J .P. Smith et al. Brain Research 887 2000 98 –109
101
positioned within 1.5 cell diameters of an astrocyte and were continuously perfused during bath exchange, and
used for rapid application of AMPA. Dose–response AMPA or TGN were added directly to the bath to
curves were generated from peak AMPA-induced inward transiently produce the desired bath concentration. Cells
current amplitudes using a least squares regression iterated were pretreated with benzamil and U73122 for no more
by the Marquardt–Levenburg method Prostat V 1.02 than 15 min during which time fluorescence ratios reached
software, Poly Software International, Salt Lake City, UT. a stable baseline, cells showed no changes in morphology,
Because A7r5 cells are well characterized for their con- and ionomycin responses remained robust indicating that
21
stitutive expression of L-type Ca channels [28], these
there were no toxic effects of these drugs at the con- cells were used as a positive control for VGCC-mediated
centrations used. currents in our patch clamp experiments.
2.6. Characterization of AMPARs
3. Results
A series of electrophysiological and pharmacological
21
3.1. AMPAR stimulation causes a [Ca ] -dependent
o
experiments were initially performed to determine whether
21
increase in astrocyte [Ca
]
i
astrocytes, under our culture conditions, fulfilled previous- ly established criteria for the expression of functional
21
To characterize their [Ca ] response to AMPA, the
i
AMPA receptors. As described by Seeburg [40], we astrocytes were loaded with Fura-2-AM and stimulated
confirmed that 100 mM AMPA produced a desensitizing with 100 mM AMPA, in the presence of 100 mM
inward current with an average amplitude of 255 pA, and cyclothiazide AMPA CTZ, to block desensitization. Ap-
that cyclothiazide CTZ, which has a specific action on proximately 80 of the astrocytes responded with signifi-
AMPARs [35,42], eliminated the desensitization and in-
21
cant increases in [Ca ] that peaked at over 360 of the
i
creased the average inward current amplitude to 295 pA.
21
resting [Ca ] n5202 cells, seven experiments. One
i
We also found that the EC for the AMPA response was
50
hundred mM AMPA alone, or 100 mM cyclothiazide alone, consistent with that for recombinant AMPARs [44,51] and
21
had no significant effect on [Ca ] Fig. 1A. To de-
i
that the AMPA response was blocked by the highly termine
whether AMPA CTZ-dependent
increases in
specific AMPAR blocker NBQX at 1 mM [56]. These
21 21
[Ca ]
required extracellular Ca , we repeated this
i
results clearly indicate that AMPA CTZ activates an
21
experiment in Ca -free medium containing 1 mM EGTA.
essentially pure population of AMPARs in these astrocytes.
21
These cells were exposed to Ca -free medium for less
than 1 min prior to recording to minimize the possibility of 2.7. Fura-2 calcium imaging
21
depleting intracellular Ca stores. As shown in Fig. 1B,
astrocytes did not respond to AMPA CTZ in the absence Semi-confluent astrocytes about 50, grown on glass
21 21
of [Ca ] , suggesting that the increased [Ca
] was most
o i
coverslips, were loaded with 4 mM Fura-2 AM Sigma in
21
likely due to influx of extracellular Ca across the plasma
0.05 pluronic acid for 1 h at 378C in growth medium and membrane see also David et al. [10].
rinsed in recording solution for 15 min prior to imaging at room temperature 22–278C. Images were acquired with
an inverted microscope Nikon Diaphot 300, Melville, 3.2. Astrocyte AMPARs contain the GluR2 subunit
NY, a cooled CCD video imaging system SenSys 1400, Photometrics, Tucson, AZ, and the Metafluor image
AMPARs are heteromers composed of one or more of processing and analysis system version 2.76 Universal
GluR1, 2, 3, and 4 subunits for review see Bettler and Imaging Corp., West Chester, PA. Cells were alternately
Mulle [3]. The presence of the GluR2 subunit substantial-
21
excited at 340 and 380 nm for 100 ms and the emitted ly reduces AMPAR Ca
permeability such that cells with fluorescence was measured at 510 nm. Background-sub-
increasing numbers of GluR2 subunits have AMPARs with
21
tracted ratio images 340 380 nm were then stored for very low Ca
permeability [12]. As shown in Fig. 2A, all off-line analysis using Prostat software Poly Software.
