably, this approach
revealed for the first time the expression
of the scaffolding protein Dlg in glia [5,
7] pointing once more to the importance
of sub-type-specific proteome analysis
for understanding brain function on the
global and on the regional level.
Conclusions
Because of its apparent limitations, none
of these methods qualifies as “the unique”
and generally applicable strategy toward
transcriptomic and proteomic approaches to unravel the molecular complexity
for example of regional glial cell populations of the brain. Unresolved issues for
proteomic approaches include the analysis of splice variants, alternative translation initiation sites, and point mutations
of proteins due to both dynamic range
and sequence coverage limitations. Finally, current proteomics is unable to simultaneously unlock all the critical determinants of cellular proteostasis because specific purification and separation procedures have to be employed for each single protein modifications making it necessary to address posttranslational modifications one by one. In parallel to the limitations mentioned for the proteomics, exploring the whole universe of RNA molecules is still challenging although sequencing methodologies have dramatically improved throughout the last decade.
Nonetheless, kinetic and structural variations as well as for example allele-specific
expression require sophisticated bioinformatics analyses, which are not yet available for all purposes.
Finally, to obtain a more complete picture and to have the opportunity to identify different technical biases, most likely several -omic and isolation strategies
should be combined and analyzed in an
integrated fashion to increase our understanding of glial heterogeneity of the
brain.
Corresponding address
D. C. Dieterich
Institute for Pharmacology and Toxicology
Otto-von-Guericke University Magdeburg
Leipziger Strasse 44, 39120 Magdeburg
[email protected]
M.J. Rossner
Laboratory of Molecular and Behavioral
Neurobiology Department of Psychiatry
Nussbaumstr. 7, 80336 Munich
[email protected]
Daniela C. Dieterich studied Biochemistry at the
University of Hannover and did her PhD. under
the supervision of Dr. Michael Kreutz in the
Neuroplasticity research group at the Leibniz
Institute for Neurobiology (LIN), Magdeburg.
She was a Leopoldina-postdoctoral fellow from
2004–2006 in Erin Schuman’s lab at the California
Institute of Technology, Pasadena, USA. In 2008 she
was granted a research group at the LIN within the
Emmy Noether program of the DFG. In 2011 she was
offered the W3 professorship in Pharmacology and
Toxicology at the Otto-von-Guericke University in
Magdeburg, where she heads the research group
Neural Communication and Plasticity as well as the
Institute for Pharmacology and Toxicology since
spring 2012. Her research group is associated with
the Leibniz Institute for Neurobiology, Magdeburg.
Moritz J. Rossner studied Biology at the University
of Heidelberg. He performed his PhD work at the
Center of Molecular Biology in Heidelberg and
worked afterwards as scientific employee at Axaron
Bioscience AG. In 2002, he moved as a group leader
to the Max-Planck-Institute of Experimental Medicine
in Göttingen and did his Habilitation in 2010 in the
field of Molecular and Cellular Neurobiology at the
Faculty of Biology of the Georg-August-University
in Göttingen. In 2013 he took over a position as W2
professor at the Ludwig-Maximilians-University in
Munich where he leads the laboratory of Molecular
Neurobiology at the Department of Psychiatry.
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Abstract
68 | e-Neuroforum 3 · 2015
Review article
e-Neuroforum 2015 · 6:69–72
DOI 10.1007/s13295-015-0014-y
Published online: 27 August 2015
© Springer-Verlag Berlin Heidelberg 2015
Leda Dimou1 · Michael Wegner2
1 Physiological Genomics, Biomedical Center (BMC),
Ludwig-Maximilians University Munich, Munich, Germany
2 Institute of Biochemistry, Emil-Fischer-Zentrum,
Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
Oligodendroglial heterogeneity
in time and space (NG2
glia in the CNS)
Introduction
Oligodendrocytes are an important cell
type in the central nervous system (CNS).
They generate myelin, which is responsible for the fast, saltatory nerve conduction.
Until a few years ago it was believed that
myelination takes place only during development and is completed at young adolescence. However, the progenitor cells that
generate oligodendrocytes during development are also present in the adult brain
and spinal cord. These oligodendrocyte
progenitor cells are also known as polydendrocytes or NG2 glia, because of their
appearance and the expression of the proteoglycan NG2. They generate not only oligodendrocytes but also astrocytes in a region-dependant manner during development [12]. Further, they are the only proliferating cell population outside the neurogenic niches in the adult brain and are able
not only to renew themselves, but also to
differentiate into mature, myelin-producing oligodendrocytes [4]. During the last
years it became obvious that NG2 glia have
even more functions in the brain. Thus,
NG2 glia express a variety of ion channels
and can form excitatory as well as inhibitory synapses with neurons (see article by
C. Steinhäuser and D. Dieterich in this issue). Despite the high number of NG2 glia
in the adult brain (5–10 % of all cells) und
their fundamental ability to generate oligodendrocytes, this maturation process is not
sufficient to ensure efficient myelin repair
after disease or injury. A targeted increase
A complete list of literature can be requested
from the authors.
in the maturation of NG2 glia under pathological conditions like Multiple Sclerosis
or injury would be beneficial, but first more
detailed information about their differentiation, the signalling processes during development and the differences between
NG2 glia in young and aged individuals
are required. Another heavily discussed
question of the last years with relevance for
repair processes in the brain concerns the
heterogeneity of the NG2 glia population.
This aspect will be investigated as part of
the SPP 1757 (Glial heterogeneity).
NG2 glia in the developing CNS
NG2 glia are first detected rather late during ontogenesis. In the murine spinal cord
NG2 glia develop after several neuronal
subpopulations have already been generated. As cells of neuroectodermal origin, NG2 glia develop from the neuroepithelial progenitor cells of the ventricular zone [8]. In the spinal cord NG2 glia
originate in the ventral area from precursor cells that express the transcription factor Olig2 and have generated motor neurons before. The Olig2 expression is sustained in NG2 glia. Remarkably additional NG2 glia are formed later in the dorsal region of the ventricular zone. Also
in other regions of the CNS NG2 glia develop in different areas of the ventricular
zone in a distinct temporal order. In the
forebrain the medial ganglionic eminence
is the earliest origin for NG2 glia, followed
by the lateral ganglionic eminence and finally the dorsal telencephalon. As a consequence NG2 glia have various origins
along the dorso-ventral and anterior-pos-
terior axes. As the cells leave the ventricular zone, the growth factor receptor Pdgfra and the transcription factor Sox10 are
induced. The expression of the proteoglycan NG2 follows with a slight delay. Despite their heterogeneity in origin most
NG2 glia are characterised by the joint
expression of Olig2, Sox10, Pdgfra and
the eponymic NG2 marker protein. NG2
glia retain their ability to undergo cell division also in the parenchyma and are
able to efficiently populate the whole CNS
from their defined site of origin, due to
their high migratory activity. As a consequence, many CNS areas are already populated by “ventral” NG2 glia, before “dorsal” NG2 glia are even generated. In line
with this, NG2 glia with dorsal origin only represent a small proportion of the total population in the spinal cord. However, in the forebrain many of the early NG2
glia with ventral origin are later replaced
by NG2 glia with dorsal origin. These observations argue that “ventral” and “dorsal” NG2 glia may have functional differences. However, an argument against
this hypothesis is that so far no differences in the properties of "ventral" and "dorsal" NG2 glia or mature oligodendrocytes
could be demonstrated and that the selective ablation of NG2 glia of a certain origin
can be compensated in the mouse brain by
remaining NG2 glia without phenotypical
consequences. However, it needs to be stated, that the missing proof of a phenotype
does not necessarily mean that this phenotype does not exist. It cannot be excluded
that NG2 glia from different origins are heterogeneous under normal conditions, but
show a high rate of plasticity in case of dise-Neuroforum 3 · 2015 | 69
Review article
Fig. 1 8 NG2 glia are a heterogeneous population under physiological (left) and pathological (right)
conditions: NG2 glia in the grey matter have a long cell cycle, differentiate slowly and only partially to
mature oligodendrocytes with most cells remaining as NG2 glia. In contrast, NG2 glia located in the
white matter have a shorter cell cycle and differentiate faster and in a higher amount into oligodendrocytes. After injury (. Fig. 1, right) NG2 glia in the grey matter show a very fast and heterogeneous
reaction. They become hypertrophic, polarize, migrate and proliferate more strongly with a shorter
cell cycle
turbances in CNS development or injury so
they are able to compensate for each other.
