Neuron Glia Biology, page 1 of 10.

# Cambridge University Press, 2012

doi:10.1017/S1740925X12000038

The role of microglia at synapses in the

healthy CNS: novel insights from recent
imaging studies
marie-e‘ve tremblay

In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile pro-
cesses. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of
synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and
synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior
including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity
and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimin-
ation of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic
spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit
reorganization.

Keywords: Motility, dendritic spine, plasticity, remodeling, elimination

INTRODUCTION promptly to any changes occurring in their environment,

Microglia were discovered almost a century ago by Pio del
Rio-Hortega. From his light microscopic observations of
silver carbonate impregnated microglia in rabbit, cat and
human brain in situ (1919–27; see Fig. 1A), del Rio-Hortega
proposed that microglia have a mesodermal origin, colonize
the brain during embryonic development, become evenly dis-
tributed, display little morphological variation in the mature
brain, but respond to pathological insults by transforming
their morphology and acquiring the capacity to migrate, pro-
liferate and phagocytose (Del Rio-Hortega, 1932; reviewed in
Kettenmann et al., 2011). Since his formulation of these
visionary postulates, which are still valid today, the changes
in microglial morphology, gene expression and functional
behavior during infection, trauma, ischemia and various neu-
ropsychiatric and neurodegenerative diseases have been
extensively studied. In all these situations, where normal
homeostasis is impaired, activated microglia were shown to
perform a variety of roles essential to the immune and inflam-
matory response of the central nervous system (CNS), includ-
ing the release of numerous factors and compounds,
presentation of antigens to T cells and phagocytosis of tissue
debris, damaged cells and microbes (reviewed in Kreutzberg,
1996; Hanisch and Kettenmann, 2007; Ransohoff and Perry,
2009; Rivest, 2009; Graeber, 2010; Prinz et al., 2011).
The roles of ‘resting’ or immunologically quiescent micro-
glia have remained relatively unknown (also see Tremblay
et al., 2011). This is largely due to the difficulties of studying
microglia in their non-activated state. Microglia respond
Corresponding author:
Marie-Ève Tremblay
Email: tremblay2@wisc.edu
and therefore experimental ex vivo and in vitro preparations
inevitably result in transformation of their normally prevailing
behavior. Recently, the development of in vivo two-photon
laser scanning microscopy (or two-photon) has for the first
time allowed the study of resting microglia in the intact
brain of living animals (Davalos et al., 2005; Nimmerjahn
et al., 2005), using CX3CR1-GFP transgenic mice in which
microglia are fluorescently labeled (Jung et al., 2000; see
Section 1 for details on these important mice). When
imaged through the skull, microglia in the superficial layers
of somatosensory and motor cortices of adult animals dis-
played small cell bodies with highly ramified thin processes
extending radially and were distributed homogeneously in
three dimensions (3D), as anticipated from previous in situ
observations (see Del Rio-Hortega, 1932). But quite unexpect-
edly, microglia were also extremely dynamic, continuously
extending and retracting processes at an average velocity of
1.5 mm min21 (2.2 mm min21 for terminal protrusions)
and maximal velocity of 4 mm min21, thereby leading to
comprehensive changes in cellular morphology on a time
scale of minutes (Davalos et al., 2005; Nimmerjahn et al.,
2005; Fig. 1B). This dynamic remodeling contrasts both to
neurons and other types of glial cells imaged in vivo in the cer-
ebral cortex of juvenile and adult mice, which showed no com-
parable restructuring of processes. Astrocytic processes
showed no motility at all over 1 h (Eom et al., 2011),
neuron–glial antigen 2 (NG2)-positive glial cells remodeled
their processes over several days (Hughes et al. (2011);
Society for Neuroscience abstract No. 548.12), while the
most motile dendritic spines only reached an average velocity
of 0.02 mm min21 (Majewska and Sur, 2003). Following
these observations, ‘resting’ microglia have emerged as the
most structurally dynamic cells of the mature CNS discovered
so far.

1
2 marie-e‘ ve tremblay

Fig. 1. Evolving views of resting microglia, from the pioneer observation of silver carbonate impregnated microglia in situ to two-photon imaging of
microglia-synapse dynamics in vivo. (A) Drawing by Pio del Rio-Hortega of ramified microglial morphology (modified from Del Rio-Hortega, 1919). (B)
Two-photon micrograph showing microglial process extensions (green) and retractions (red) in a CX3CR1-GFP mouse over the course of 20 min (reproduced
from Nimmerjahn et al., 2005). (C) Electron micrograph showing direct contacts of an Iba1-immunostained microglial process with dendritic spines (pink),
axon terminals (blue), synaptic cleft (arrow) and perisynaptic astrocytic processes (green). Note the extracellular space pockets surrounding the microglia
(asterisks). (D) 3D reconstruction of proximal (cut in transverse) and distal (longitudinal) microglial processes uncovering simultaneous contacts with
dendritic spines varying in shape and size (pink). Extracellular space pockets are displayed in white, and a phagocytic inclusion within the proximal microglial
process is in purple. (E) Two-photon time-lapse micrographs showing a dynamic microglial process (yellow for presentation purposes) transiently interacting
with dendritic spines (green) in a CX3CR1-GFP/Thy1-YFP mouse over the course of 20 min. Each frame was captured 5 min apart. Red arrowheads indicate
non-targeted spines, and white arrowheads targeted ones. (C) to (E) reproduced from Tremblay et al. (2010b).

