JBC Papers in Press. Published on April 15, 2014 as Manuscript M114.557132 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.557132 TRPV4+ astrocytes modulate synaptic transmission
A novel subtype of astrocytes expressing TRPV4 regulates neuronal excitability via release of gliotransmitters Koji Shibasaki1#, Kazuhiro Ikenaka2,5, Fuminobu Tamalu6, Makoto Tominaga3,4,5, Yasuki Ishizaki1
1
Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of
Medicine, Maebashi 371-8511, Japan; 2Division of Neurobiology Neuroinformatics, 3Division of Cell Signaling, National Institute for Physiological Sciences, 4Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki 444-8787, JAPAN; 5
Department of Physiological Sciences, The Graduate University for Advanced Studies, 6
Department of Physiology, Saitama Medical University,
Moroyama 350-0495, JAPAN. Running title:
TRPV4+ astrocytes modulate synaptic transmission
To whom correspondence should be addressed: Koji Shibasaki, Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan. Tel: +81-27-220-7951; Fax: +81-27-220-7951; E-mail:
[email protected] Keywords: TRPV4, astrocyte, ATP, glutamate, gliotransmitter Ca2+
Background: The functional differences
Neuronal
among
transients in astrocytes, and these Ca2+
astrocyte
subtypes
are
uncharacterized. Results:
Only
a
subset
of
astrocytes
expresses TRPV4 and regulates neurons.
excitation
transients
can
excitability.
While
can
evoke
modulate only
a
neuronal subset
of
astrocytes appears to communicate with
+
Conclusion: TRPV4 astrocytes release ATP
neurons, the types of astrocytes that can
and glutamate to regulate neurons.
regulate neuronal excitability are poorly
Significance: Astrocytes can be classified by
characterized. We found that ~30% of
TRPV4 expression and by function.
astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be
Abstract Astrocytes
play
regulation
of
active synaptic
the
classified on the basis of their expression
transmission.
patterns. When TRPV4+ astrocytes are
roles
in
1
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Okazaki 444-8585, JAPAN;
TRPV4+ astrocytes modulate synaptic transmission
activated by ligands such as arachidonic
processing by releasing gliotransmitters such
acid,
to
as ATP and glutamate (8). Increases in
gap
intracellular Ca2+ levels induce glutamate
the
activation
neighboring
propagates
astrocytes
through
release
junctions and by ATP release from the +
-
astrocytes
(9).
Glutamate
increases synaptic activity and modulates
TRPV4 astrocytes. Following activation, +
from
astrocytes
behaviors such as epilepsy and anxiety (2,10).
release glutamate, which acts as an
An increase in intracellular Ca2+ also induces
excitatory
astrocytes to release ATP, and this suppresses
both TRPV4
and TRPV4
gliotransmitter
to
increase
synaptic
synaptic transmission through type 1
activity
in
the
form
of
heterosynaptic depression (11). Dysfunction
astrocytes constitute a novel subtype of the
of astrocytes affects spatial working memory
population and are solely responsible for
and pain perception (2). Although it is
initiating excitatory gliotransmitter release
becoming clear that astrocytes contribute to
to enhance synaptic transmission. We
neuronal function, the detailed functional
mGluR. Our results indicate that TRPV4
+
propose that TRPV4 astrocytes form a
characteristics of astrocytes remain poorly
core of excitatory glial assembly in the
understood. In particular, the identity of the
brain and function to efficiently increase
astrocytes that regulate neuronal activity is
neuronal
unknown. Furthermore, there is controversy
excitation
in
response
to
as to whether astrocytes are functionally
endogenous TRPV4 ligands.
homogeneous or heterogeneous, although astrocytes can be clearly classified as either Introduction
protoplasmic or fibrous on the basis of their
Astrocytes provide metabolic support and
morphology (12,13).
eliminate waste products from extracellular
TRPV4 is a non-selective cation
spaces (1). The astrocyte-mediated clearance
channel that was first described as an
+
of extracellular glutamate and K
osmosensor
and
for
detection
of hypotonic
regulation of extracellular volume are known
stimuli (14-18). TRPV4 can also be activated
to be perfectly coupled with synaptic
by heat (> 27°C-34°C), the phorbol ester
activities (2,3). These cells also regulate
derivative 4-phorbol 12, 13 didecanoate
blood flow within the brain in response to
(4PDD), or metabolites of arachidonic acid
neuronal activity (4,5). In addition to their
(19-23). We have previously reported that
basic physiological roles, astrocytes are
physiological temperatures activate TRPV4
essential for bidirectional communication
in some subtypes of hippocampal neurons
with neurons. Astrocytes can detect, respond
and that this activation enhances neuronal
to, and modulate neuronal activity (6,7).
activity (19). Moreover, we and others have
Specifically, they actively regulate neural
reported 2
that,
in
addition
to
neurons,
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+
TRPV4+ astrocytes modulate synaptic transmission
astrocytes also express TRPV4 (19,24-26).
reports (19). The following antibodies were
Calcium signaling is crucial for astrocyte
used: mouse monoclonal anti-GFAP antibody
2+
function. An increase in the intracellular Ca
(1:500, Sigma, CA, USA) and mouse
concentration
inositol
monoclonal anti-S100 antibody (1:500,
mediated
1,4,5-triphosphate
by
the
receptor
in
Sigma).
the
To
determine
the
ratio
of
endoplasmic reticulum drives the release of
TRPV4-positive astrocytes, we used 12 slides
gliotransmitters such as ATP and glutamate.
of stained hippocampal tissue (each slide
Astrocytic TRPV4 is thought to be the
contained 6 brain slices). We used a
upstream molecule that regulates intracellular
microscope
2+
Ca
count
the +
+
+
number
TRPV4 /astrocyte-marker
Ca levels, because it is a highly permeable 2+
to
-
of and
TRPV4 /astrocyte-marker astrocytes in the
TRPV4 expression is restricted to a specific
CA1 region. To count the cells, we combined
subtype of astrocytes.
