Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Li, Yang,
Lynne A. Holtzclaw, and
James T. Russell.
Müller Cell Ca2+ Waves Evoked by Purinergic
Receptor Agonists in Slices of Rat Retina.
J. Neurophysiol. 85: 986-994, 2001.
We have measured
agonist evoked Ca2+ waves in Müller cells
in situ within freshly isolated retinal slices. Using an eye cup dye
loading procedure we were able to preferentially fill Müller glial cells in retinal slices with calcium green. Fluorescence microscopy revealed that bath perfusion of slices with purinergic agonists elicits Ca2+ waves in Müller
cells, which propagate along their processes. These
Ca2+ signals were insensitive to tetrodotoxin
(TTX, 1.0 µM) pretreatment. Cells were readily identified as
Müller cells by their unique morphology and by subsequent
immunocytochemical labeling with glial fibrillary acidic protein
antibodies. While cells never exhibited spontaneous
Ca2+ oscillations, purinoreceptor agonists, ATP,
2 MeSATP, ADP, 2 MeSADP, and adenosine readily elicited
Ca2+ waves. These waves persisted in the absence
of [Ca2+]o but were
abolished by thapsigargin pretreatment, suggesting that the purinergic
agonists tested act by releasing Ca2+ from
intracellular Ca2+ stores. The rank order of
potency of different purines and pyrimidines for inducing
Ca2+ signals was 2 MeSATP = 2MeSADP > ADP > ATP
meATP = uridine triphosphate (UTP) > uridine diphosphate (UDP). The Ca2+
signals evoked by ATP, ADP, and 2 MeSATP were inhibited by reactive blue (100 µM) and suramin (200 µM), and the adenosine induced signals were abolished only by 3,7-dimethyl-1-propargylxanthine (200 µM) and not by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine) or
8-cyclopentyl-1,3-dipropylxanthine at the same concentration. Based on
these pharmacological characteristics and the dose-response relationships for ATP, 2 MeSATP, 2 MeSADP, ADP, and adenosine, we
concluded that Müller cells express the P1A2 and
P2Y1 subtypes of purinoceptors. Analysis of
Ca2+ responses showed that, similar to glial
cells in culture, wave propagation occurred by regenerative
amplification at specialized Ca2+ release sites
(wave amplification sites), where the rate of
Ca2+ release was significantly enhanced. These
data suggest that Müller cells in the retina may participate in
signaling, and this may serve as an extra-neuronal signaling pathway.
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INTRODUCTION |
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Glial cells, like neurons,
participate in signaling in the mammalian nervous system. Glial cells
monitor and respond to neuronal activity and transmit such activity
over long distances by way of propagating Ca2+
waves (Smith 1992). Moreover, bi-directional
glial-neuronal communication has received increasing attention as a
potential mode of cell-cell signaling in the CNS (Dani et al.
1992
; Duffy and MacVicar 1995
; Grosche et
al. 1999
; Mennerick and Zorumski 1994
;
Nedergaard 1994
; Newman and Zahs 1997
;
Pasti et al. 1997
; Porter and McCarthy
1996
; Wu and Barish 1994
). Propagation of
Ca2+ waves through a network of
intercommunicating astrocytic processes in the CNS could serve as a
conduit for extraneuronal, long-distance intercellular communication.
The cellular mechanisms that support the temporal and spatial
characteristics of such glial Ca2+
waves in situ, however, are not fully understood. We have recently shown that in glial cells in culture, Ca2+ wave
propagation in processes occurs by regenerative
Ca2+ release at specialized release sites or
"wave amplification sites" (Simpson and Russell
1998
; Simpson et al. 1997
; Yagodin et al. 1994
). It remains to be seen whether these observations in
cultured cells reflect glial responses in situ.
Müller cells, the principal glial cells in the retina, provide an
ideal experimental model to study the cell biology of wave propagation.
These cells span almost the entire depth of the retina from the outer
photoreceptor layer to the inner border of the retina, where they
terminate in expanded end-feet adjacent to the vitreous humor (see
review by Newman and Reichenbach 1996). Müller
cells have long, relatively linear processes, which allows for precise
analysis of Ca2+ wave propagation. To utilize
their unique morphology, we developed a novel dye loading method in the
eyecup, in which Müller cells are preferentially filled with
calcium green. Wave propagation in identified single Müller cells
was recorded using a conventional wide-angle fluorescence microscope.