This unexpected behavior suggested that resting or surveil-
lant microglia may continuously survey the brain parenchyma
as part of their immune function, which would justify the sub-
stantial expenditure of energy required to continuously main-
tain microglial dynamics in the normal brain, without
excluding the possibility of an additional, distinct contribution
to normal brain physiology. Although the field of research
addressing the roles of microglia under non-pathological con-
ditions is still very young and comprises only a few studies, for
the purpose of this special issue of Neuron Glia Biology, I will
discuss these emerging roles of microglia at synapses in the
mammalian CNS. Within this limited scope, I will focus on
non- or minimally invasive in situ and in vivo imaging
approaches, and discuss recent observations describing the
structural interactions between microglia and synaptic
elements. I will also examine the evidence supporting a
regulation of microglial behavior by neurotransmission, neur-
onal activity and sensory experience, and conversely, for an
involvement of microglia in the elimination of synaptic struc-
tures during experience-dependent plasticity.
Characterization of microglial interactions
with synaptic elements
Electron microscopy (EM) enables the investigator to accu-
rately visualize the fine structural intricacies between all the
cells and their various compartments, and the extracellular
matrices binding them together, thereby uncovering their
immensely complex array of possible functional interactions
at the local level within any tissue, at the best resolution
(approximately 1 nm) so far achieved by a morphological

technique. Over the last 60 years, ultrastructural examinations
of the CNS have provided unprecedented insights into the
functional relationships of neurons and glial cells (astrocytes,
NG2-positive cells, oligodendrocytes and microglia in most
regions of the mature CNS) under various conditions of
health and disease. For example, EM contributed to the estab-
lishment of astrocytes as synaptic partners modulating neuro-
transmission (see Ventura and Harris, 1999; reviewed in
Theodosis et al., 2008) and to the recent discovery of synaptic
interactions between NG2-positve cells and neurons
(reviewed in Bergles et al., 2010). Some 40 years ago, a possible
but still controversial role of activated microglia in synaptic
stripping also emerged from EM observations showing that
microglial processes structurally intervene between pre-
synaptic axon terminals and post-synaptic dendrites or neur-
onal cell bodies under pathological conditions (Blinzinger and
Kreutzberg, 1968; reviewed in Graeber, 2010; Perry and
O’Connor, 2010; see also Trapp et al., 2007).
Microglial cell bodies under healthy, non-pathological con-
ditions were also described with EM just as long ago (Herndon,
1964; Mori and Leblond, 1969), but since microglial processes
are rarely contiguous to their cell bodies in single ultrathin sec-
tions, and these processes have few distinctive ultrastructural
features, their discrimination from other subcellular elements
(such as small unmyelinated axons, dendrites and other
small glial processes) required the development of selective
labeling approaches. In particular, an enzymatic staining for
thiamine pyrophosphatase (TPPase) allowed examination of
microglial processes in situ for the first time 30 years ago,
revealing their direct apposition with excitatory synapses in
the cerebral cortex of mature rats (Murabe and Sano, 1982).
Since TPPase labels not only microglia, but also neurons, astro-
cytes and oligodendrocytes (Kumamoto et al., 1976; Castellano
et al., 1989), a more specific approach was needed to validate
these observations. To this end, immunocytochemical EM
with a specific antibody directed against ionized calcium
binding adaptor molecule 1 (Iba1), a protein only expressed
by microglia within the healthy CNS (Ito et al., 1998), was
recently performed, showing that microglial processes can
interact with both pre-synaptic axon terminals and post-
synaptic dendritic spines in the somatosensory cortex of
adult mice (Wake et al., 2009). Contrary to these interactions
with excitatory synapses, the existence of microglial inter-
actions with inhibitory synapses under normal physiological
conditions remains yet unknown.
Extending this characterization, Tremblay et al. (2010b)
revealed with a similar immunocytochemical EM approach
(see Tremblay et al. (2010a) for methodological details) that
almost all microglial processes localize to the vicinity of excit-
atory synapses in the superficial layers of juvenile mouse visual
cortex: 94% of microglial processes directly contacted term-
inals, spines, perisynaptic astrocytic processes and synaptic
clefts, in decreasing order of frequency, and 68% of these
processes contacted more than one synaptic element
(Fig. 1C). Conversely, the proportion of each of those synaptic
elements contacted by microglial processes could not be deter-
mined, because not all microglial processes were positively
identified, since only a subset were stained for Iba1 under
these stringent immunocytochemical conditions. Recently,
microglial interactions with terminals, spines, astrocytic pro-
cesses and synaptic clefts were also confirmed throughout
adulthood and normal aging, in the superficial layers of
mouse visual and auditory cortices (Tremblay et al., 2012).