NBT/BCIP-mediated detection of mRNA
In this study, we examined the
with DAB-mediated detection of protein.
expression pattern of TRPV4 in astrocytes and characterized the physiological function
Culture of dissociated astrocytes and neurons
+
of TRPV4 astrocytes.
Cortical astrocytes and hippocampal neurons were prepared from postnatal day 0 (P0) wild type (WT) or TRPV4KO mice as previously
Experimental procedures
described (19). Hippocampal neurons from
Animals
TRPV4KO mice were co-cultured with
C57BL/6J was utilized as the background
astrocytes (WT or TRPV4KO) to examine
strain for TRPV4-deficient (TRPV4KO) mice.
the changes in neuronal activity induced by
All animal care procedures and experimental
TRPV4-positive astrocytes.
protocols were performed in accordance with the guidelines of the National Institute of
Fluorescent
Health,
electrophysiology
the
National
Institute
Physiological
Sciences,
and
for
Fura-2
Gunma
measurements
fluorescence
was
and
measured
by
Fura-2-AM (Molecular Probes, Carlsbad, CA,
University.
USA) in a standard bath solution containing Immunohistochemical
analysis,
in
140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2
situ
mM CaCl2, 10 mM HEPES, and 10 mM
hybridization, and cell counts Immunohistochemistry
and
in
situ
glucose, pH7.4. The 340:380 nm ratio was
hybridization were performed as previously
recorded. Because spontaneous Ca2+ spikes
described (19,27). We used a TRPV4 mRNA
were
always
below 2+
probe that has been described in previous
TRPV4-activated Ca 3
0.1,
we
defined
spikes as those at or
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channel (20). The possibility exists that
TRPV4+ astrocytes modulate synaptic transmission
above a value of 0.1. The standard bath
USA),
an
image
intensifier
(C8600,
solution for the patch-clamp experiments was
Hamamatsu Photonics, Hamamatsu, Japan),
the same as that used for fluorescence
and a 34x (NA 0.28) dry objective. Images of
measurements. The reversal potential was
ATP release (512 X 512 pixels) were
measured by using voltage ramps (-100 to
acquired
every
500
+100 mV in 5-s intervals). Pipette solutions
averaged
over
10
for whole-cell recordings contained 120mM
Meta-Morph (version 6.5; MDS Inc., Toronto,
K-gluconate, 20mM KCl, 0.5mM EGTA,
Canada). All experiments were performed at
2mM Mg-ATP, 2mM K2-GTP, and 10mM
30°C to 34°C in a perfectly dark room.
milliseconds frames
by
and using
HEPES, pH7.4. Whole-cell recordings were sampled at 10 kHz and filtered at 5 kHz for
Analysis of glutamate release in cultured
analysis
astrocytes
(Axon
200B
amplifier
with
Standard bath solutions with or without
City, CA, USA). Data were statistically
4PDD were applied to cultured astrocytes
analyzed by using the unpaired t test,
for 10 minutes, and then the conditioned
ANOVA or Duncan’s multiple range test.
media were collected. After filtration, the amino acid composition of the media was
Visualization of ATP dynamics in cultured
determined with an automatic amino acid
astrocytes
analyzer (Hitachi, Ibaraki, Japan).
Cultured
astrocytes
poly-L-lysine-coated
were
seeded
on
glass
slides
at
confluence. The glass slides were placed in 3
Results
x 10-mm chambers made from silicon. The
We and others have previously reported that
culture media in the chambers were replaced
TRPV4 is an important regulator of neuronal
with
a
excitability in hippocampal neurons and that
luciferin-luciferase mixture (Checklite HS set,
TRPV4 is expressed in both astrocytes and
Kikkoman, Noda, Japan). Since changing the
neurons
medium causes excessive stress to astrocytes,
sought to determine which types of astrocytes
our experiments were performed 20 minutes
express
after the medium was exchanged to allow
hybridization and immunohistochemistry in
ATP release from the astrocytes to stabilize.
adult mouse hippocampus. TRPV4 expression
Bioluminescence
a
Ringer
solution
containing
(19,24,28,29). TRPV4
by
Therefore,
combining
in
we situ
by
the
(arrowheads in Fig. 1A-B) was restricted to a
reaction
was
subset of astrocytes distinguished by GFAP
detected by an upright microscope (BX51WI,
expression (Fig. 1A, arrowheads) or S100
Olympus, Tokyo, Japan) equipped with a
expression (Fig. 1B). Interestingly, only 30%
cooled CCD camera (Evolve 512, Roper,
of GFAP-positive astrocytes (323 of 1155
emitted
ATP/luciferin-luciferase
4
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pCLAMP software, Axon Instruments, Foster
TRPV4+ astrocytes modulate synaptic transmission
cells, n=12 slides) expressed TRPV4. We also
oscillations with a spike-like appearance
quantified
the
were observed in the majority of the
astrocytes
to
ratio
of
TRPV4-positive astrocytes.