four GluR subunit mRNAs were expressed in our cultures,
21
Approximate [Ca ] was determined using the method of
as assessed by non-quantitative rtPCR followed by size
i
Grynkiewicz [16]. At the end of each experiment, 1 mM analysis of restriction enzyme digests. The restriction assay
ionomycin was added to the bath as a positive control. that we used to identify each subunit results in two
21
Ionomycin caused [Ca ] to increase in each experiment
restriction fragments that uniquely identify each subunit
i 21
mean 613 in which there was Ca in the bathing
[25]. Importantly, the GluR2 subunit mRNA was readily
21 21
solution. With 0 Ca in the bathing solution, [Ca
] detectable in these astrocyte cell cultures.
i
decreased by 50 in the presence of ionomycin. Western blot analysis also indicated expression of
During measurements, the bath solution was constantly abundant GluR2 subunit protein Fig. 2B. Immuno-
exchanged at the rate of 2 ml min through the 1-ml bath cytochemical analysis of the cultures demonstrated a
producing a 95 exchange every 3 min. CTZ, Bay K8644, uniform distribution of immunostaining, such that the vast
1
benzamil, U73122 or depolarizing solution 55 mM K majority of astrocytes were immunopositive for the GluR2
102 J
mine is known to inhibit only AMPAR-mediated currents in cells that lack functional edited GluR2 subunits [53].
Taken together, these electrophysiological, molecular, and immunocytochemical data strongly suggest that the as-
trocyte AMPARs contained functional GluR2 subunits and, therefore, would be expected to have less than maximal
21
Ca permeability. It is important to note that the maximal
permeability of AMPARs that completely lack GluR2 subunits is itself thought to be extremely low [12].
21
The low AMPAR Ca permeability predicted by these
experiments was further supported by our experiments with benzamil and U-73122. As described below, these
21
agents completely eliminated AMPAR-mediated Ca responses, but they are not known to affect AMPARs
21
directly. Thus it is highly questionable whether Ca influx
through AMPAR channels accounted for the entirety of the
21
large AMPA CTZ-dependent [Ca ] responses that we
i
observed in our cultures.
21
3.3. AMPAR-dependent increases in [Ca ] were not
i
mediated by voltage-gated calcium channels VGCCs
One possible mechanism to explain the AMPA CTZ-
21 21
dependent increase in [Ca ] , without substantial Ca
i
influx through AMPARs, is via activation of VGCCs following AMPAR-mediated depolarization. Since the
21
Fig. 1. A Fura-2 Ca imaging experiment showing the mean change in
expression of VGCCs in cultured astrocytes may vary [1],
21
[Ca ] 6S.E.M. for a representative example of 33 cells. Similar results
i
it was important to determine whether VGCCs were
were observed in six additional experiments involving a total of 202 cells.
expressed under our culture conditions. We were unable to
In control bath solution, addition of 100 mM AMPA caused no change in
21
[Ca ] . With the addition of 100 mM CTZ to the bath, the same dose of
detect VGCC expression using a number of different
i 21
21
AMPA elicited a large increase in [Ca ] that fell to baseline over a
i
detection methods. First, inward Ba currents were not
21
period of about 5 min. B Fura-2 Ca imaging experiment showing
detectable in whole-cell patch clamped astrocytes Fig.