The goals of this study were as follows: 1) to investigate
whether the cellular mechanism of Ca2+ wave
propagation in situ mirrors that described in cultured glial cells,
2) to describe the temporal and spatial characteristics of
Ca2+ wave propagation within Müller cells
in intact retinal slices, and 3) to characterize receptor
systems that evoke Ca2+ waves in Müller
cells. Although Ca2+ waves between Müller
cell end-feet have been recorded across the vitreal surface of
flat-mounted whole retina (Newman and Zahs 1997), these waves were visualized by
confocal microscopy in different optical sections through the thickness
of the retina. In contrast, we studied wave propagation along single
Müller cell processes to investigate the underlying cellular
mechanisms that support intracellular Ca2+ waves.
We prepared slices of retina where the Müller cells were discretely loaded with the Ca2+ indicator dye,
calcium green, and recorded Ca2+ waves through
the entire length of processes in the plane of the microscopic field
using fluorescence microscopy. We show that Ca2+
waves in Müller cells are elicited by purinergic activation. In
addition, like in cultured glial cells, Müller cell waves propagate by regenerative Ca2+ release at a
series of wave amplification sites along the length of processes.
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METHODS |
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Indicator loading
Müller cells in the adult rat retina were selectively
loaded with the Ca2+ indicator dye, calcium
green, using an eyecup loading technique. Retinae were isolated from
male Sprague Dawley rats maintained under a 12-h light-dark cycle using
procedures authorized by the Animal Use and Care Committee, National
Institute of Child Health and Human Development, National Institutes of
Health (protocol number 98-017). Following decapitation, the eye lid
and other connective tissue covering the eye were peeled away, and the
eye was gently rolled out of the socket and transferred to a dish with
fresh artificial cerebral spinal fluid (ACSF; in mM: 117.4 NaCl, 2.0 KCl, 1.4 MgSO4, 2.5 CaCl2,
1.0 KH2PO4, 26.2 NaHCO3, and 11.0 glucose), which was continuously
aerated with 95% O2 containing 5%
CO2. The anterior chamber of the eye was opened, and the iris, lens, and vitreous humor were removed. To make a useful
eyecup for subsequent dye loading, it was essential to cut and trim the
tissue above the ora serrata, since the retina was firmly attached to
the pigment epithelium in only two areas, the optic disk and the ora
serrata. The isolated eyecup was placed in ACSF containing collagenase
(2 mg/ml) and DNase (0.1 mg/ml) and incubated at 30°C for 15 min to
ensure complete removal of the remaining vitreous humor. This treatment
improved dye access to the Müller cell endfeet (Coleman
and Miller 1989; Gottesman and Miller 1992
;
Newman and Zahs 1997
). The eyecup was then incubated for
2 h at 30°C in oxygenated ACSF containing calcium green AM (70 µg/ml; Molecular Probes) dissolved in DMSO containing pluronic acid
(4.7 mg/ml). At this stage the retina was intact ensuring that only
Müller cell end feet were exposed to indicator containing solution. This precaution prevented cells other than Müller cells such as astrocytes from taking up the dye. Retinae were then isolated from the eyecup, laid photoreceptor layer down, and chopped into 100-µm slices using a McIlwain tissue chopper. Slices were submerged immediately in oxygenated ACSF and maintained in the dark at room temperature. Slices were used within 4-5 h after loading.
Solution perfusion and drugs
Dye-loaded retinal slices were transferred to a Leiden cover slip chamber and secured with specimen grips and a metal weight. Slices were submerged in ACSF and continuously aerated with 95% O2 in 5% CO2. The volume of the recording chamber was approximately 0.3 ml. Slices were perfused at the rate of 2.5 ml/min. The drugs used were disodium ATP, tetrodotoxin, 2 MeSATP, 2 MeSADP (RBI), uridine triphosphate (UTP), phenylephrine, and glutamate and reactive blue (Sigma Chemical), suramin, (Calbiochem), and thapsigargin (LC Service).