role of microglia at synapses in the healthy cns 3
Serial section EM (SSEM) with 3D reconstruction further
revealed the geometry of various contacts between a single
microglial process and numerous excitatory synapses
(Fig. 1D) in juvenile animals (Tremblay et al., 2010b). While
microglial contacts generally occurred en passant, one of the
contacts displayed morphological specialization in the form
of finger-like protrusions wrapping around a spine, suggesting
immediate and functional interactions. Clathrin-coated pits
were also observed at interfaces between microglial processes
and neuronal or astrocytic elements of synapses, on either side
of the interaction, suggesting reciprocal exchange of molecular
signals through clathrin-mediated endocytosis of membrane-
bound receptors and their ligands (see Le Roy and Wrana
(2005) for details on clathrin-mediated endocytosis). Even
though the nature of the exchanged signals remains to be
determined, these two types of morphological specializations
(finger-like protrusions and clathrin-coated pits) indicate
that surveillant microglia can functionally interact with synap-
tic structures under non-pathological conditions.
Inspired by the extreme dynamism of surveillant microglia
in the intact CNS (Davalos et al., 2005; Nimmerjahn et al.,
2005), recent studies have additionally explored the dynamic
nature of microglial interactions with synaptic structures
using two-photon in vivo imaging. The imaging of microglia
and synaptic structures required the development of trans-
genic mice in which both microglia and neurons are fluores-
cently labeled. For this purpose, Wake et al. (2009) crossed
Iba1-GFP (Hirasawa et al., 2005) with Thy1-GFP mice
(Feng et al., 2000) for visualizing microglia and neuronal
elements including axons, dendrites, terminals and spines in
the same color (green). More recently, other investigators
crossed the CX3CR1-GFP mice from Jung et al. (2000),
which provide exceptional in vivo visualization of microglial
morphology including the distal processes and fine protru-
sions (Fig. 2), with Thy1-YFP mice (Feng et al., 2000 or
Hirrlinger et al., 2005), enabling simultaneous visualization
of microglia and neurons in two different colors (green and
Fig. 2. Imaging of surveillant microglia in vivo with the CX3CR1-GFP mice
from Jung et al. (2000). Two-photon micrograph (maximum z-projection of
20 slices captured 1 mm apart) showing the density and morphology of
microglial cell bodies, processes and terminal protrusions, with or without
bulbous endings (shown by arrowheads), when imaged through a thinned
skull in somatosensory cortex.
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yellow respectively, see Fuhrmann et al. (2010); Tremblay
et al. (2010b) and Dibaj et al. (2010) for examples in cerebral
cortex and spinal cord). It should be noted that CX3CR1-GFP
heterozygous mice generally used for imaging may be partially
deficient in fractalkine signaling, a pathway that was pre-
viously shown to modulate microglial activation and cytokine
release, neuron–microglia communication and neuronal sur-
vival in different contexts of disease and CNS regions (for
example see Harrison et al., 1998; Cardona et al., 2006;
Fuhrmann et al., 2010; Cho et al., 2011; Donnelly et al.,
2011) due to the replacement of one of the two alleles encod-
ing microglial CX3CR1 with a GFP reporter. Nevertheless,
many aspects of microglial morphology, process motility
(Nimmerjahn et al., 2005; Wake et al., 2009) and interactions
with synaptic elements (Wake et al., 2009; Tremblay et al.,
2010b) that were examined in vivo were comparable
between the CX3CR1-GFP heterozygous mice and Iba1-GFP
mice from Hirasawa et al. (2005) where CX3CR1 is not
deleted.
Using Iba1-GFP/Thy1-GFP mice and CX3CR1-GFP/
Thy1-YFP mice, respectively, Wake et al. (2009) and
Tremblay et al. (2010b) recently explored microglial inter-
actions with axon terminals and dendritic spines from
subsets of cortical layer V pyramidal neurons in vivo. With
great caution, a non-invasive thinned-skull preparation was
performed to prevent brain injury and microglial activation
(see Tremblay et al. (2010b), Yang et al. (2010) and Marker
et al. (2010) for methodological details). In this manner,
Wake et al. (2009) first showed that microglial processes tran-
siently interact with terminals and spines, in the somatosen-
sory and visual cortices of adult animals. Microglial contacts
with terminals generally lasted 5 min and occurred at a fre-
quency of 1 per hour and per terminal. During the contacts,
microglial processes displayed enlarged extremities or bulbous
endings (Wake et al., 2009) resembling the morphological
specializations described with SSEM and 3D reconstruction
in situ (Tremblay et al., 2010b). Additionally, Tremblay
et al. (2010b) observed microglial contacts with terminals
and spines that varied in duration between 5 and 30 min in
the visual cortex of juvenile animals, using time-lapse
imaging every 5 min, thereby adding a new dimension to
the modalities of microglial interactions with synaptic
elements occurring in vivo under non-pathological conditions.
In the mature healthy CNS, neuronal networks are con-
tinuously remodeled through the formation, modification
and elimination of synaptic structures (see Fortin et al.