21
mean change in [Ca ] 6S.E.M. for 15 cells. In a bath solution
i 21
3A, nor were they induced by a 1-h pretreatment with the
containing 100 mM CTZ, and zero [Ca ] , plus 1 mM EGTA, the
o
adenylate-cyclase activator, forskolin 10 mM, which has
addition of 100 mM AMPA caused no response.
been previously reported to enhance VGCC-mediated
21
currents in astrocytes [1]. Likewise, using Fura-2 Ca subunit data not shown. Taken together, these data
imaging, we did not detect depolarization-dependent in-
21 1
strongly suggest abundant GluR2 subunit expression in creases in [Ca
] during stimulation with 55 mM K ,
i 21
most if not all astrocytes within the cultures, suggesting even in the presence of the L-type Ca
channel agonist, that calcium permeability of the astrocyte AMPARs would
Bay K 8644 1 mM. These negative results are most be very low [12].
likely due to the absence of VGCC expression, rather than Evidence for the function of GluR2 subunits in astrocyte
to the presence of silent channels, since we did not detect AMPARs was derived from electrophysiological whole-
mRNA encoding the pore-forming a subunit of the L-
1 21
cell patch recordings. Previous studies of recombinantly type Ca
channel in astrocytes using rtPCR. a subunit
1
expressed AMPARs in mammalian cells have shown that mRNA was, however, readily detectable in smooth muscle
the current–voltage I –V curves of AMPARs lacking A7r5 cells, which displayed inward currents Fig. 3C.
functional edited GluR2 subunits are doubly rectifying Taken together, these data strongly suggest that few, if
with reversal potentials near 230 mV, whereas the I –V any, VGCCs were expressed in the astrocyte cultures thus
relationship for GluR2 subunit-containing AMPARs are making it highly unlikely that VGCCs made any signifi-
21
linear with reversal potentials near 0 mV [50]. We observed cant contribution to AMPAR-mediated Ca
responses. a linear I –V relationship for AMPAR-induced currents in
21
our astrocytes linear regression R50.9955, that inter- 3.4. AMPAR-dependent increases in [Ca
] require the
i 1
21
cepted the voltage axis at 0.53 mV data not shown. In reverse-mode operation of the Na Ca
exchanger addition, administration of 500 mM HPP-spermine at a
21 21
holding potential of 260 mV had no effect on the Since neither Ca
currents through AMPARs nor Ca magnitude of the AMPA CTZ-induced currents. HPP-sper-
current through VGCCs appeared to be necessary to have
J .P. Smith et al. Brain Research 887 2000 98 –109
103
Fig. 2. A rtPCR results for astrocytes. GluR1, 2, 3, and 4 mRNA were detected in these cerebral astrocytes as indicated by the presence of restriction products that appear below 600 bp in each lane that is labeled GluR. Unrestricted products are shown for comparison. Also included are 100 bp size
standards that show 100 bp increments around 600 bp, which appears as the brightest band. B ECL Western blot SDS–PAGE results. As shown by both the GluR2 and the GluR2 3 antibodies, GluR2 subunits were expressed in relatively high proportions in cultured cerebral astrocytes. Brain tissue
containing neurons and astrocytes was used as a positive control. COS-7 cells, which do not express GluRs, were used as a negative control. GluRs migrate near 110 kDa during SDS–PAGE, therefore the region around 110 kDa is shown for each experiment.
1 1
produced the observed AMPAR-mediated increases in ing [Na ] directly affects both the function of the Na
o 21
21
[Ca ] , we examined the contribution of the reverse-mode
Ca exchanger as well as of AMPARs. Therefore, to test
i 1
21 1
21
operation of the Na Ca exchanger [5], apparently
the involvement of the Na Ca exchanger, we exposed
1
driven by Na influx through the AMPAR. Indeed, Gold-
the cells to AMPA CTZ after a 10-min pretreatment in 100
1 21
man et al. [14] have previously demonstrated the reverse- mM benzamil, a well known inhibitor of the Na Ca
mode operation of this transporter following increased exchanger [22,24,26]. As shown in Fig. 4A, benzamil
1
[Na ] in astrocytes. Additionally, David et al. [10]
completely eliminated the AMPA CTZ-dependent increase
i 1
21
showed that removal of [Na ] completely blocks AMPA in [Ca
] in 94 of the astrocytes. Benzamil pretreatment
o i
21
CTZ-dependent increases
in protoplasmic
astrocyte caused an increase of resting [Ca
] to 147 of that in
i 21
1 21
[Ca ] , suggesting the possibility that AMPAR activity
untreated cells, consistent with inhibition of the Na Ca
i 1
21 21
and the Na Ca exchanger are functionally linked. It is,
exchanger; however, this [Ca ] level was still well below
i 1
21
however, not possible to alter [Na ] in order to test the the average AMPAR-mediated increase in [Ca
] . These
o i
1 21
1 21
possible functional relationship between the Na Ca results strongly suggest that the Na Ca
exchanger was
21
exchanger and AMPARs, with respect to understanding the involved in the [Ca
] response to AMPA CTZ.