Relation of NG2 glia in the
developing and adult CNS
During CNS development most NG2 glia
differentiate to oligodendrocytes. Due to
this, NG2 glia are usually referred to as oligodendrocyte progenitor cells (OPCs) in
developmental studies. Disturbances in
development and differentiation of NG2
glia are related to demyelinating diseases and leukodystrophies. Part of the NG2
glia population does not differentiate, but
remains as an autonomous cell popula-
70 | e-Neuroforum 3 · 2015
tion in the adult CNS. At the moment it
is unclear, which signals and mechanisms
decide, whether a NG2 glia cell differentiates further or stays at a progenitor state.
Further, NG2 glia have to adapt to the
modified situation in the adult brain in
various characteristics and abilities. How
this is achieved on mechanistic and molecular levels is currently unclear and part
of our research in the SPP 1757 (Glial heterogeneity).
NG2 glia in the adult brain
In the adult brain NG2 glia are able to
proliferate and to renew themselves, but
in contrast to developmental states, the
cell cycle is rather slow. In the last years
it has been shown, that NG2 glia generate mature, myelin-generating oligodendrocytes also in adulthood and hence at a
time point, when myelination has already
been completed. The role of these newly
generated oligodendrocytes is not known.
Recent studies lead to the assumption,
that these oligodendrocytes could be important for complex, motor learning tasks
[7]. Interestingly, for adult NG2 glia the
ability to proliferate and divide is strongly region-dependent. While the majority
of NG2 glia in the white matter of the cerebral cortex (e.g. in the corpus callosum)
generates mature, myelin-producing oligodendrocytes, most of the NG2 glia in
the grey matter keep their identity as
progenitor cells. Homo- and heterotopic transplantations in the brain of adult
mice point to a substantial heterogeneity of NG2 glia in different brain regions,
which can either be explained by different
intrinsic determinants or by divergent environmental stimuli [9]. Further, the proportion of proliferating NG2 glia is smaller in the grey matter and cell cycle length
is increased, compared to NG2 glia residing in the white matter (. Fig. 1). Proliferation and differentiation can also be influenced by neuronal modulation. This
can be explained by the fact that NG2 glia
interact closely with synapses and nodes
of Ranvier and some NG2 glia are also in contact with neurons via synapses.
All of these observations underline, that
under physiological conditions NG2 glia
act as a heterogeneous population, which
can react differently to a variety of stimuli. This heterogeneity needs to be understood, before therapeutics based on NG2
glia can be developed and applied to remyelination and repair (for a review article see [2]).
Also within the same region heterogeneity of NG2 glia was observed. The transcription factor Mash1/Ascl1 was only detected in a subpopulation of NG2
glia in the cerebral cortex. It remains unclear, whether Ascl1-positive and –negative NG2 glia show differences in proliferation- and differentiation behaviour. Also
the G-protein coupled membrane receptor 17 (GPR17) is only expressed in a subpopulation of NG2 glia in the brain. Sur-
Abstract
prisingly, NG2 glia expressing GPR17 do
not develop to mature oligodendrocytes
in an investigated period of 3 months. As
part of SPP1757, the molecular differences
between GPR17-positive and GPR17-negative NG2 glia will be analysed to identify
molecules and signalling pathways that are
important and necessary for the differentiation of NG2 glia.
Regulatory mechanisms
of NG2 glia
Different properties of NG2 glia in the developing and in the adult brain are caused
by alterations of the gene regulatory network. For NG2 glia in the developing
CNS the main components of the network are known [5]. Among those are the
two transcription factors and marker proteins for NG2 glia Olig2 and Sox10. Both
are supported in their function by closely related proteins (Olig1 for Olig2; and
Sox8, Sox9 support Sox10), but are functionally dominant compared to the corresponding paralogous proteins. Their
effect is further modulated by additional transcription factors. The helix-loophelix proteins Id2 and Id4 inhibit Olig2
and the transcription factor Sox10 is regulated in its activity by the related proteins Sox5 and Sox6. Additional crucial
regulators are the helix-loop-helix protein Mash1/Ascl1, the zinc finger protein Sip1 and the homeodomain protein
Nkx2.2. New studies give first insights into the complex interactions of these transcription factors inside the gene-regulatory network that involves antagonistic as
well as synergistic and inductive relations
and establish a variety of control circuits,
which additionally are influenced by the
exact amount of the corresponding transcription factor, post-translational modifications and epigenetic factors [10, 11].
Currently, there is insufficient knowledge
how the gene regulatory network in NG2
glia during development differs from the
adult CNS. However, current research
hints at some functionally relevant differences. The influence of Olig2 and Olig1
on the differentiation of NG2 glia appears
to be interchanged between developing
and adult CNS. Whereas Olig2 is responsible for many developmental aspects und
properties of NG2 glia in the developing
CNS and can not be substituted by Olig1,
regeneration and differentiation of adult
NG2 glia depends on Olig1 and is drastically reduced if only Olig2 is present [1].
By using comparative genomics to investigate the regulatory network we hope to
receive important information about the
causes for differences and similarities between NG2 glia in the developing and in
the adult brain. Further, we will tackle the
question, how the modulation of the network influences the heterogeneity of NG2
glia during adulthood.
Response of NG2 glia after injury
NG2 glia react towards injurý or other
pathological conditions with a change in
their morphology and proliferation rate.
The type and time course of this reaction is
dependent on the nature of the insult. After demyelination NG2 glia become hypertrophic, more cells proliferate with a shortened cell cycle and differentiate finally to
mature oligodendrocytes to repair myelin.
NG2 glia react similarly in neurodegenerative diseases such as Morbus Alzheimer or
Amyotrophic Lateral Sclerosis (ALS), but
no hypertrophy can be detected in these
cases. It remains unclear, whether the reaction of NG2 glia also differs between different kinds of injury and other pathological states. Using repetitive in vivo 2-photon microscopy, first evidences for a heterogeneous reaction of NG2 glia towards
acute injury (e.g. focal laser lesion and stab
wound) was detected ([6]; von Streitberg,
Dimou et al., unpublished data). Some
NG2 glia polarize and migrate towards the
site of injury, while others proliferate and/
or become hypertrophic (. Fig. 1). All these
events lead to an accumulation of NG2 glia
at the lesion site. The relevance of this behaviour remains unclear (for a review see
[3]). Speed and strength of the reaction lead
to the assumption that NG2 glia are responsible for wound closure and scar formation.