(2011) for molecular mechanisms of structural plasticity) in
relation with behavioral and sensory experience. In particular,
two-photon in vivo imaging in Thy1-YFP or Thy1-GFP mice
revealed that spines, especially small ones, are structurally
dynamic and transient in mouse visual, somatosensory,
motor and frontal cortices (Trachtenberg et al., 2002; Zuo
et al., 2005; De Paola et al., 2006; Majewska et al., 2006; also
see Alvarez and Sabatini, 2007; Holtmaat and Svoboda,
2009). To determine a possible role of surveillant microglia
in the structural remodeling of synaptic structures under
normal physiological conditions, Tremblay et al. (2010b)
also examined the size changes of spines and terminals
before, during and after microglial contacts. Spines contacted
by microglial processes during imaging (30–120 min sessions)
were found to be smaller initially than those which remained
non-contacted. Spines, but not terminals, also underwent
transient increases in size during microglial contact, with
smaller spines showing the most pronounced changes.
Surprisingly, chronic imaging over 2 days further revealed a
statistically significant difference in the elimination rate of
microglia-contacted spines: spines contacted by microglia
were more frequently eliminated than non-contacted spines
(24 versus 7%; P  0.05), and in all cases, only the small
spines were seen to disappear. These observations suggest
that despite an apparently random sampling of the parench-
yma, microglial processes specifically target a subset of
small, structurally dynamic and transient dendritic spines.
Altogether, these immunocytochemical EM, SSEM with 3D
reconstruction and two-photon in vivo imaging observations
indicate that as part of their normal surveillance, microglial
processes frequently interact with a subset of synaptic struc-
tures. This specificity raises the intriguing possibility that
microglia could perceive neuronal activity or structural
changes at individual synapses.
Regulation of microglial behavior by
neurotransmission, neuronal activity
and sensory experience
Cultured microglia were shown to express various receptors
for acetylcholine, glutamate, GABA, serotonin, dopamine,
noradrenaline, ATP, cannabinoids, opioids and neuropeptides
among other neurotransmitters. Each of these neurotransmit-
ters induced changes in the electrophysiological properties of
microglia (reviewed in Biber et al. (2007), Pocock and
Kettenmann (2007), Kettenmann et al. (2011); and see Liu
et al. (2011) for details and receptors involved) while ATP
and glutamate also influenced microglial motility in vitro
(Honda et al., 2001; Haynes et al., 2006; Liu et al., 2009; but
also see Wu and Zhuo, 2008; Chen et al., 2010). But impor-
tantly, since microglial resting state is believed to differ
between in vitro and in vivo environments, given the
absence of in situ evidence it remains uncertain whether sur-
veillant microglia also express receptors for these neurotrans-
mitters in the intact CNS (aside from ATP, see below).
Therefore, how these demanding immune cells normally
respond to the various neurotransmitters released by
neurons and glial cells remains to be seen.
To determine whether surveillant microglia can detect
neuronal and synaptic activity under physiological conditions,
recent studies investigated microglial response to neurotrans-
mission, neuronal activity and sensory experience in the intact
CNS. Specifically, microglial sampling of the parenchyma,
basal velocity of their processes and dynamic interactions
with axon terminals and dendritic spines were examined.
neurotransmission
Even though it remains unknown whether changes in neuro-
transmission at individual synapses can influence microglia in
the intact CNS, two imaging studies have revealed conse-
quences of globally modulating glutamatergic, GABAergic
or purinergic neurotransmission on microglial morphology
and dynamic behavior in vivo or ex vivo (Davalos et al.,
2005; Fontainhas et al., 2011).
The first evidence for a regulation of surveillant microglia
by neurotransmission in vivo was provided by the pioneer
study from Davalos et al. (2005). In this study, most of the
imaging was performed through a thinned skull, but the appli-
cation of pharmacological agents onto the cortical surface

required a more invasive procedure, i.e. the removal of the
skull and dura mater. Nonetheless, microglial morphology
and motility seemed unaffected by the procedure at the corti-
cal depths that were imaged. In this manner, cortical surface
application of apyrase which catalyzes the hydrolysis of extra-
cellular ATP and ADP reduced the basal velocity (length
changes over time) of microglial processes. In contrast, appli-
cation of carbenoxolone (an inhibitor of connexin and
pannexin-1) or flufenamic acid (an inhibitor of connexin)
nearly abolished their velocity within 30 min (Davalos et al.,
2005), suggesting that ATP released from astrocytes via con-
nexin channels, such as forming gap junctions, or hemichan-
nels could mediate the effects of extracellular purines on
microglial process velocity (see Fields and Burnstock, 2006;
Inoue et al., 2007; Kang et al., 2008; Halassa and Haydon,
2010 for details; also see discussion below).