astrocytes (Fig. 2A-B, excluding the pink
Consistent with the findings of TRPV4
trace; supplementary movie 1). This finding
expression in astrocytes expressing GFAP,
indicates
only 20% of S100-positive astrocytes (209
astrocytes was initiated after TRPV4 was
of 952 cells, n=12 slides) expressed TRPV4
activated. Under normal conditions in our
(Fig. 1B, arrowheads). To confirm that
assay
TRPV4 mRNA is expressed in a subset of
exhibited very rare (1-2 times/10 min) and
astrocytes, we examined its expression in
very small (0.1Δ
TRPV4-dependent Ca2+ oscillations were
expressed TRPV4 (Fig. 1C, arrowheads). We
never observed in TRPV4KO astrocytes after
propose that astrocytes can be classified into
exposure to 4PDD (Fig. 2C-D). The
functionally heterogeneous groups on the
presence of 3mM octanol, a gap junction
basis of the expression of markers such as
blocker, significantly reduced the later Ca2+
TRPV4.
oscillations (Fig. 2B, excluding the pink trace, Next, we examined the response of
and Fig. 2D), although the initial, presumably
astrocytes to chemical ligands of TRPV4,
TRPV4-mediated responses (pink traces in
since the use of heat is not suitable for
Fig. 2B and 2C) were not affected by
analysis of TRPV4 function in astrocytes (29)
exposure to octanol.
unlike
hippocampal
neurons
(19).
To
prove
that
the
astrocytes
Application of a TRPV4-specific ligand,
producing the acute responses to the TRPV4
4PDD,
in
agonist were TRPV4-positive, we combined
+
Ca2+ imaging and whole-cell patch-clamp
astrocytes (less than 20% of the total number
experiments (Fig. 3). First, we treated cells
of astrocytes) within 1 minute after 4PDD
with another TRPV4-specific chemical ligand,
was
caused
an
2+
intracellular
acute 2+
Ca
([Ca ]i)
increase in
TRPV4
2A-B,
pink
trace;
GSK1016790A (GSK, 1 M) then performed
1).
These
acute
Ca2+ imaging. This approach allowed us to
resulted
from
identify both the astrocytes producing acute
activation of TRPV4 because they were not
responses to GSK and those producing
detected in astrocytes from TRPV4KO mice
non-acute responses. The results of these
(Fig. 2C). Following the initial increase in
experiments (Fig. 3A and supplementary
applied
supplementary responses
2+
(Fig. movie
most
+
likely
2+
[Ca ]i in TRPV4 astrocytes, substantial Ca
movie 2) were equivalent to those obtained 5
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only a subset of cultured S100 astrocytes
+
TRPV4+ astrocytes modulate synaptic transmission
trace;
released ATP is very difficult to detect
supplementary movie 1). These data indicate
(exoATPases immediately degrade released
with
4PDD
(Fig.
2A-B,
pink
2+
that the increase in [Ca ]i is a specific
ATP). In this system, ionotropic receptors
response to TRPV4 activation. After the
expressed in HEK293 cells detect molecules
2+
imaging,
released from adjacent astrocytes that have
we applied 3 M GSK then performed
been stimulated with ligand (Fig. 4A).
whole-cell patch-clamp recordings from the
During
identified cells. To avoid desensitization of
TRPV4 in WT astrocytes at a holding
TRPV4 by repeated exposure to agonist, we
potential of -60 mV, P2X2-like currents were
increased the dose of GSK in the patch-clamp
recorded only from whole-cell patch-clamped
recordings. Consistent with our expectation,
HEK293 cells that were in close proximity to
all of the astrocytes that produced an acute
the astrocytes (located within 10 m; Fig.
response to GSK evoked TRPV4 currents,
4B; n = 6 of 43 trials). Using a whole-cell
but astrocytes that did not respond acutely
patch-clamp recording technique, we applied
did not evoke any such currents (Fig. 3B).
ramped pulses from -80 mV to +80 mV (Fig.
These findings strongly indicate that the
4B) at 5-second intervals. Exposure to
astrocytes that produce an acute response to
4PDD
TRPV4 agonists express TRPV4.
current-voltage relationship (Fig. 4B, red
astrocytes were identified by Ca
4PDD-mediated
an
inwardly
of
rectified
The failure of octanol to inhibit all
arrowhead and the red trace labeled b in the
+
graph on the right), although the basal
astrocytes (Fig. 2B, arrowheads, and 2D)
current-voltage relationship had a linear
indicates that gliotransmitters are released
pattern (Fig. 4B, blue arrowhead and the blue
2+
trace labeled a in the graph on the right). The
oscillations were completely abolished when
expression of functional P2X2 receptors in
octanol and 100 M suramin (an ATP
the HEK293 cells was confirmed by applying
receptor blocker, Fig. 2D) were applied
100 M ATP to HEK293 cells (to avoid the
simultaneously, although TRPV4 remained
possible effects of ATP-induced ATP release
activated (Fig. 2C). These results strongly
from astrocytes, these cells were not in close
2+
Ca
oscillations
caused
by
TRPV4
from the astrocytes. The residual Ca
+
suggest that excitation of TRPV4 astrocytes
proximity to any astrocytes). In addition, we
is transmitted through gap junctions and ATP
determined that no P2X2-like inwardly
release.
whether
rectifying currents were recorded from
activation of TRPV4 causes ATP release from
TRPV4KO astrocytes. Exposure to ATP
astrocytes, we utilized an ATP bio-sensor
evoked an inwardly rectified current-voltage
system (30). We have previously utilized this
relationship (Fig. 4B, green arrowhead and
bio-sensor technique to monitor ATP release
the green trace labeled c in the graph on the
from skin keratinocytes (30) since locally
right). Taken together, these results indicate
To
directly
determine
6
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evoked
activation
TRPV4+ astrocytes modulate synaptic transmission
that ATP is released from TRPV4+ astrocytes
modulate neuronal functions (32). Since
when the channel is activated.