Corresponding address
L. Dimou
Physiological Genomics, Biomedical Center
(BMC)
Ludwig-Maximilians University Munich
80336 Munich
[email protected]
e-Neuroforum 2015 · 6:69–72
DOI 10.1007/s13295-015-0014-y
© Springer-Verlag Berlin Heidelberg 2015
L. Dimou · M. Wegner
Oligodendroglial
heterogeneity in time and
space (NG2 glia in the CNS)
Abstract
NG2 glia represent a neural cell population
that expresses the proteoglycan NG2 and is
distinct from other cell types of the central
nervous system. While they generate oligodendrocytes and a subset of astrocytes during development, their progeny in the adult
brain solely consists of oligodendrocytes and
further NG2 glia. In the last years, it has become clear that NG2 glia represent a heterogeneous population of cells with different
properties and potential. In this review we
will first discuss the similarities and differences between NG2 glia of the developing and
adult CNS, before we will describe the regulatory mechanisms in these cells to finally concentrate on the heterogeneity of NG2 glia
under physiological and pathological conditions.
Keywords
Heterogeneity · Differentiation · Proliferation ·
Transcription factors · Injury
Leda Dimou studied biology at the RuprechtKarls-University Heidelberg and did her PhD at the
Center for Molecular Biology (ZMBH) in Heidelberg
and at the Max-Planck-Institute for Experimental
Medicine in Göttingen focussing on the homologs
of the main myelin protein PLP in the brain. After
a postdoctoral fellowship at the Institute for Brain
Research in Zurich she moved to the Department
of Physiological Genomics at the Biomedical
Center of the Medical Faculty (LMU) in Munich,
where she leads her own research group since
2012. Her research group investigates the role of
NG2 glia in the adult brain under physiological
and pathological conditions using molecular and
cell biological- as well as imaging methods.
Michael Wegner studied biology at the universities
of Münster and Würzburg and did his PhD from 1987
to 1990 at the Institute of Biochemistry in Würzburg.
After postdoctoral work at the University of California
at San Diego (UCSD) he took over a position as
leader of a junior research group at the Center for
Molecular Neurobiology at Hamburg University
(ZMNH) in 1994 and was appointed to the chair of
Biochemistry and Pathobiochemisty at the Institute of
Biochemistry of the Medical Faculty at the FriedrichAlexander-University Erlangen-Nürnberg in 2000.
His research is dealing with the transcriptional and
epigenetic control of glial cells and myelin formation.
e-Neuroforum 3 · 2015 | 71
Review article
Acknowledgment. The work of the authors is
supported by the DFG (We1326-8, We1326-11 and
We1326-12 for MW & DI 1763/1–1 and SFB870 for
LD) and the EU/DLR (01EW1306A for LD). We thank
Frau Dr. Francesca Vigano for the graphic illustration.
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72 | e-Neuroforum 3 · 2015
Review article
e-Neuroforum 2015 · 6:73–77
DOI 10.1007/s13295-015-0010-2
Published online: 25 July 2015
© Springer-Verlag Berlin Heidelberg 2015
Christian Steinhäuser1 · Dirk Dietrich2
1 Institute of Cellular Neurosciences, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany
2 Experimental Neurophysiology, Neurosurgery Hospital, University of Bonn, Bonn, Germany
Neuron–glia synapses in the
brain: properties, diversity
and functions of NG2 glia
Introduction
NG2 glia represent a subgroup of glial cells
in the brain which express the NG2 proteoglycan. Earlier research assumed that
these cells are immature astrocytes, but it
is now well established that they comprise
a distinct cell type. Owing to their specific functional and morphological properties, NG2 glial cells have also been termed
complex cells, oligodendroglial precursor
cells (OPCs), GluR cells or polydendrocytes [1]. NG2 glial cells make up approximately 5–10 % of the total cell number in
the brain, and many of these cells undergo cell division throughout postnatal life.
During brain development, the vast majority of white matter NG2 glia differentiate into NG2-negative oligodendrocytes
and thus can be considered immature precursors. In contrast, in gray matter, many
cells do not become oligodendrocytes but
keep their NG2 phenotype throughout life
(see article by L. Dimou and M. Wegner in
this issue). Upon injury to the adult brain,
NG2 glia have been observed to differentiate into oligodendrocytes or astrocytes,
that is, they show properties characteristic of progenitors. However, it is unclear
whether all NG2 glial cells can undergo
such a type of differentiation. NG2 glia express a plethora of ion channels and plasma membrane receptors with properties
similar to those found in neurons. In contrast to astrocytes, which form abundant
inter-cellular networks among each other and with oligodendrocytes (so-called
panglial networks), in most brain regions,
NG2 glial cells lack coupling. Intriguingly, these glial cells receive direct synaptic
input from GABAergic and glutamatergic neurons.
NG2 glia possess heterogeneous
functional properties
Ion channels
So far, NG2 glial cells have been electrophysiologically analysed only in a few
brain regions, such as the cerebellum,
neocortex, hippocampus and corpus callosum. These cells possess voltage-gated
Na+ channels, but they cannot fire regenerative action potentials. The lack of action potentials is due to the low number
of plasma membrane Na+ channels and
the much higher densities of voltage-gated delayed rectifier and transient K+ channels because opening of the latter on depolarization produces outward currents
that largely compensate the Na+ inward
currents. NG2 glia also express inwardly
rectifying K+ (Kir) channels (. Fig. 1a).
While Kir channels are instrumental in
stabilizing the cells’ negative resting potential, the density of the delayed rectifier K+ channels (subunits Kv1.3, Kv1.5) was
reported to correlate with cell division:
High expression of the channels was observed in proliferating cells, and the block
of Kv1.3 inhibited the transition from
the G1 to the S phase of the cell cycle [2].
Analyses of mice with gene deletions, antibody staining and single-cell transcript
profiles have shown that the Kir channels
in NG2 glia are mainly composed of the
Kir4.1 subunit, which is also abundantly expressed by astrocytes. In the hippocampus, Na+ channels are downregulated
during postnatal development while the
density of Kir channels increases.
In addition to Na+ and K+ channels,
different types of voltage-gated Ca2+ channels have been described in NG2 glia,
which together with transmitter receptors
(see below) might be important to regulate Ca2+ -dependent cellular processes,
such as motility, proliferation or migration
[3, 4]. Independent of the developmental
stage, NG2 glia in gray and white matter
differ with respect to their morpho­logical
and physiological properties. For example, compared to subcortical white matter, NG2 glial cells in the neocortex express a lower density of delayed rectifier K+ channels but higher densities of Kir
and Na+ channels [5]. The physiological
impact of this diversity is still unclear.