Building on these observations, Fontainhas et al. (2011)
recently discovered a modulation of microglial dynamics by
glutamatergic, GABAergic and purinergic neurotransmission
ex vivo in adult CX3CR1-GFP mice acute retinal explants, a
preparation that minimizes CNS injury because the retina is
imaged as a whole. In this preparation, application of gluta-
mate receptor agonists (AMPA and kainate) into the record-
ing chamber increased the size (area and length), complexity
and basal velocity of microglial processes, whereas GABA or
GABAA receptor antagonist bicuculline (BCC) had opposite
effects. ATP also increased the size, complexity and velocity
of microglial processes within a few minutes of application,
while opposite changes were observed with apyrase, suramin
(a purinergic P2 receptor antagonist) or probenecid (a
pannexin-1 inhibitor; Silverman et al., 2008), suggesting
ATP release from pannexin-1 channels, which are expressed
by excitatory neurons, inhibitory neurons and astrocytes
under non-pathological conditions (see Inoue et al., 2007;
MacVicar and Thompson, 2010; also see Wong et al., 2011).
Additionally, voltage-clamp recordings failed to reveal any
inward or outward currents in microglia during local appli-
cation of AMPA or GABA, although application of ATP did
induce large inward currents (Fontainhas et al., 2011).
During brain injury, purinergic signaling was shown to
regulate microglial process dynamics in vivo, with ATP
released from the damage tissue and/or the surrounding
cells inducing process extension in surveillant microglia via
P2Y12 microglial receptors, and adenosine (produced from
the extracellular degradation of ATP into ADP, AMP and
adenosine notably by microglial ectonucleotidases; see
Kettenmann et al., 2011) inducing process retraction in acti-
vated microglia via A2A receptors (Davalos et al., 2005;
Haynes et al., 2006; Orr et al., 2009). Under normal physio-
logical conditions, since pre-synaptic release of glutamate
and GABA is regulated by neuronal activity, and ATP is
released from connexin and pannexin-1 channels in an
activity-dependent manner (see Fields and Burnstock, 2006;
Inoue et al., 2007; MacVicar and Thompson, 2010), ATP
could additionally represent an indirect mechanistic link
between glutamatergic and GABAergic neurotransmission
and microglial process dynamics. The particular subtypes of
microglial purinergic receptors recruited by ATP in these con-
ditions are yet to be determined, but P2Y12 receptor is prob-
ably not involved since surveillant microglia displayed a
normal basal process velocity when imaged through the
skull in the cerebral cortex of P2Y12 receptor-deficient adult
CX3CR1-GFP mice in vivo (Haynes et al., 2006).
role of microglia at synapses in the healthy cns 5
neuronal activity
Two additional in vivo studies correlated the changes in spon-
taneous or evoked neuronal activity, induced by pharmacologi-
cal manipulations of neuronal excitability or neurotransmission,
eye enucleation or reduction of body temperature and moni-
tored by electrophysiological recording or calcium imaging,
with the changes in microglial sampling, process velocity and
dynamic interactions with synaptic elements in the intact CNS
(Nimmerjahn et al., 2005; Wake et al., 2009). Contrary to the
previously described modulations of neurotransmission in vivo
or ex vivo, these manipulations influenced excitatory and
inhibitory neurons simultaneously within extensive networks.
In particular, Nimmerjahn et al. (2005) examined the
changes in microglial dynamics during pharmacological
manipulations of spontaneous neuronal activity in vivo.
With a methodology analogous to Davalos et al. (2005),
enhancing excitatory neuronal activity by surface application
of BCC increased microglial sampling of the parenchyma
over 1 h, as evidenced by differences in fluorescence intensity
between the outer (distal microglial processes) and inner rings
(proximal processes) surrounding individual microglial cell
bodies. However, silencing excitatory and inhibitory neuronal
activity with TTX produced no significant outcome over 1 h,
suggesting that microglial sampling proceeds at a basal rate
that is unaffected by neuronal activity (Nimmerjahn et al.,
2005). In both cases, the changes in neuronal activity were
confirmed by electrocorticogram recording using minimally
invasive electrodes. Moreover, both BCC and TTX were inef-
fective at modifying the basal velocity of microglial processes
(Nimmerjahn et al., 2005), suggesting that neuronal activity
increases the rate of branching or turnover of individual
microglial processes without influencing their basal velocity.
The discrepancy in BCC-induced changes in microglial
process velocity between Nimmerjahn et al. (2005) and
Fontainhas et al. (2011) is unclear and could result from
differences between CNS regions or between in vivo and ex
vivo environments, as microglial process basal velocity
was approximately two-fold higher in retina ex vivo than in
cerebral cortex or spinal cord in vivo (Liang et al., 2009
versus Nimmerjahn et al., 2005; Wake et al., 2009; Dibaj
et al., 2010).
Wake et al. (2009) further examined the influence of
sensory-evoked and spontaneous neuronal activity on micro-
glial interactions with terminals in vivo. In this study, binocu-
lar enucleation, injection of TTX into both eyes and reduction
of body temperature from 37 to 328C each reduced neuronal
activity over 2 h, which was confirmed by calcium imaging in
separate experiments, and subsequently induced a retraction
of microglial processes over 4–6 h (eye enucleation), while
also halving the frequency of microglial contacts with individ-
ual terminals (TTX and body temperature). These obser-
vations provided the first evidence for a modulation of
microglial interactions with synaptic structures by neuronal
activity. Furthermore, microglial process basal velocity
remained unchanged during these manipulations (Wake
et al., 2009), in line with the observations from Nimmerjahn
et al. (2005) during cortical surface application of TTX. The
discrepancy between the other effects of TTX reported by
these two studies, i.e. a retraction of microglial processes
(Wake et al., 2009) versus an unchanged microglial sampling
(Nimmerjahn et al., 2005), could result from differences in
experimental paradigms, such as the application of TTX
onto the brain surface versus into the eyes, differences in the
6 marie-e‘ ve tremblay
anesthetics used for in vivo imaging (isoflurane for
Nimmerjahn et al. (2005) versus ketamine and xylazine for
Wake et al. (2009)), and in the durations of the experiments
and imaging sessions during which the resting state of micro-
glia could vary (2 h for Nimmerjahn et al. (2005) versus
8 h for Wake et al. (2009)).