neurons also express TRPV4 (19), we could bio-sensor
not use patch-clamp recordings of slices from
system does not indicate which astrocytes
WT or TRPV4KO mice to examine this
release ATP. To obtain this information, we
possibility. To eliminate the contributions
used the luciferin-luciferase system to image
from
ATP release in real time (31). In this imaging,
prepared a unique co-culture system. First,
we
we
Unfortunately,
put
cultured
the
astrocytes
(on
glass
TRPV4 cultured
expressing astrocytes
from
or
made from silicon. Then, the culture medium
hippocampal neurons were then added to the
was
solution
astrocytes (Fig. 5A). When 4PDD was
containing a luciferin-luciferase mixture.
applied to the cultures, mEPSCs increased
Bioluminescence
ATP
significantly in neurons cultured with WT
luciferin-luciferase reaction was detected by
astrocytes, but not in those cultured with
an upright microscope equipped with a
TRPV4KO
cooled-CCD camera in dark room. Under
addition, the amplitude of evoked mEPSCs
basal conditions, very rare spontaneous ATP
was 2 times larger in cultures with WT
release was observed (Fig. 4C). In contrast,
astrocytes than in those with TRPV4KO
when TRPV4 was activated in astrocytes
astrocytes (Fig. 5B-C; WT: basal, 12.1 0.7
exposed to 4PDD, this activation evoked
pA vs 4PDD, 25.1 1.8 pA; TRPV4KO:
strong ATP release in many astrocytes (Fig.
basal, 13.2 0.8 pA vs 4PDD, 13.7 1.2
4C and supplementary movie 3). The amount
pA; n=16 for each genotype). As with the
and duration of ATP release differed between
amplitude, the frequency of mEPSCs evoked
astrocytes (Fig. 4C-D). Consistent with the
by treating the cultures with 4PDD was 7
results of our experiments with the bio-sensor
times larger in cultures with WT astrocytes
system (Fig. 4B), some astrocytes displayed
than in those with TRPV4KO cells (Fig.
both weak/sustained and high/transient ATP
5C-D; WT: basal, 7.5 0.5 Hz vs 4PDD,
release (Fig. 4D, shown by the brown trace).
49.3 1.2 Hz; TRPV4KO: basal, 6.1 0.6
Astrocytes from TRPV4KO mice did not
Hz vs 4PDD, 6.8 0.7 Hz; n=16 for each
release any ATP (Fig. 4D). This finding
genotype).
indicates that ATP release by WT astrocytes
reported to be an endogenous ligand for
upon
a
TRPV4 (23). Therefore, we tested the effects
TRPV4-dependent response and leads us to
of arachidonic acid in our co-culture system.
application
a
Ringer
emitted
of
by
the
4PDD
was
+
astrocytes
Arachidonic
(Fig.
acid
TRPV4KO
5B-D).
has
In
been
hypothesize that TRPV4 astrocytes might
Similar to the results obtained with 4PDD,
regulate
excited
mEPSCs in cultures with WT astrocytes
astrocytes have been shown to directly
increased significantly after exposure to
neuronal
activity,
as
7
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TRPV4KO
with
and
WT
we
coverslips) into a tiny chamber (3X10 mm) replaced
mice,
neurons,
TRPV4+ astrocytes modulate synaptic transmission
arachidonic acid (10 M) , but an increase
activated. ATP is not a likely candidate, as it
was not observed in cultures containing
has been reported that ATP release from
TRPV4KO astrocytes (Fig. 5E). Since we
astrocytes actually inhibits synaptic function
opted to use a co-culture system to evaluate
(33),
the function of TRPV4 in astrocytes, we
lyophilization. Therefore, we searched for
cannot exclude the possibility that our results
another gliotransmitter with the ability to
might be artificial. Therefore, we recorded
enhance
mEPSCs
slices
activation of TRPV4. We focused on amino
prepared from WT and TRPV4KO mice. Puff
acids and utilized an amino acid analyzer to
application of arachidonic acid (10 M) to
examine the contents of conditioned media
neighboring patch-clamped cells significantly
from cultures of WT and TRPV4KO
increased mEPSCs in slices from WT mice,
astrocytes. Surprisingly, glutamate release
but not in those from TRPV4KO mice (Fig.
was significantly enhanced in cultures with
5F).
WT astrocytes after exposure to 4PDD,
in
results
hippocampal
described
ATP
is
synaptic
not
retained
function
after
following
although the release of other amino acids was
above
strongly suggest that activation of TRPV4 in
not
astrocytes causes them to release factors that
glutamate release was not enhanced in
enhance synaptic function. To confirm this
cultures of TRPV4KO cells treated similarly
possibility, we prepared conditioned media
(Fig. 6C-D). These results strongly indicate
from
4PDD.
that activation of TRPV4 in astrocytes causes
Unfortunately, TRPV4KO neurons exposed
them to release glutamate. To identify the
to conditioned medium did not exhibit any
cells that release glutamate, we performed
changes in the frequency or amplitude of
bio-sensor experiments of the GluN1/N2B
evoked mEPSCs. We hypothesized that the
NMDA receptor in cultures containing
gliotransmitters in the conditioned media
TRPV4+ or TRPV4- astrocytes, which we
were too dilute, because they were secreted
identified by the Ca2+-imaging after exposure
outside of the normally restricted synaptic
to 4PDD. Both TRPV4+ and TRPV4-
spaces.