i 21
1 21
origin of AMPA-dependent [Ca ] responses, since alter-
The reverse-mode operation of the Na Ca exchanger
i
104 J
Fig. 3. A Whole-cell patch clamp results from cerebral astrocytes grown in FCS. Recordings were made in 10 cells and no inward currents were observed
21
within the sensitivity of our measurements. Error bars represent S.E.M.s. The extracellular recording solution contained Ba -TEA, and the results were
21 21
adjusted by linear leak subtraction see Section 2. B Fura-2 Ca imaging results in astrocytes showing no increase in [Ca
] in depolarizing solution
i 1
21 21
55 mM K , or in depolarizing solution with the L-type Ca channel agonist Bay K 8466 1 mM. Average [Ca
] 6S.E.M. is shown from data pooled
i 21
from nine groups of 10–30 cells. C rtPCR results for astrocytes grown in FCS and for A7r5 cells. A7r5 cells lane 1 express L-type Ca channel a
1
subunit mRNA 932 bp. L-Type calcium channel a subunit mRNA was not detected in astrocytes lane 2. b-Actin sequences 300 bp were amplified
1
from astrocyte cDNA in parallel reactions as a positive control to confirm the integrity of the rtPCR reactions.
1 21
is known to require phospholipase-C PLC-activation and reverse-mode operation of the Na Ca
exchanger lead-
21
hydrolysis of
phosphoinositol-bis-phosphate PIP
ing to elevated [Ca ] in astrocytes exposed to AMPA
2 i
1 21
CTZ. [2,7,17]. Therefore, as a second measure of the Na Ca
21
exchanger involvement in AMPAR-mediated [Ca ] re-
i
3.5. AMPA CTZ depletion of thapsigargin TGN- sponses, we pretreated astrocytes in 0.5 mM U-73122, an
21
sensitive Ca stores
inhibitor of PLC in astrocytes, and subsequently exposed the cells to AMPA CTZ [47,49]. As shown in Fig. 4B,
Because of the many instances of interactions between U-73122 completely eliminated the AMPAR-dependent
21 21
21
transmembrane Ca flux and the release of Ca
from increase in astrocyte [Ca
] . In this experiment, resting
i 21
intracellular stores, we investigated the possibility of a role [Ca
] was again elevated, consistent with an inhibition
i 1
21 21
for thapsigargin-sensitive stores in the AMPAR-dependent of the Na Ca
exchanger, but again this [Ca ] level
i 21
increase in [Ca ] . Thus we treated astrocytes with
was well below the average AMPAR-mediated increase in
i 21
21
thapsigargin, to deplete intracellular TGN-sensitive Ca [Ca
] .
i
stores both prior to and after AMPA CTZ treatment see One possible explanation for the block of AMPAR-
21
Wictome et al. [55]. As shown in Fig. 5A, AMPAR- mediated Ca
responses following PLC inhibition is
21 21
dependent increases in [Ca ] were not attenuated by
through the inhibition of Ca release from internal stores.
i
pre-treatment with TGN. However, as shown in Fig. 5B, This is, however, unlikely because benzamil had a similar
21
pre-treatment with AMPA CTZ severely decreased the blocking effect to U-73122 and since release of Ca
from
21
TGN-dependent [Ca ] response.
internal stores has not been linked previously to AMPAR
i
Fig. 5C summarizes the results of 17 experiments stimulation. Thus, the simplest explanation of our data is
1
involving approximately 800 cells and shows that the that Na
influx through AMPARs can stimulate the
J .P. Smith et al. Brain Research 887 2000 98 –109
105
21
regulating internal Ca stores and where it may also help
21
to regulate other Ca phenomena [14,15,46]. Despite its
potential importance in astrocytes, very little is known
1
about physiological mechanisms that can stimulate Na
21
Ca exchange and its role in astrocyte-brain physiology.