Transmitter receptors
NG2 glial cells possess different types of
ionotropic glutamate receptors (mainly AMPA subtype), which mediate depolarization and Ca2+ influx into the cell
[1]. Significant regional differences have
been reported regarding the expression
strength and properties of glial AMPA receptors. For example, Ca2+ permeability
and density of the receptors in NG2 glia
of the cerebellum are much higher compared with those of the hippocampus, entailing a higher efficiency of neuron–glia
synaptic signaling. The question whether
differences in the molecular composition
of the receptors add to the observed functional heterogeneity is addressed by our
research project within the framework of
the DFG Priority Program 1757. We have
found that hippocampal glial AMPA receptors mainly express the GluA2 subunit,
which is less Ca2+ permeable. The Na+ influx through the receptors leads to a block
of Kir channels and enhanced receptormediated depolarization of the postsynaptic glial membrane [1].
e-Neuroforum 3 · 2015 | 73
Review article
Fig. 1 8 Properties of synaptic inputs to NG2 cells in acute slices of the hippocampus from juvenile transgenic hGFAP/EGFP
mice. a The morphology of NG2 cells was visualized by Texas Red dextran filling during whole-cell recording. Subsequent confocal analysis and 2D maximum projection showed the extensive arborisation of their processes. Note the typical nodules appearing as dots along the fine processes (bar 10 µm). A typical current pattern is given in the middle panel. Current responses were evoked by de- and hyperpolarizing the membrane between + 20 and − 160 mV (holding potential − 80 mV, 10 mV
steps, bars 1 nA, 10 ms). Post-recording immunostaining and triple fluorescence confocal analysis were applied to test for
NG2 immunoreactivity. The middle panel shows three separated colour channels of one confocal plane. Texas Red dextran labelling is given in green (g), NG2 immunoreactivity in red (r) and EGFP expression in blue (b). The superimposed RGB picture
(right) shows the membrane-associated distribution of the NG2 immunoreactivity of the recorded cell (yellow details). b Postsynaptic currents were recorded from an NG2 cell upon short single pulse stimulation. At least two types of presynaptic neurons, glutamatergic CA3 pyramidal neurons and GABAergic interneurons, innervate hippocampal NG2 cells. Corresponding
glial postsynaptic currents can be distinguished by their current kinetics and pharmacological profiles. [1]
NG2 glia bear GABAA receptors which
also mediate depolarization of the membrane through the efflux of Cl−. This ef-
74 | e-Neuroforum 3 · 2015
flux is due to a relatively high intracellular
Cl− concentration resulting from the absence of transporters (e.g. KCC2), which
move Cl− outwardly in neurons. Combined pharmacological and transcript
analyses in single cells unraveled the sub-
Abstract
unit composition of the glial GABAA receptors in the hippocampus and neocortex. In addition to various α and β isoforms, the cells also have the γ2 subunit,
although its expression is confined to
the postsynaptic membrane; on the other hand, extrasynaptic GABAA receptors
lack this subunit [6]. In the neocortex, γ2
is downregulated during postnatal development and this change is accompanied
by a loss of the GABAergic synaptic innervation of NG2 glia [7].
Besides ionotropic receptors, NG2 glial cells also carry metabotropic glutamate
receptors (mGluR1, mGluR5), whose activation entails Ca2+ -induced release of intracellular Ca2+ [3]. Other studies have reported functional expression of purinergic and nicotinic acetylcholine receptors
in NG2 glia, which also mediate increase
in the intracellular Ca2+ concentration.
Synaptic signaling between
neurons and NG2 glial cells
NG2 glia receive synaptic
input from neurons
Over the past years, NG2 glial cells have
attracted much attention for two main
reasons: (i) As described earlier, these cells
express a plethora of voltage-gated ion
channels, and (ii) intriguingly, almost all
NG2 glial cells receive synaptic input from
neighboring neurons. The synaptic input
is mediated through classical vesicular
transmitter release at morphological contact points, similar to classical neuron–
neuron synapses (. Fig. 2). The properties of postsynaptic currents in NG2 glia
are also indistinguishable from those recorded in neurons (. Fig. 1b). NG2 glial
cells express a variety of neurotransmitter
receptors (cf. above), but synaptic release
activates virtually exclusively AMPA and
GABAA receptors, as could be demonstrated by the complete block of the postsynaptic receptors with specific antagonists (. Fig. 1b). Dwight Bergles was the
first to report functional synapses on NG2
glia in the hippocampus [8]. Since then,
several studies have shown that in various gray and white matter regions, for example, the neocortex, hippocampus, cerebellum, corpus callosum and optic nerve,
NG2 glial cells receive glutamatergic and
GABAergic synaptic inputs. Quantal synaptic currents in NG2 glia (due to exocytosis of a single transmitter-filled vesicle)
have amplitudes of approximately 10 pA
and very fast (millisecond range) rising
and decaying phases. As in neurons, glutamatergic synaptic currents reverse direction at approximately 0 mV, while GABA-induced postsynaptic currents reverse
at approximately − 40 mV (cf. above). Accordingly, in NG2 glia, GABA is not an
inhibitory transmitter but depolarizes the
cells similarly to glutamate. NG2 glial cells
receive much fewer synapses than neurons, with the innervation in the cerebellum being stronger than that in the hippocampus. Glial glutamatergic synapses
outnumber GABAergic synaptic inputs
by approximately fivefold.
Synaptic innervation as a signal
regulating glial differentiation
Increasing evidence suggests that synapses on NG2 glia might adjust the development of oligodendrocytes to neuronal activity. This idea is still speculative because
convincing experimental evidence is still
missing, and there are many possibilities
regarding how neuronal activity might
influence the differentiation of NG2 cells.
However, considering the enormous biological effort needed to establish and sustain this non-conventional signal pathway
between neurons and NG2 cells, it appears
likely to assume a specific impact of this
mechanism. Actually, (i) synapses on NG2
glial cells exist in white and gray matter
throughout the life span; (ii) establishing synapses, particularly with projecting
axons, requires significant transport efforts; (iii) synapses are already established
shortly after birth; (iv) surprisingly, neuron–NG2 glia synapses are retained during cell division and passed on to daughter cells; (v) during differentiation into oligodendrocytes, the release machinery of
NG2 cells is rapidly degraded; (vi) even
adult-born NG2 glia, which upon white
matter injury migrate to the lesion site and
contribute to regeneration, receive synaptic input.
So far, most properties of neuron–glia
communication have been determined
in rodents during the first two postnatal weeks. It is possible that in the third
e-Neuroforum 2015 · 6:73–77
DOI 10.1007/s13295-015-0010-2
© Springer-Verlag Berlin Heidelberg 2015
C. Steinhäuser · D. Dietrich
Neuron–glia synapses in the
brain: properties, diversity
and functions of NG2 glia
Abstract
Although NG2 glial cells represent a frequent glial cell type in the brain, characterized by expression of the NG2 proteoglycan, the functional impact of these cells is
still enigmatic. A large proportion of NG2
glia are proliferatively active throughout
life. These cells express a plethora of ion
channels and transmitter receptors, which
enable them to detect neuronal activity. Intriguingly, NG2 glial cells receive synaptic input from glutamatergic and GABAergic neurons. Since these postsynaptic glial currents are very small, their spatial and temporal integration might play
an important role. In white matter, most
NG2 glial cells differentiate into oligodendrocytes and this process might be influenced through the activity of the aforementioned neuron–glia synapses. Increasing evidence suggests that the properties
of NG2 glia vary across brain regions; however, the impact of this variability is not
understood yet.
Keywords
NG2 cells · Neuron–glia synapse · Synaptic
integration · Glial differentiation
and fourth weeks, when myelination culminates, the electrical impact of released
transmitter molecules increases owing to
enhanced synaptic strength. This correlation suggests that the synaptic input to
NG2 glia modulates oligodendrogenesis
to adjust it to the needs of the neuronal
electrical circuitry.