sensory experience
In addition to these manipulations of neurotransmission and
neuronal activity, the effects of environmental experience on
microglial interactions with synaptic elements were recently
examined in vivo, during binocular light deprivation
induced by housing the animals in complete darkness
throughout their fourth postnatal week (Tremblay et al.,
2010b). This time point coincides with the beginning to the
peak of the critical period for experience-dependent plasticity
in the visual system, where neuronal circuit remodeling in
relation with visual experience is most pronounced (see
Hooks and Chen, 2007). Binocular deprivation is known to
increase spine motility and turnover in the visual cortex of
juvenile and adult mice (Majewska and Sur, 2003; Keck
et al., 2008), making this paradigm ideal for correlating micro-
glial behavior with the experience-dependent modification of
synaptic structures under non-pathological conditions.
In this manner, comparing differences in microglial mor-
phology over time revealed a reduced microglial sampling of
the parenchyma that was accompanied by a sparsening and
thickening of microglial processes during visual deprivation,
suggesting a relationship between microglial morphology and
surveillance behavior. Even though microglial contacts with
spines were not significantly altered in duration and frequency
during visual deprivation, microglial processes were found to
change their preference of contact among the small and struc-
turally dynamic spines, from transiently growing spines during
normal experience, to persistently shrinking spines during
deprivation. All these changes reversed during reexposure to
a normal 12-h light/dark cycle for 2 days (Tremblay et al.,
2010b). These observations indicate that microglial inter-
actions with dendritic spines are regulated by sensory experi-
ence in the healthy CNS. Since microglial processes
preferentially interact with a subset of small, structurally
dynamic and frequently eliminated spines during normal
sensory experience (Tremblay et al., 2010b), and small and
shrinking spines, such as those contacted by microglia
during visual deprivation, have been shown to gradually disap-
pear over a span of days in the somatosensory cortex of adult
mice in vivo (Holtmaat et al., 2006), these findings suggest that
microglial contacts could facilitate the elimination of small and
structurally dynamic spines. Furthermore, since Knott et al.
(2006) revealed that small and recently formed spines generally
lack synaptic contacts in the somatosensory cortex of adult
mice in situ, using two-photon in vivo imaging over 1 month
combined with retrospective SSEM with 3D reconstruction,
it also remains uncertain whether the small spines contacted
by microglia receive synaptic contacts.
Altogether, by examining different CNS regions and stages
of the lifespan, using different experimental paradigms for
modulating neurotransmission, neuronal activity or sensory
experience, and different methodologies for assessing the
changes in microglial dynamics in all these conditions, these
studies have revealed that microglia do not behave randomly
in the absence of pathological insults. In light of the combined
observations, resting microglia can now be viewed as
continuously and differentially modulating their morphology,
basal process velocity, sampling of the parenchyma and
dynamic interactions with synaptic structures under normal
physiological conditions.
Microglial elimination of synaptic structures
The specificity of microglial interactions with structurally
dynamic and frequently eliminated dendritic spines raises
the intriguing likelihood that surveillant microglia may facili-
tate the elimination of dendritic spines, with or without synap-
tic contacts, thereby preventing the formation of new synapses
or promoting the elimination of existing ones. In this way,
they could contribute to the experience-dependent remodel-
ing of neuronal networks that is required for learning and
memory storage throughout the lifespan. Mechanisms by
which surveillant microglia may eliminate synaptic structures
were recently proposed and will be described here.
microglial phagocytosis of axon
terminals and dendritic spines
Like macrophages, activated microglia contribute to preser-
ving normal homeostasis by their phagocytosis and clearance
of cellular debris (see Neumann et al. (2009) and Napoli and
Neumann (2009) for details on the mechanisms of microglial
phagocytosis in different contexts). Under normal physiologi-
cal conditions, Nimmerjahn et al. (2005) observed a few
examples of microglial processes engulfing unidentified
tissue components which were subsequently transported to
the cell bodies in vivo. More recently, Tremblay et al.