the
astrocytes evoked inward, NMDA-derived
conditioned media by lyophilization then
currents in the bio-sensor cells (Fig. 6F),
reconstituted the media in a standard bath
although the mock bio-sensor cells did not
solution. Both the amplitude and frequency
produce such currents (Fig. 6E). These results
of mEPSCs were significantly increased in
indicate that both TRPV4+ and TRPV4-
cultures exposed to this solution (Fig. 6A-B).
astrocytes
astrocytes
Hence,
exposed
we
to
concentrated
changed
(Fig.
release
6C-D).
glutamate,
Moreover,
although
+
These results support the hypothesis that a
TRPV4 astrocytes are solely responsible for
gliotransmitter acting as a positive regulator
initiating glutamate release.
is released from astrocytes when TRPV4 is
The lack of ATP or glutamate 8
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The
acute
and
TRPV4+ astrocytes modulate synaptic transmission
release from astrocytes from TRPV4KO mice
results indicate that the glutamate released
(Figs. 4 and 6) might be responsible for the
from astrocytes in response to activation of
functional abnormalities of these cells. To
TRPV4 signals through the type 1 mGluR.
confirm this idea, we applied ATP (1 M) to
Taken together, we conclude that astrocytic
WT and TRPV4KO astrocytes and then
TRPV4 is an important regulator of synaptic
2+
performed Ca
transmission (Fig. 7G).
imaging. Both WT and
TRPV4KO cells responded to ATP (Fig. 7A-B), and the amplitudes of the responses Discussion
These results indicate that the mutant cells
The present study revealed that a unique
are fully functional and that the observed lack
subtype of astrocytes regulates neuronal
of ATP and glutamate release from the
activity. We examined TRPV4 expression in
mutant astrocytes is caused by a deficiency of
both brain slices and cultured astrocytes and
TRPV4.
glutamate
is
a
major
found that expression of this ion channel was
capable
of
positively
restricted to 20% to 30% of the astrocyte
regulating synaptic activity, then the effects
population (Fig. 1). Using Ca2+ imaging and
should be inhibited by antagonists of type 1
whole-cell
mGluR, as previously described (34). To
confirmed
examine this issue, we pretreated co-cultures
expression of TRPV4 to 20% of astrocytes
of TRPV4KO neurons and WT astrocytes
(Figs. 1-3). Astrocytes have been reported to
with CPPG, a type 2/3 mGluR antagonist, or
be
MPEP, a type 1 mGluR antagonist, then
although
applied 4PDD. Treatment with CPPG had
expression of biochemical markers are
no
The
different (12). We agree with these findings,
amplitudes of mEPSCs evoked in cultures
because the in vitro electrophysiological
exposed to both CPPG and 4PDD were not
properties
of
significantly different from those of mEPSCs
astrocytes
are
evoked in cultures exposed to 4PDD alone
preparation). Hence, we hypothesize that all
(CPPG+4PDD: 24.5 1.5 pA; 4PDD
astrocytes have the same electrophysiological
alone: 25.2 1.9 pA; n=6). In contrast, the
properties regardless of their morphological
potentiation of synaptic activity (both the
differences; however, subsets of astrocytes
frequencies and amplitudes of EPSCs) was
might synthesize different collections of
significantly inhibited in cultures exposed to
gliotransmitters. Thus, we propose that the
both MPEP and 4PDD, and washout of
expression of markers such as TRPV4 can be
MPEP
increased
used to classify astrocytes into functionally
4PDD-evoked EPSCs (Fig. 7E-F). These
heterogeneous groups. This idea is at odds
If
gliotransmitter
inhibitory
effects
(Fig.
significantly
7D).
9
patch-clamp the
recordings,
restricted
electrophysiologically their
functional
homogeneous,
morphologies
protoplasmic similar
we
and
and
the
fibrous
(manuscript
in
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were similar in both types of cells (Fig. 7C).
TRPV4+ astrocytes modulate synaptic transmission
with data from previous studies. Benfenati et
stronger than those of ATP (Fig. 6). If this is
al. (2007) reported that most astrocytes
the case, then ATP-mediated regulation
express TRPV4; however, these results were
might only be required for astrocyte-astrocyte
obtained with a commercial antibody, and the
communication (Figs. 2-4). We also found
specificity of the antibody was never
that glutamate release enhanced presynaptic
assessed in samples from TRPV4KO mice.
activity by signaling through type 1 mGluRs
Additionally, Benfenati and colleagues did
(Fig. 7D-F). This type of positive regulation
2+
of synaptic activity by astrocyte-derived
TRPV4 ligands affect astrocytes. In contrast
glutamate and the presynaptic expression of
to their experiments, ours used an in situ
type 1 mGluRs are both supported by other
hybridization method that has previously
reports (6,34). In addition to these reports,
been confirmed by our group and others
glutamate released from astrocytes located
(35,36) and which has been demonstrated to
near axons has recently been reported to
produce no background in neural samples
modulate the duration of action potentials
from TRPV4KO mice. Given the functional
and enhance presynaptic neurotransmitter
evidence (Figs. 1-4), it is clear that only a
release (13,38). Furthermore, an important
restricted subset of astrocytes express TRPV4.
finding of our study is that activation of
Moreover, sonic hedgehog has recently been
TRPV4 in a subset of astrocytes elicits an
shown to regulate the development of a
increase
subtype of astrocytes (37), and this finding
gliotransmitter release (Figs. 2-7) and that
demonstrates that astrocyte subtypes are
TRPV4+
specified early in development.