Synaptic integration in NG2 glia
Electrical integration of synaptic depolarisation through spatial and temporal summation of postsynaptic events represents
a classical mechanism of signal processing
in neurons. These cells integrate the input in their dendrites and conduct the resulting signal to their axon hillock, where
eventually action potentials are generated. However, NG2 glial cells lack axons
and do not generate action potentials, ine-Neuroforum 3 · 2015 | 75
Review article
tenth that of typical principal neurons, we
could recently show that the spatial voltage spread is similar in both cell types. [9].
Accordingly, regarding electrical compactness, NG2 glia might be considered a
miniature edition of neurons and able to
process the input, which could be crucial
for initiating activity-dependent myelination. Within this DFG Priority program,
we will investigate the role of ion channels and altered intracellular Ca2+ concentrations and their subcellular localization in the integration of synaptic signals
in NG2 glia.
Functions of NG2 glial cells
and neuron–glia synapses
Fig. 2 9 Morphological properties of neuron–glia synapses in
acute brain slices of
the juvenile hippocampus. a Electron microscopic visualization of
an asymmetric neuron–glia synapse. The
cell was first functionally characterized (inset, pulse protocol as
in . Fig. 1b), thereby filled with biocytin,
and then fixed and analysed ultrastructurally. Note the neuronal
presynaptic vesicles,
synaptic cleft and glial postsynaptic structure. b is an enlarged
view of the boxed area;
the arrow heads indicate vesicles. Scale bar:
200 nm. [3]
dicating that their signal integration fundamentally differs from neurons. Despite
the lack of action potentials, the fast glial Na+ channels may amplify or accelerate
synaptic depolarisation. It remains to be
shown whether these Na+ channels open
rapidly enough and if their number is sufficient to amplify synaptic depolarisation.
In reality, the large number of voltage-gated K+ channels counteracts this depolarisation, and the net effect will depend on
the activation kinetics and amplitudes of
the latter.
76 | e-Neuroforum 3 · 2015
NG2 glia possess a complex process
tree with a surface area of approximately 2000 µm2. Assuming that the diameter
of the cell body is 6–7 µm, it follows that
the processes make up more than 90 % of
the total cell surface and probably receive
most of the synaptic input. This raises the
question whether NG2 glial cell processes are competent of local synaptic integration. This has not yet been investigated.
However, using a simple “ball and stick”
cable model and considering that the
length of NG2 glia processes is only one
The consequences of receptor activation
in NG2 glia through presynaptic transmitter release are largely unknown until
now because the resulting depolarisations
are very small (only a few millivolts in the
hippocampus). However, most analyses so
far were confined to the cell body and it
is possible that receptor activation in the
thin peripheral processes entails much
stronger depolarization or intracellular
Ca2+ increase. Accordingly, it is tempting
to speculate that NG2 glial cells command
complex local signal integration, which allows them to respond to the specific activity patterns of single presynaptic axons.
Indeed, increasing evidence suggests that
these non-conventional synapses between
neurons and NG2 glia encode important
information for cell differentiation. It can
be expected that identifying the functional role of these synapses will be crucial for
our general appreciation of the enigmatic NG2 cell population within the central
nervous system.
Corresponding address
C. Steinhäuser
Institute of Cellular Neurosciences
Rheinische Friedrich-Wilhelms-Universität Bonn
Sigmund-Freud Str. 25, 53105 Bonn
[email protected]
Christian Steinhäuser studied physics at the
Friedrich-Schiller Universität Jena. He received a
PhD at the Institute for Neurobiology and Brain
Research, Academy of Sciences, Magdeburg and
the Faculty of Biology, University of Jena on the
functional analysis of Na+ channels in pyramidal
neurons freshly isolated from the hippocampus.
After habilitation in physiology, in 1997 he moved to
Bonn as a professor for Experimental Neurobiology
at the Medical Faculty of the Rheinische FriedrichWilhelms-Universität. In 2007, he founded the
Institute of Cellular Neurosciences at University of
Bonn that he chairs since. He employs molecular,
electrophysiological and imaging techniques to
investigate the role of glial cells in information
processing in the normal and epileptic brain
Dirk Dietrich studied medicine and informatics in
Bonn, Hagen and London and received an MD at the
Medical Faculty of the Rheinische Friedrich-WilhelmsUniversität Bonn. After habilitation in neurophysiology
he accepted a W2 professorship for Experimental
Neurophysiology at Neurosurgery Hospital, Bonn.
Since 2014 he is a Research Chair at the Neurosurgery
Hospital. His research is focused on the analysis
of neuron-glia signaling, pre- and extrasynaptic
regulation of glutamatergic neurotransmission,
Ca2+ -induced vesicular release and synaptic plasticity.
Acknowledgements. Work of the authors is supported by Deutsche Forschungsgemeinschaft (STE
552/5, STE 552/4, DI 853/5-1, DI 853/3-1, SFB 1089).
We thank Dr. Ines Heuer for technical support.
References
1. Bergles DE, Jabs R, Steinhäuser C (2010) Neuronglia synapses in the brain. Brain Res Rev 63:130–
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2. Chittajallu R, Chen Y, Wang H, Yuan X, Ghiani CA,
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3. Haberlandt C, Derouiche A, Wyczynski A, Haseleu
J, Pohle J, Karram K, Trotter J, Seifert G, Frotscher
M, Steinhäuser C, Jabs R (2011) Gray matter NG2
cells display multiple Ca-signaling pathways and
highly motile processes. PLoS One 6:e17575
4. Hughes EG, Kang SH, Fukaya M, Bergles DE (2013)
Oligodendrocyte progenitors balance growth with
self-repulsion to achieve homeostasis in the adult
brain. Nat Neurosci 16:668–676
5. Chittajallu R, Aguirre A, Gallo V (2004) NG2-positive cells in the mouse white and grey matter display distinct physiological properties. J Physiol
561:109–122
6. Passlick S, Grauer M, Schäfer C, Jabs R, Seifert G,
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e-Neuroforum 3 · 2015 | 77
Review article
e-Neuroforum 2015 · 6:79–83
DOI 10.1007/s13295-015-0011-1
Published online: 11 August 2015
© Springer-Verlag Berlin Heidelberg 2015
Christian Henneberger1,2 · Gabor C. Petzold3
1 Institute of Cellular Neurosciences, University of Bonn Medical School, Bonn, Germany
2 UCL Institute of Neurology, London, UK
3 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany
Diversity of synaptic
astrocyte–neuron signaling
Introduction
Synaptic transmission is the primary
means of information transfer in the nervous system, and its long-term plasticity is thought to underlie acquisition and
storage of new information. While these
processes are primarily neuronal, they do
occur in an environment that contains
both extracellular matrix and nonneuronal cells such as glia. Especially astrocytes,
a subtype of glia cell with a characteristic
star-like morphology, have been the subject of intense study over the past decades.
An important feature of astrocytes is that,
unlike neurons, the branches of an individual cell occupy clearly delineated territories with little overlap with their neighbors under normal conditions. Within
these domains, fine astrocyte branches
approach and enwrap synapses (. Fig. 1,
[20]). A single astrocyte can cover a total
of approximately 100,000 synapses arising
from many different neuronal connections in the rat hippocampus [6]. As astrocytes are capable of sensing and modulating synapse activity, they are potentially powerful modulators of large synaptic
populations. Therefore, their role as key
players in neuronal network function has
received a lot of attention but is also intensely debated.