21
It has been previously suggested that Ca influx through
1 21
reverse mode function of the Na Ca exchanger might
result from ionotropic glutamate receptor iGluR activa- tion in astrocytes [10,21,22]. Demonstration of this cou-
pling would greatly improve our understanding of the
1 21
Na Ca exchanger in astrocytes. In contrast, modulation
21
of [Ca ] by ionotropic glutamate receptor stimulation of
i 1
21
reverse mode operation of the Na Ca exchanger has
been demonstrated in Bergmann glia, oligodendrocyte progenitors, and rat forebrain neurons, clearly demon-
21
strating that these two transmembrane Ca influx path-
ways can be functionally related [19,23,26]. In this report we provide several lines of evidence to suggest that
1 21
AMPAR activation and reverse-mode Na Ca exchange
are indeed coupled in cultured astrocytes leading to
21 1
21
AMPAR-dependent Ca influx through the Na Ca
21
exchanger and an associated release of Ca from intracel-
lular stores.
21
21
4.1. AMPAR stimulation causes a [Ca ] -dependent
Fig. 4. A Fura-2 Ca imaging experiment showing the mean change in
o 21
21
increase in astrocyte [Ca
]
[Ca ] 6S.E.M. for 64 cells from three separate experiments. In a bath
i
i
solution containing 100 mM CTZ, and 100 mM benzamil, the addition of
21
100 mM AMPA caused no response. B Fura-2 Ca imaging experiment
It is well established that application of glutamate,
21
showing the mean change in [Ca ] 6S.E.M. for 55 cells from two
i
quisqualate, kainate, and or AMPA to astrocytes can lead
separate experiments. In a bath solution containing 100 mM CTZ, and 0.5
21 21
to the
[Ca ] -dependent
elevation of
[Ca ]
mM U-73122, the addition of 100 mM AMPA caused no change in
o i
21
[10,13,18,21,22]. Fewer studies have specifically focused
[Ca ] .
i
on the role that the AMPA-preferring subtype of glutamate receptor might play in these responses. Our studies re-
produce the results of David et al. [10] who showed the
21 21
21
average peak [Ca ] response evoked by AMPA CTZ
[Ca ] -dependent elevation of [Ca
] in astrocytes fol-
i o
i
alone, or AMPA CTZ after a pretreatment with TGN, were lowing AMPAR stimulation Fig. 1B. This result raises
21
not significantly different from one another. Treatment of the question: how does AMPAR-stimulation lead to Ca
the cells with TGN after pretreatment with AMPA CTZ, influx across the plasma membrane? Since some AMPARs
21
however, resulted in the significant attenuation of the TGN are known to have a limited permeability to Ca
, the
21
response to less that 66 of its original magnitude. The simplest conclusion is that the Ca
enters directly through
21
results of Fig. 5 indicate that TGN-sensitive Ca stores
the AMPAR; however, our results bring this interpretation are partially depleted by AMPA CTZ, but that release of
into question in our cells.
21
Ca from these stores does not significantly add to the
21
AMPA CTZ-stimulated increase in [Ca ] . Thus AMPAR
i
stimulation appears to be linked to the reverse mode 4.2. Astrocyte AMPARs contain functional GluR2
1 21
operation of the Na Ca exchanger and to an associated
subunits
21
release of Ca from TGN sensitive stores, to achieve a
21
much more complex intracellular [Ca ] response than
The molecular biological data shown in Fig. 2 clearly
i 21
would be expected by the function of either of these Ca indicate the presence of the GluR2 subunit mRNA and
effectors alone. protein in our astrocytes, moreover, by determining that
the I –V relationship for AMPA CTZ induced currents was linear, reversed very close to zero mV, and was unrespon-
4. Discussion sive to HPP-spermine, we were able to confirm the