throughout the brain. The unique function
synaptic
astrocytes
events
may
be
through present
and localization of TRPV4+ astrocytes might
In this study, we determined that TRPV4+
in
with
form a localized core that triggers neural
surrounding astrocytes through gap junctions
excitation in response to the release of one
and/or ATP release (Figs. 2-4). Although it
type of neurotransmitter (Fig. 7G). Thus,
has been reported that astrocyte-derived ATP
TRPV4+
is inhibitory and is rarely a positive regulator
neuronal excitability and help increase the
of synaptic function (31,33), we did not
diversity of synaptic information and brain
observe such an effect in our experiments. In
function.
addition to ATP, we identified glutamate as a
pathophysiological function of TRPV4 in
TRPV4-induced excitatory gliotransmitter in
astrocytes has been reported (25,26,28).
astrocytes (Fig. 6C-D). Opposing actions for
According to these reports, TRPV4 signaling
astrocytic ATP and glutamate have been
in astrocytes prevents the progression of
reported (7,33). Our results indicate that the
brain
effects of glutamate on synaptic function are
importance of astrocytic TRPV4 under
astrocytes
communicate
10
astrocytes
Very
damage,
but
might
synchronize
recently,
the
the
physiological
Downloaded from http://www.jbc.org/ by guest on April 10, 2016
imaging to determine how
not use Ca
TRPV4+ astrocytes modulate synaptic transmission
normal conditions has remained unclear. We
which types of astrocytes are involved (42).
have demonstrated that an endogenous
Our findings might help to identify the
TRPV4 ligand, arachidonic acid, is sufficient
characteristics of ATP-releasing, purinergic
to enhance synaptic activity (Fig. 5E-F). We
astrocytes that are related to brain function.
speculate that the homeostatic temperature of
In astrocytes, TRPV4 is thought to enhance
the brain weakly and constitutively activates
vasodilation and contribute to local Ca2+
TRPV4 in astrocytes (29) and that activation
oscillations in endfeet (43). Since TRPV4 can
of TRPV4 by endogenous chemical ligands
be activated by the metabolites of many
such as arachidonic acid can modulate
lipids, the contribution of ATP release to
synaptic activity through the release of
vasodilation might depend on the status of
gliotransmitters (Fig. 7G). In a separate
these
study, we have shown that another
Furthermore, we recently reported that
thermo TRP member, TRPV2 (39,40), is
activation of TRPV4 promotes water efflux
expressed in all astrocytes (29) and that
in the choroid plexus (45). Since astrocytes
astrocytic
to
express various members of the AQP family
(LPC).
(1), TRPV4 might function similarly in all
responds
lysophosphatidylcholine Arachidonic
acid
is
generated
at
as
reported
(44).
astrocytes that express this channel.
postsynaptic sites at the same time as
In our study, treating astrocytes
LPC and might excite astrocytes by
with MPEP (a type 1 mGluR antagonist)
activating TRPV4. Therefore, astrocytic
partially blocked the enhanced synaptic
TRPV4 might be activated in response
transmission resulting from activation of
to increased lipid metabolism near
TRPV4 (Fig. 7E-F), although CPPG (a type
synapses.
2/3 mGluR antagonist) did not have an
We visualized TRPV4-dependent
inhibitory effect (Fig. 7D). Furthermore, we
ATP release from astrocytes by using both
have
the bio-sensor and ATP-imaging methods
gliotransmitter
(Fig. 4). These results are very similar to
MALDI-TOF mass spectrometry analysis to
those of our other reports, in which urinary
compare conditioned media from cultures of
bladder epithelial cells and esophageal
WT and TRPV4KO astrocytes (manuscript in
keratinocytes released ATP in response to
preparation). These results strongly suggest
activation of TRPV4 (36,41). However, this
that other gliotransmitters exist. Thus, we
study provides information about the unique
hypothesize that TRPV4+ astrocytes might
features of ATP release from astrocytes.
release other excitatory gliotransmitters to
Reduced ATP release from hippocampal
positively regulate neuronal activity. Future
astrocytes has recently been reported to
studies
trigger depression, but, it remains unknown
gliotransmitters 11
identified
will
another candidate
identify and
excitatory by
these
characterize
using
new their
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TRPV2
metabolites,
TRPV4+ astrocytes modulate synaptic transmission
contribution to synaptic function.
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Footnotes We would like to thank Drs. T. Mori (NIBB, Okazaki), N. Inamura (NIPS, Okazaki), Mrs. S. Mizuno (Gunma Univ.), and the technical division of NIBB for technical assistance, and Drs. K. Yamada (Hirosaki Univ.), B. MacVicar (British Columbia Univ.), and our lab members for helpful discussions. The TRPV4KO mice were kindly provided by Dr. A. Mizuno (Jichi Medical Univ.). This research was supported by Grants-in-Aid for Scientific Research 26117502 to K.S., 23650159 to Y.I., and 18077012 to M.T.) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology; by grants from the Uehara Memorial Foundation, Sumitomo Foundation, Brain Science Foundation, Narishige Neuroscience Research Foundation, and Salt Science Research Foundation No.13C2 (all to K.S.); and by a grant from the Takeda Science Foundation (to K.S. and Y.I.).