The possibility of fast reciprocal signaling between neurons and astrocytes was
first suggested in the 1990s. Among the
many important findings was the observation of Ca2+ responses in astrocytes induced by the neurotransmitter glutamate
in cultured astrocytes and in situ [7, 35].
At the same time, an increase of cytosolic Ca2+ was shown, for example, to trigger release of glutamate from astrocytes,
thereby exciting nearby neurons [31].
These and other findings challenged the
classical view that the electrically passive
astrocytes only act as scaffolding for neurons, maintaining ion homeostasis and
providing metabolic support and neurotransmitter clearance, and gave rise to
the concept of the tripartite synapse [3].
A vast number of signaling cascades enabling neuron–astrocyte communication
is well documented now. They are triggered by neuronal activity and appear
to converge on astrocyte Ca2+ signaling
while astrocyte Ca2+ signaling was shown
to have a plethora of reciprocal effects on
neuronal network function (for review
[36] and references therein). For this reason, understanding astrocyte Ca2+ signaling is critical for establishing the functional and computational role of astrocyte–
neuron interactions, although astrocytes
have other means of sensing and integrating neuronal activity, sodium signaling for
instance [16, 36].
Astrocyte Ca2+ signaling
in astrocyte–neuron
communication
Metabotropic neurotransmitter receptors
represent a common link between neuronal activity and astrocyte Ca2+ signaling.
In the juvenile hippocampus for instance,
group I metabotropic glutamate receptors
mediate somatic store-dependent Ca2+
transients in response to Schaffer collateral stimulation [35]. Similar pathways exist
in hippocampal astrocytes for other presynaptically released neurotransmitters
such as acetylcholine [25], while multiple
mechanisms underlie astrocyte Ca2+ responses to the inhibitory neurotransmitter GABA [21]. These examples highlight
that astrocytes appear to respond with a
transient Ca2+ increase regardless of the
identity of the neurotransmitter and its
excitatory or inhibitory action on neurons. It raises the question if and how astrocytes discriminate between activity of
the various different presynaptic terminals they cover. In addition, astrocyte Ca2+
transients also occur in response to postsynaptic depolarization. For instance, endocannabinoid release after depolarization of hippocampal CA1 pyramidal cells
activates store-dependent Ca2+ signaling
in astrocytes [23]. This reduction of many different incoming signals into a single type of response, a Ca2+ transient of a
hippocampal astrocyte, implies that strikingly different neuronal network activity
may be translated into apparently similar
astrocyte Ca2+ signals.
This convergence onto a seemingly uniform Ca2+ signaling is contrasted by highly diverging effects that an astrocyte Ca2+ increase can have on synaptic transmission (. Fig. 2). Targets of astrocyte–neuron communication include
axonal action potential propagation. For
instance, action potentials of CA3 pyramidal cells are broadened while traveling
along their axons and release from their
synapses is facilitated if the Ca2+ concentration in nearby astrocytes is increased
by Ca2+ uncaging [38]. Neurotransmitter
release from these synapses is also under
direct control of astrocyte Ca2+ signaling.
Increasing astrocyte Ca2+ levels can transiently or permanently potentiate the release probability of CA3-CA1 synapses
by triggering glutamate release from astrocytes [24, 32] or by adenosine receptor signaling [30]. However, intact astrocyte Ca2+ signaling is also required for the
transient postburst depression of release
from these synapses [2] and spontaneous
e-Neuroforum 3 · 2015 | 79
Review article
Synaptic specificity of astrocyte–
neuron communication
Fig. 1 8 Synapses covered by a single astrocyte. Three-dimensional (3D) reconstruction after serial section electron microscopy of a small astrocyte fragment reveals the elaborate 3D fine structure of
astrocytes with their leaf-like terminal processes. (a turquoise, rat hippocampus). Postsynaptic spines
(b, white and green) with their postsynaptic densities (red) are embedded in the meshwork created by
fine astrocyte processes. Terminal astrocyte branches approaching and covering synaptic contacts are
often as thin as 100–200 nm. (scale cube, 1 µm3, figure adopted from [20]). An individual hippocampal
astrocyte covers approximately 100,000 synapses [6].
Fig. 2 8 Astrocyte Ca2+ signaling—one signal
with too many targets? Cytosolic Ca2+ rises in astrocytes are a potent trigger of astrocyte to neuron communication. A vast body of experimental evidence reports a highly diverse set of neuronal targets. For instance, glutamatergic excitatory synaptic transmission (orange, upper half)
received by CA1 pyramidal cells of the hippocampus is modulated presynaptically at the level of axonal action potential propagation and
neurotransmitter release and by activation of
postsynaptic receptors. At the same time, GABAergic transmission (green, lower half) is also altered in response to similar Ca2+ transients. The
net effect on the synaptic or cellular level will
depend on the specific set of mechanisms available at an individual synapse, the spatial extent
of the astrocyte Ca2+ signal and the range of action of a signaling molecules released from astrocytes
excitatory synaptic transmission can be
inhibited by a Ca2+-dependent boost of
astrocyte potassium uptake [43]. Further-
80 | e-Neuroforum 3 · 2015
more, postsynaptic neuronal N-methylD-aspartate receptors (NMDARs) are activated by Ca2+-dependent release of glutamate from astrocytes [23] and supported by Ca2+-dependent NMDAR co-agonist supply by astrocytes [13]. As a consequence of this diversity, the net effect of an astrocyte Ca2+ signal on excitatory synaptic transmission is far from
clear (. Fig. 2, upper half). Another target of astrocyte–neuron communication
is GABAergic inhibition (for review [18]).
For instance, induction of astrocyte Ca2+
transients increases GABAergic transmission pre and postsynaptically ([15],
. Fig. 2, lower half). These examples of
astrocyte modulation of synaptic transmission highlight the diverse and possibly opposing effects an astrocyte Ca2+ elevation could trigger locally at the synapses received by a CA1 pyramidal cells. Developmental changes of glutamate receptor expression by astrocytes [40] and profound alterations of astrocyte Ca2+ signaling in an animal model of Alzheimer’s
disease [9] are just two important examples of additional layers of complexity of
astrocyte–neuron communication that also emphasize the need to carefully consider experimental conditions.
This puzzling diversity of astrocyte–neuron communication at synapses received
by a single neuron subtype, here the CA1
pyramidal cell, poses questions about its
physiological and computational function. Are individual pathways spatially
segregated? Are they simultaneously recruited at single synapses and across synapse populations? For instance, a global astrocyte Ca2+ transient propagating
through an entire astrocyte and possibly
invading neighbouring gap-junction coupled astrocytes is likely to engage astrocyte–neuron signaling at a vast number
of different synapses and thus may represent a homeostatic mechanism. In contrast, a local and spatially confined Ca2+
transient could serve to fine-tune information transfer at single synaptic connections or gate their plasticity. Indeed, astrocyte processes can generate Ca2+ transients locally and independently as demonstrated more than a decade ago in situ [26]. More recent advances in Ca2+ imaging techniques and development of genetically encoded Ca2+ sensors allowed
detection of a striking degree of compartmentalization of astrocyte Ca2+ transients in situ and in vivo [10, 14, 39]. The
vast majority of these Ca2+ transient were
highly localized events often not extending more than a micrometre and only a
minority of events invaded larger parts
of the astrocyte or its soma. Importantly, local Ca2+ transients could be induced
by synaptic stimulation and depended
on synaptic activity [10, 30]. This implies
that astrocytes can translate neuronal activity into the small scale Ca2+ transients
that could locally modulate the function
of single or few synapses through a specific mechanism. Demonstrating that this
is indeed the case will be a significant experimental challenge for several reasons.