(2010b) also noticed cellular inclusions with ultrastructural
features of axon terminals and dendritic spines, i.e. synaptic
vesicles and post-synaptic densities, respectively, within
microglial processes and cell bodies in juvenile mouse somato-
sensory cortex in situ. These putative terminals and spines
occasionally displayed synaptic contacts, and invariably pre-
sented ultrastructural features of healthy cellular elements
such as an electron-lucent cytoplasm (see Schmechel (1999)
for additional features of apoptotic and necrotic cells), unlike
apoptotic cells that are phagocytosed by resting microglia
during adult neurogenesis (Sierra et al., 2010). In vivo, micro-
glial processes with phagocytic specializations were also
observed, sometimes encircling neuronal elements, but since
these structures were stable throughout the 30–120 min
imaging sessions, episodes of microglial engulfment followed
by a full disappearance of the synaptic elements were not com-
pletely visualized. Both in situ and in vivo, microglial phagocy-
tic structures became more prevalent during light deprivation,
and still persisted after reexposure to light for 2 days (Tremblay
et al., 2010b), in a context of sensory experience where dendri-
tic spine remodeling and elimination is also increased
(Majewska and Sur, 2003; Keck et al., 2008).
Additionally, Tremblay et al. (2012) recently observed pha-
gocytic inclusions within microglial cell bodies and processes
throughout adulthood and normal aging, using immunocyto-
chemical EM. This study revealed that microglial cell bodies
and processes accumulate large amounts of phagocytic
inclusions, which often resemble terminals and spines, with or
without an electron-lucent cytoplasm, but also lysosomal lipo-
pigments, large vesicles, vacuoles and lipid droplets, in the
visual and auditory cortices of two stains of mice normally
undergoing complementary age-related loss of vision (CBA/

CaJ mice) or hearing (C57Bl/6J mice) during their second year
of life. Surprisingly, this accumulation was exacerbated by the
age-related loss of visual or auditory function, with nearly all
microglia containing phagocytic inclusions and 20% of micro-
glial cells becoming almost completely filled by inclusions, akin
to fat granule cells or gitter cells, in this context of sensory depri-
vation. Also, microglial processes extending between axon term-
inals and dendritic branches were encountered, suggesting an
early stage of phagocytosis or an evidence of synaptic stripping
under non-pathological conditions. Together, these obser-
vations during adolescence, adulthood and normal aging
suggest that microglial phagocytosis is regulated in an
experience-dependent manner in the mature CNS.
In line with these observations, activated microglia have
been shown to be involved in the apoptotic and phagocytic
elimination of excess neurons during early postnatal develop-
ment (see Bessis et al. (2007) and Pont-Lezica et al. (2011) for
details and additional roles during development), and
recently, Paolicelli et al. (2011) showed that microglia can
also eliminate synaptic elements during the second and
third postnatal weeks in CX3CR1-GFP mouse hippocampus
in situ. In that study, puncta immunopositive for either
PSD95 (marker of dendritic spines) or SNAP25 (axon term-
inals) were found to be colocalized at nanometer resolution
within GFP-positive microglial processes using stimulated
emission depletion microscopy (see Willig et al. (2006) for
technical details). Microglial colocalization with PSD95 was
more prevalent in homozygous CX3CR1-GFP mice, where
fractalkine signaling is completely eliminated (Jung et al.
(2000); see Section 1 for details about these mice), and was
accompanied by a reduced density of microglial cells and an
increased density of PSD95 puncta with or without microglial
colocalization, suggesting that reduced microglial numbers
could impact the pruning of supernumerary spines
(Paolicelli et al., 2011). Providing additional molecular
insights, recent observations from Schafer et al. (2011;
Society for Neuroscience abstract No. 663.03) also revealed
that microglia can phagocytose terminals in a complement
C3-dependent manner (Stevens et al., 2007) during early post-
natal development in mouse thalamus in situ.
Altogether, these studies propose a novel physiological role
for microglial phagocytosis, in the elimination of individual
synaptic structures and quite likely of entire excitatory
synapses, throughout the lifespan.
regulation of the perisynaptic
extracellular space
Another proposed mechanism by which surveillant microglia
could eliminate dendritic spines was suggested by the same
study of Tremblay et al. (2010b). Immunocytochemical EM
and SSEM with 3D reconstruction revealed that microglial
cell bodies, proximal and distal processes are distinctively sur-
rounded by pockets of extracellular space, which consists of
interstitial fluid supplemented with various extracellular
matrix proteins (see Syková and Nicholson, 2008), in stark
contrast to all other cellular and subcellular elements in the
visual cortex of juvenile mice in situ. This tight association
indicates that microglial processes could dynamically influ-
ence the volume and geometry of the extracellular space,
and thus the concentration of neurotransmitters and signaling
molecules in the vicinity of synapses. These pockets of extra-
cellular space were regulated in an experience-dependent
role of microglia at synapses in the healthy cns 7
manner, undergoing expansion during light deprivation and
shrinkage during light reexposure. More recently, pockets of
extracellular space juxtaposing microglial cell bodies and pro-
cesses were also observed in mouse auditory and visual cor-
tices throughout adulthood and normal aging (Tremblay
et al., 2012). Interestingly, more than 40 years ago, Herndon
(1964) mentioned that a ‘small amount of extracellular
space may be seen in a few places about the cell, particularly
about its thick processes’, when describing for the first time
microglial cell bodies and contiguous, proximal processes at
the ultrastructural level under normal physiological con-
ditions in adult rat cerebellum. Whether microglia create
these extracellular spaces remains unknown.