Figure legends Figure 1 TRPV4 is expressed in a restricted subpopulation of astrocytes. A, B: TRPV4 mRNA (green) and GFAP (red) expression (A) or TRPV4 mRNA (green) and S100 (red) expression (B) in adult mouse hippocampus (CA1). White arrowheads indicate TRPV4+/GFAP+ or TRPV4-/S100+ astrocytes. Scale bars: 100 m. C: TRPV4 mRNA expression (green) in cultured astrocytes. Astrocytes were identified by S100 expressions (red). White arrowheads indicate TRPV4 + astrocytes. Scale bar: 50 m. Figure 2 A specific subpopulation of astrocytes responds to a chemical TRPV4 agonist and initiates Ca2+ oscillations. A: Representative pictures of Ca2+ imaging experiments (see supplementary movie 1). Cultured astrocytes were exposed to 4PDD. Ca2+ influx was observed only in TRPV4+ populations (at 50 s). Following the observed increase in [Ca2+]i exclusively in TRPV4+ astrocytes, significant Ca2+ oscillations were observed in most of the other astrocytes (at 620 s – 720 s). B: 16
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(KAKENHI Project Nos. 21200012, 20399554, 24111507+26111702< Brain Environment> and
TRPV4+ astrocytes modulate synaptic transmission
Representative traces from cells exposed to 4PDD (10M) are shown in the upper graph. The pink trace is from a TRPV4+ cell. Representative traces from cells exposed to both 4PDD (10 M) and octanol (3 mM) are shown in the lower graph. The pink trace is from a TRPV4+ cell. Arrowheads indicate Ca2+ spikes in TRPV4- cells. C: Quantification of 4PDD-evoked activation of TRPV4 compared with the response in WT astrocytes. D: Quantification of the number of Ca2+ spikes evoked by exposure to 4PDD compared with the number evoked in WT astrocytes. * P < 0.01, t test (vs WT). Figure 3 Only TRPV4+ astrocytes display an acute increase in [Ca2+]i in response to a chemical TRPV4 agonist. A: Representative pictures from Ca2+ imaging experiments (see supplementary movie 2). The the late Ca2+ oscillations (oscillation) are shown in representative pictures (see supplementary movie 2). White arrowhead indicates a cell that responds acutely to GSK, and red arrow indicates a cell with a non-acute response to GSK. B: Representative whole cell currents (at -60 mV holding potential) after application of 3 M GSK in acute GSK responder cells (white arrowhead in panel A) or the non-acute GSK responder cells (red arrow in panel A). During whole-cell patch-clamp recordings, we applied ramp pulses from -100 mV to +100 mV at 5-second intervals. The ratios indicate the number of astrocytes that displayed GSK-evoked currents per the total number of sampled cells, with the percentages in parentheses. Figure 4 Activation of TRPV4 causes ATP release from cultured astrocytes. A: Cartoons representing the biosensor system. HEK293 cells expressing P2X2 (the bio-sensor) are placed in close proximity to the astrocytes, then 4PDD (10 M) is applied to the astrocytes. At the end of each experiment, the reliability of the bio-sensors is confirmed by directly applying ATP to a location that is distant to the astrocytes. B: Representative traces showing that application of 4PDD evokes whole-cell current responses (left panel) with inward rectification (red traces labeled “b” in the right panel) in HEK293 cells transfected with a P2X2 cDNA. Application of ATP (100 M) was used to confirm P2X2-mediated responses (left panel) with inward rectification (green traces labeled “c” in the right panel). Blue traces labeled “a” in the right panel represent control currents. Ramp pulses (from -80 mV to +80 mV for 500 msec) were applied at 5-second intervals. The holding potential was -60 mV. The traces are representative of a typical experiment (6 of 43 trials). C: Real-time ATP imaging was performed in cultured astrocytes (almost confluent). We stacked all of the images after the experiments 17
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initial time point (basal), the moment just following the application of 1 M GSK (GSK) and
TRPV4+ astrocytes modulate synaptic transmission
were completed. The ratio ATP release is represented as pseudo color images. In this figure, the height and color (as shown by the scale bars) represent the amount of ATP that is released. Representative images showing ATP release after application of 4PDD (right panel) and in basal conditions (left panel). D: Quantification of ATP release in 3 representative cultures of WT (left panel) and TRPV4KO (right panel) astrocytes after application of 4PDD. Figure 5 Activation of TRPV4 in astrocytes enhances synaptic activity. A: Co-culture of TRPV4KO neurons with WT or TRPV4KO astrocytes to analyze the physiological significance of TRPV4 signaling in astrocytes. B: Quantification of the amplitudes of mEPSCs in cultures with WT or TRPV4KO astrocytes (basal conditions or after application of 10 M 4PDD). * P < 0.01, t test (vs basal). C: Representative traces of a typical potential of –60 mV (n=16 for each genotype). D: Quantification of the frequency of mEPSCs in cultures with WT or TRPV4KO astrocytes (basal conditions or after application of 10 M 4PDD). * P < 0.01, t test (vs basal). E: Representative traces of mEPSCs in cultures with WT or TRPV4KO astrocytes after application of 10 M arachidonic acid (AA) at a holding potential of -60 mV. F: Representative traces of mEPSCs in hippocampal slices from WT or TRPV4KO mice after localized application of 10 M arachidonic acid (AA) at a holding potential of -60 mV. Figure 6 Activation of TRPV4 in astrocytes enhances synaptic activity through release of excitatory glutamate. A: A representative trace of mEPSCs in TRPV4KO hippocampal neurons evoked after application of conditioned media from cultured astrocytes. The conditioned media was prepared after 4PDD (10 M) was applied. B: Comparison of the amplitude and frequency of evoked mEPSCs to those detected in basal conditions. * P < 0.01, t test (vs basal). C: A representative trace of the analysis of amino acids present in the conditioned media from WT or TRPV4KO astrocytes exposed to 10 M 4PDD. D: Quantification of glutamate release (in basal media or conditioned media from cells exposed to 4PDD) from WT or TRPV4KO astrocytes. * P < 0.01, t test (vs WT basal). E, F: Currents from NMDA receptor (NMDA-R) biosensor cells (GluN1- and GluN2B-expressing HEK293 cells). The bio-sensor cells sense glutamate release from TRPV4-positive or -negative astrocytes, which are identified by Ca2+ imaging after exposure to 10 M 4PDD (F). As a negative control, the mock biosensor cells (HEK293 cells transfected with the pCAG vector) did not display inward currents after 10 M 4PDD was 18
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experiment that measured mEPSCs in cultures with WT or TRPV4KO astrocytes at a holding
TRPV4+ astrocytes modulate synaptic transmission
applied to TRPV4+ astrocytes (E). Figure 7 Glutamate released following activation of astrocytic TRPV4 enhances synaptic activity by signaling through type 1 mGluR. A, B: Representative traces evoked by exposing cells to ATP (1 M) are shown for WT and TRPV4KO astrocytes. C: Comparison of ATP-evoked activation in WT and TRPV4KO astrocytes. D: A representative trace of 4PDD-evoked mEPSCs in TRPV4KO hippocampal neurons with or without CPPG (50 M). E: A representative trace of 4PDD-evoked mEPSCs in TRPV4KO hippocampal neurons with or without MPEP (1 M). F: Comparison of mEPSC amplitude and frequency in cells exposed to MPEP, 4PDD, or MPEP and 4PDD. * P < 0.01, Duncan’s multiple range test (vs MPEP). # P < 0.01, Duncan’s multiple range test (vs the blue color, are specifically localized in the brain; activation of TRPV4 in these astrocytes causes excitation in neighboring astrocytes through gap junctions and ATP release, shown as red arrows. These cells form a unit of excitatory astrocytes that release glutamate, shown as green arrows. The glutamate that is released signals through type 1 mGluRs at presynaptic sites to enhance neurotransmitter release.
19
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MPEP+4PDD). G: Schematic representation of our findings. TRPV4+ astrocytes, shown by
TRPV4
GFAP
Merged
B
TRPV4
S100
Merged
C
TRPV4
S100
Merged
Figure 1
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A
A
10 M 4PDD
basal
2.0
5s
50 s
620 s
680 s
700 s
720 s
0.5
B
10 M 4PDD + 3 mM octanol ΔF/F0.5
Figure 2
TRPV4 activation by 4PDD
D
calcium spikes after TRPV4 activation
100
100 75 50 25 0
ratio vs WT spike numbers
C
ratio vs WT 4PDD response
30 s
N.D. WT
octanol
octanol suramin
TRPV4KO
75 50
*
25 0
WT
octanol
N.D.
octanol suramin
N.D.
TRPV4KO
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10 M 4PDD
A
B
basal
GSK
oscillation
non-acute GSK responder (Arrow)
0/14 (0%)
GSK
14/14 (100%)
5s
Figure 3
nA Downloaded from http://www.jbc.org/ by 0.5 guest on April 10, 2016
acute GSK responder (Arrowhead)
A B
ATP 100 M
4PDD
b
a
(nA) 1 (mV) -80
c
-60
-40
-20
20
40
80
a -1
4 nA
b
basal 4PDD ATP 100 M
-2
c -3
4PDD
basal
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20 s
C
60
High
Low
D
WT
TRPV4KO
intensity
4PDD
4PDD
60000
60000
40000
40000
20000
20000
0
Figure 4
300
600 (sec)
0
300
600 (sec)
B mEPSC amplitude (pA)
A
*
30 20 10 0
WT
C
TRPV4KO
D 4PDD
20 s
40 20 0
WT
E
basal 4PDD
*
60
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20 pA
TRPV4KO
mEPSC frequency (Hz)
WT
basal 4PDD
TRPV4KO
F WT hippocampal slice
WT
AA
AA
20 pA 20 s
TRPV4KO
TRPV4KO 20 pA
Figure 5
20 s
B
CM (+4PDD)
20 pA 20 s
C
WT
(%)
ratio vs basal
A
basal +4PDD
600
*
300
0
amplitude
frequency
D
TRPV4KO 0.06
0.06
0.01
0.01 0
10
20
30
time (min)
4
2
0
0
10
20
30
basal 4PDD
basal 4PDD
WT
TRPV4KO
time (min)
F
Mock bio-sensor
TRPV4-positive
NMDA-R bio-sensor
TRPV4-positive
4PDD
4PDD
0.5 nA 30 s
Figure 6
(n mol/mg protein)
Glu release
0.03
0.03
*
TRPV4-negative
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A.U.
6
E
*
A
B
WT ATP
Fura-2 ratio
1.5 1.0 0.5 0
150
300
450
1.5 1.0 0.5 0
600
Time (sec)
C
150
300
450
600
Time (sec)
D CPPG
4PDD
0.5
E MPEP
WT
20 pA
0
TRPV4KO
F
4PDD
20 s
(%)
20 pA
G
ratio vs basal
600
20 s
MPEP MPEP+4PDD +4PDD
#
**
amplitude
frequency
* *
300
0
GAP junctions
Neuronal transmission
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ΔFura-2 ratio
ATP
2.0
Fura-2 ratio
2.0
TRPV4KO
Astrocytic transmission GAP junctions
Figure 7
A novel subtype of astrocytes expressing TRPV4 regulates neuronal excitability via release of gliotransmitters Koji Shibasaki, Kazuhiro Ikenaka, Fuminobu Tamalu, Makoto Tominaga and Yasuki Ishizaki J. Biol. Chem. published online April 15, 2014
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