It requires methods to manipulate and
monitor synapses and astrocytes simultaneously at the micrometre scale. In addition, it is for instance unclear if all astrocytes and all processes of a single astrocyte are equipped with the same molecular machinery for detection of synaptic activity and its modulation. Thus, the question arises which astrocyte and which part
Abstract
of the astrocyte to probe for local synaptic
astrocyte–neuron signaling. While differences of astrocyte–neuron signaling between brain regions have been established
[19], it is largely unexplored if individual astrocytes in a subregion like the CA1
stratum represent a homogeneous population or could be as diverse as, for instance, interneurons [17]. The gradient of
astrocyte gap junction coupling anisotropy and morphology within the CA1 stratum radiatum could indicate that such
local heterogeneity exists [1]. Also, astrocytes can potentially control the supply of
the NMDAR co-agonist D-serine individually rather than acting as a gap junctioncoupled functional syncytium [13]. It remains unknown however what the physiological granularity of NMDAR co-agonist supply is and if variability of astrocyte morphology is functionally relevant
for astrocyte–neuron communication.
Significant heterogeneity could also exist on the level of a single astrocyte.
It is conceivable that certain compartments of an astrocyte would only be capable of modulation of inhibitory or excitatory synapse or be exclusively sensitive to their activity. Identification of
such subcellular specializations in astrocytes is complicated by the lack of a clearly definable cellular polarity. In neurons,
localization of a protein already gives a
strong clue as to its functions on the cellular level. Presence or absence of a particular signaling cascade in synaptic spines,
dendritic shafts, soma, axon, or synaptic
boutons guides interpretation of experimental results regarding the computational processes it might be relevant for.
Such structural features with firmly established functions are largely undefined
for astrocyte with the exception of astrocyte endfeet. These are astrocyte processes that contact and encase blood vessels
and are thought to play a role in neurovascular coupling [34]. Interestingly, astrocyte endfeet are compartments where diffusion is slowed considerably compared
with the rest of the cell thus limiting the
spread of diffusible signals [27]. They are
also regions of the astrocyte enriched with
Ca2+-permeable TRPV4 channels [4] that
trigger store-amplified Ca2+ transients in
endfeet and may contribute to neurovascular coupling [11]. Whether other simi-
larly specialized compartments exist within single astrocytes that control astrocyte–
neuron signaling on a synaptic scale remains to be established. Several recent
studies indicate that at least the signaling
cascades that generate Ca2+ transients are
unevenly distributed throughout the astrocytes. Genetic deletion of the IP3 receptor expressed by astrocytes revealed
that a significant fraction of spontaneous
Ca2+ transients do not rely on IP3-dependent signaling, especially those generated
in the astrocyte periphery [14, 39]. The localization of astrocyte Ca2+ transients triggered by synaptic stimulation also determines their dependence on metabotropic
glutamate receptors [41]. Thus, the spatial
distribution of different astrocyte Ca2+
signaling mechanisms could ensure specific neuron–astrocyte communication on
the synaptic scale.
Astrocyte coverage of synapses
Most of the well-established mechanisms of astrocyte–neuron signaling rely on diffusion of signaling molecules between neurons and astrocytes. As a consequence, the efficiency of interactions is
determined by the distance between a site
of release and the site of action and therefore the spatial configuration of synapses
and the neighbouring astrocyte branches
(. Fig. 1). Physiological changes of astrocyte coverage of neurons during lactation
affect glutamate clearance and supply of
the NMDAR co-agonists D-serine in the
supraoptic nucleus [28, 29]. In the hippocampus, the coverage of excitatory synapses by astrocytes also varies considerably between individual synapses [20, 42].
A functional implication could be that astrocyte–neuron communication shows a
similar degree of variability or selectivity,
thereby increasing its synaptic specificity. In that regard, it is of particular interest that the morphology of astrocytes and
their fine terminal branches is not static
but dynamic [5, 12, 33, 44]. Such dynamic changes of astrocyte morphology and
therefore reconfiguration of the spatial
relationship between astrocyte branches
and synaptic contacts could be a critical
determinant of astrocyte–neuron communication. For example, an astrocyte
process retraction from a synaptic con-
e-Neuroforum 2015 · 6:79–83
DOI 10.1007/s13295-015-0011-1
© Springer-Verlag Berlin Heidelberg 2015
C. Henneberger · G.C. Petzold
Diversity of synaptic
astrocyte–neuron signaling
Abstract
Fast signal exchange between neurons and
astrocytes at the synaptic level has attracted
considerable attention. Astrocytes often respond with Ca2+ transients to widely different neuronal synaptic activity. At the same
time, astrocyte Ca2+ elevations trigger profound and diverse changes of both excitatory and inhibitory synaptic transmission. Here,
we briefly review examples of the heterogeneity of Ca2+-dependent astrocyte–neuron
communication in the rodent hippocampus
and discuss mechanisms that could maintain
specificity of synaptic astrocyte–neuron signaling in the face of its diversity.
nection could affect synaptic transmission
in a number of ways. The astrocyte’s ability to sense activity of that particular synaptic connection could be impaired while
transmitters released from the astrocyte
may reach a much lower concentrations
at this synapse. On the other hand, astrocytes mediate most of the glutamate uptake [8]. An astrocyte process retraction
could therefore reduce clearance of neurotransmitter released from neurons and
as a consequence effectively boost synaptic transmission through extrasynaptic high affinity glutamate receptors. It is
noteworthy that induction of long-term
potentiation (LTP) appears to be a particularly robust trigger of astrocyte morphology changes [5, 12, 33, 44]. Coordinated morphology changes of presynaptic boutons and postsynaptic spines in
parallel to LTP [22] accompanied by astrocyte restructuring could therefore persistently enable or disable astrocyte–neuron communication at potentiated synapses. Indeed the diffusion weighted distance from a postsynaptic density to the
surrounding astrocyte processes is smaller at thin spines compared to mushroom
spines, which possibly have undergone
previous potentiation in the dentate gyrus [20]. While this suggests that synaptic plasticity can profoundly alter astrocyte–neuron communication on the level of single synapses by structural change
e-Neuroforum 3 · 2015 | 81
Review article
such modulation could also occur as a result of, for instance, TNFα signaling [37].
Conclusions
Astrocytes sense ongoing neuronal activity by various mechanisms that appear to trigger qualitatively similar Ca2+
transients. At the same time, Ca2+ signaling in astrocytes is a powerful modulator
of synapse function with diverse and potentially opposing net effects on neuronal activity even within a subregion like
the CA1 stratum radiatum. To what degree and how synapse specificity of astrocyte–neuron communication is maintained in the face of such diversity is only beginning to emerge. Establishing if
regional astrocyte heterogeneity or subcellular compartmentalization of signaling mechanisms within single astrocytes
does constrain neuron–astrocyte communication will be a technical challenge
and conceptually demanding because
principles established for neurons may
not fully apply. The specific geometry of
the tripartite synapse and its dynamic
changes are additional important determinants of signal exchange that will require careful consideration. Characterizing the spatial scale of a documented signaling pathway, if it tunes single synaptic
connections or homeostatically modulates a vast number of synapses, and the
context in which it is activated appears
to be required before the computational
role of astrocyte–neuron communication
can be fully appreciated.