Among candidate molecules for microglial involvement,
the polysialylated form of neural cell adhesion molecule
(PSA-NCAM) increases the size of the extracellular space
(Yang et al., 1992; see Bonfanti, 2006; Syková and
Nicholson, 2008) and was recently found to be expressed by
microglia in vitro (Wang and Neumann, 2010). Proteases
degrading extracellular matrix proteins which are expressed
by activated microglia in vitro might also be involved (see
Nakanishi, 2003; Choi et al., 2010). Application of proteases
in vitro and in vivo has been shown to promote spine motility
and pruning, as well as activity-dependent and experience-
dependent plasticity in the visual cortex of adult mice
(Pizzorusso et al., 2002; Mataga et al., 2004; Oray et al.,
2004), thereby suggesting a possible link between the regu-
lation of the extracellular space and the elimination of the
small and structurally dynamic spines preferentially contacted
by microglial processes in vivo (Tremblay et al., 2010b).
SUMMARY AND CONCLUSION
Over the last century, the development of selective staining
approaches and concomitant refinement of imaging tech-
niques, from conventional light and electron microscopy to
two-photon laser scanning microscopy, have provided unpre-
cedented insights into microglial morphology, dynamic be-
havior and interactions with excitatory synapses in the
intact CNS. In particular, recent imaging studies have revealed
that microglia are extremely dynamic in their presumed
‘resting’ state, continuously surveying their local environment
with highly motile processes which transiently contact subsets
of synaptic structures in vivo, especially the small, dynamic
and frequently eliminated dendritic spines. Surveillant micro-
glia also perceive ongoing changes in neurotransmission,
neuronal activity and sensory experience, and respond by
modulating their behavior including their interactions with
dendritic spines, their phagocytic elimination of axon term-
inals and dendritic spines and their remodeling of the perisy-
naptic extracellular space. As resident immune cells, microglia
are not directly bound by the constraints imposed by the con-
tinuous flow of information processing in the brain, and may
uniquely contribute to remodeling neuronal circuits in
relation with behavioral and sensory experience in the
mature CNS. Together, these observations have opened
many functional possibilities beyond immune surveillance
for these once presumed ‘resting’ cells.
At this very early stage of investigation in this field, our
knowledge about surveillant microglia almost exclusively
comes from observing the superficial layers of cerebral cortex
and cannot be confidently extrapolated to the entire CNS.
8 marie-e‘ ve tremblay
Since the recent studies have barely scratched the surface (of
the brain in this case), the modalities of microglial interactions
with excitatory and inhibitory synapses throughout the CNS,
much as their functional significance and particular cellular
and molecular mechanisms still remain undetermined. For
example, in which contexts do quiescent microglia directly
phagocytose axon terminals and dendritic spines, use other
mechanisms such as proteolytic remodeling of the extracellular
space, or refrain from intervening? How do surveillant micro-
glia recognize and respond to the various molecular signals in
their environment, including dynamic changes in neurotrans-
mission and neuronal activity at individual synapses? How do
these immune cells cooperate with other glial cells, as well as
peripheral myeloid cells, in maintaining or shaping neuronal
architecture and activity? And, as in the case of microglial
memory of past immune challenges (see Bilbo et al., 2012),
do surveillant microglia somehow remember their previous
behavioral states, the flux of information processing in the
brain, or the structural changes of synaptic elements in
recent and not so recent windows of intervention?
Even though most essential questions remain unanswered
at present, the novel insights about microglia have established
integrative in vivo and in situ imaging approaches as a valu-
able methodological way, alongside genetics, electrophysi-
ology and animal behavior, for studying these demanding
immune cells in the healthy CNS. Promising and increasingly
integrative approaches for modulating or monitoring cellular
activity in a non-invasive way are now emerging, with the
ongoing development of optogenetics and imaging of func-
tional markers, sophistication of transgenic mice in the post-
genomic era and improvement in the spatiotemporal
resolution of microscopy techniques. Therefore, in addition
to these innovative recent observations, we can optimistically
envision an explosion of discoveries regarding the roles of sur-
veillant microglia across the various CNS regions including
the cerebellum and spinal cord, stages of the lifespan and
states of normal brain physiology, in awake and normally
behaving animals. These discoveries and their underlying
technical approaches will facilitate a broader understanding
of microglial involvement in various neuropsychiatric and
neurodegenerative diseases in the near future.
ACKNOWLEDGEMENTS
I am extremely grateful to Andreas Buchmann, Harris A.
Gelbard, Ania K. Majewska, Richard M. Ransohoff and
Cynthia D. Rittenhouse for their comments and suggestions.
This work was supported by Fonds de la Recherche en
Santé du Québec (FRSQ) and Canadian Institutes of Health
Research (CIHR) postdoctoral fellowships.
Statement of interest
None.
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AUTHOR’S ADDRESS
Department of Psychiatry, University of Wisconsin-Madison,
Madison, WI 53719, USA
Correspondence should be addressed to:
Marie-Ève Tremblay
Department of Psychiatry
University of Wisconsin-Madison
6001 Research Park Boulevard
Madison, WI 53719
USA
phone: 585-474-8300
email: tremblay2@wisc.edu