Corresponding address
C. Henneberger
Institute of Cellular Neurosciences
University of Bonn Medical School
Sigmund-Freud-Str. 25, 53105 Bonn
[email protected]
Christian Henneberger studied medicine at the
Humboldt and Free University Berlin (Germany)
where he obtained his degree and defended his
thesis in neurophysiology both in 2003. He continued
his work on the properties of synaptic transmission
in the developing visual system as a postdoctoral
fellow at the Institute of Neurophysiology at the
Charité (Berlin, AiP research fellowship). After moving
to the UCL Institute of Neurology (London, UK), he
focused on hippocampal synaptic transmission and
plasticity and its dependence on components of the
82 | e-Neuroforum 3 · 2015
extracellular matrix and astrocyte Ca2+ signaling.
Obtaining a UCL Excellence Fellowship allowed him
to continue this line of work as a principal investigator
at UCL before establishing his own lab in Bonn in
2011. Funded by the NRW-Rückkehrerprogramm,
DFG and HFSP his lab (www.henneberger-lab.com,
Institute of Cellular Neuroscience) investigates how
dynamic changes of astrocyte morphology affect
astrocyte–neuron interactions at the cellular and
synaptic level in healthy brain tissue and in disease.
Gabor C. Petzold studied medicine in Düsseldorf,
Budapest, New York and London. He worked as
a postdoc and resident in clinical neurology at
the Departments of Neurology and Experimental
Neurology, Charité Berlin. He continued to work as
a postdoc, funded by an EU Marie Curie fellowship
and the DFG, at the Department of Molecular and
Cellular Biology, Harvard University. He returned
to Berlin to work as a principal investigator on the
role of astrocytes in neurovascular coupling and
neurodegenerative diseases, and as a senior clinical
fellow in clinical neurology. In 2011, he joined the
German Center for Neurodegenerative Diseases
(DZNE) in Bonn as a group leader, and the University
Hospital Bonn as a consultant. In 2013, he was
jointly appointed full tenured professor for vascular
neurology by the DZNE and Bonn University. His
lab investigates the contribution of astrocytes,
neuron–astrocyte communication and blood flow
changes to neurodegenerative diseases and stroke.
Acknowledgments. Work was supported by DFG
(SFB1089 B03 to Christian Henneberger, SPP1757
HE6949/1 to Christian Henneberger, PE1193/2-1 to
Gabor C. Petzold), NRW-Rückkehrerprogramm (Christian Henneberger), Human Frontiers Science Program
(Christian Henneberger), Else-Kröner Fresenius
Foundation (Gabor C. Petzold), Network Of Centres
Of Excellence In Neurodegeneration—CoEN (Gabor
C. Petzold) and the DZNE (Gabor C. Petzold).
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e-Neuroforum 3 · 2015 | 83
Special Issue Glia
e-Neuroforum 2015 · 6:85–86
DOI 10.1007/s13295-015-0013-z
Published online: 31 July 2015
© Springer-Verlag Berlin Heidelberg 2015
Kazuhiro Ikenaka
Centre for Multidisciplinary Brain Research and Department of Molecular Physiology,
National Institute for Physiological Sciences (NIPS), Okazaki, Japan
Funding of a glial research
program in Japan: the
glial assembly project
The Japanese government represented by
the Japan Society for the Promotion of
Science supports research in special programs. The Grant-in-Aid for Scientific
Research on Innovative Areas program
supports research projects upon proposal by a group of scientists. It is expected
to strengthen the scientific standard of
Japan on emerging fields and to develop
collaborations within the group. It is also expected to educate and train young
researchers who will further develop this
scientific field in the future. The support
will be given for 5 years with an expected budget of up to 300 million Yen (about
2.2 million EUR) per year. The approval
rate is about 10 %, and each year about 20
projects will be funded.
In 2013 the Grant-in-Aid program selected the proposal Glial Assembly: A new
regulatory machinery of brain function
(http://square.umin.ac.jp/glialassembl/
en/) for funding, thereby supporting research of glia biology. The Glial Assembly
research consortium will receive 1.2 billion Yen (8.7 million EUR) for 5 years.
The general aim of the research project
is a better understanding of neuron glia
interactions in brain function (. Fig. 1).
Neurons communicate with one another by forming neuronal circuits, which
play a major role in expressing the brain
function. Similarly, glial cells also communicate with one another and form glial circuits. The communication pathways
among neurons and those among glial
cells are intrinsically different: glial communication being much slower and more
gradual than neuronal communication.
Long-range communication by glial cells
often covers a macroscopic brain area, interacts with and affects the activity of neu-
ronal networks and circuits, and thereby
controls the brain function.
The purpose of the project’s work program is to clarify the mechanisms that underlie the formation of the gigantic glial
circuits, the glial assembly, and to understand how they control the brain function.
Furthermore, it is aimed to clarify how abnormalities of the glial assembly are related to the pathophysiology of neuropsychiatric disorders.
Annual activities of the consortium
are technical workshops, progress reports,
general symposia and young investigator
workshops. Financial support is allocated
to foster collaborations within the consor-
neurons
neuronal circuits
fast, firing/resting, polarity
tium and the attendance of young scientists at international glia meetings.
Thereby, the Grant-in-Aid appears to
be very similar to the German DFG Priority Programs.
List of core members:
IKENAKA, Kazuhiro: National Institute for Physiological Science
IINO, Masamitsu: The University of
Tokyo
KOIZUMI, Shuichi: University of Yamanashi
OHKI, Kenichi: Kyushu University
OKABE, Shigeo: University of Tokyo
FUKUYAMA, Hidenao: Kyoto University
glial cells
glial assembly
slow, gradual, bidirectional
Fig. 1 8 Neuronal circuits and the glial assembly cooperate in distinct pattern for proper brain function
e-Neuroforum 3 · 2015 | 85
Special Issue Glia
KOHSAKA, Shinichi: National Institute of Neuroscience
TAKEBAYASHI, Hirohide: Niigata
University
OZAKI, Norio: Nagoya University
INOUE, Kazuhide: Kyushu University
KANBA, Shigenobu: Kyushu University
KIRA, Junichi: Kyushu University
Corresponding address
Prof. K. Ikenaka
Centre for Multidisciplinary Brain Research and
Department of Molecular Physiology
National Institute for Physiological
Sciences (NIPS)
38 Nishigonaka Myodaiji
444–8585 Okazaki, Aichi
[email protected]
Prof. Kazuhiro Ikenaka 1975 Graduated from
Faculty of Science, Osaka University. 1980
Graduated from the doctoral course at Osaka
University, PhD. 1980 Instructor at Institute for
Protein Research, Osaka University. 1991 Associate
Professor at Institute for Protein Research,
Osaka University. 1992 Professor, NIPS. Major
subject of research: Molecular Neurobiology.
86 | e-Neuroforum 3 · 2015