[Ca2+]i measurements and data analysis
Calcium green fluorescence was imaged with an inverted
microscope on a vertical optical bench using a Nikon ×40/1.3 NA CF Fluor DL objective lens. The slice preparation was illuminated with a
mercury arc lamp (Oriel Optics), with quartz collector lenses. A
shutter (Uniblitz), an excitation filter (495 nm), appropriate dichroic mirror, and a long pass filter (515 nm) were mounted in the light path such that fluorescence could be imaged through a
microchannel plate intensifier (Model KS-1380, Videoscope
International, Washington, DC) using a charge-coupled device (CCD)
camera (Pulnix). Images were digitized and integrated (2 frames per
image) on a Macintosh computer running Synapse, an image acquisition
and analysis program (Synergy Research, Silver Spring, MD). The time
interval between images varied depending on the required temporal
resolution and ranged from 2 to 30 images/s. After all the images were
acquired, Müller cells were outlined with a region of interest
(ROI) tool, and fluorescence intensity within the whole cell or
sub-regions of cells was extracted and analyzed. Wave propagation and
local Ca2+ release rates were analyzed in
consecutive cellular sections as described previously (Simpson
et al. 1997; Yagodin et al. 1994
). Images of
Müller cell processes were divided for analysis into 0.8- to
2.0-µm-wide regions sequentially along the longitudinal axis, and
fluorescence intensity values in the nonzero pixels within each slice
were averaged (F) and plotted as normalized fluorescence
intensities (
F/F0)
against time.
Immunohistochemistry
In some experiments, cells were verified as Müller cells by immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP). Immediately following a recording, retinal slices were fixed in 4% paraformaldehyde for 30 min, and then rinsed in PBS (pH 7.3). The fixed sections were then permeabilized and blocked in PBS containing 0.3% Triton X-100, 1% BSA, and 20% normal goat serum (pH 7.4) for 1 h at room temperature. The sections were then incubated in anti-GFAP antibody (Boehringer Mannheim) for 18 h at 4°C in the same solution as above, but containing 1% normal goat serum. Sections were washed extensively in PBS and incubated with a fluorescence-conjugated goat anti-mouse antibody for 90 min and then washed again. After a brief PBS rinse, the sections were dried and mounted with Mowiol onto gelatin-coated glass slides. Cells were imaged using a fluorescence microscope with a digital cooled CCD camera (Photometrics, Tucson, AZ).
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RESULTS |
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Specific labeling of Müller cells in retinal slices
Fluorescent indicator dyes were preferentially taken up by Müller cells by the use of the eyecup loading technique. Müller cells were easily distinguished within the tissue by their intense fluorescence signal and elongated bipolar morphology, which spanned the entire thickness of the retinal slice (Fig. 1). The end-feet and the soma appeared intensely fluorescent compared with the processes (Fig. 1B). To examine cellular morphology in detail, Müller cells were loaded with calcein using the same eyecup loading procedure. Calcein-loaded Müller cell processes appeared regular with very fine hairlike branches (Fig. 1A). In some experiments, calcium green-loaded Müller cells were identified by subsequent immunohistochemistry using anti-GFAP antibodies. Figure 1C shows one example where Müller cells in a retinal slice were labeled immunocytochemically following [Ca2+]i measurements and were identified as GFAP-positive glial cells. Examination under confocal optics revealed that only Müller glial cells were loaded with calcium green as no fluorescence was detected in astrocytes or ganglion cells (data not shown). Thus all the fluorescence measured in our experiments originated from within Müller cells. Damaged preparations, where fluorescence was observed within astrocytes and neurons (<15% of retinae) were routinely discarded.
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Purinergic receptor-mediated [Ca2+]i transients in Müller cells
In the absence of stimulation, Müller cells in retinal slices did not show spontaneous [Ca2+]i changes. Bath application of purinergic agonists, however, increased [Ca2+]i in all the Müller cells examined as indicated by the increase in calcium green fluorescence (Fig. 2). The P2 purinoreceptor agonists, ATP (100 µM, Fig. 2A), 2-methylthio-ATP (2 MeSATP, 10 µM, Fig. 2B), ADP (10 µM, Fig. 2C), and the P1 receptor agonist adenosine (10 µM, Fig. 2D) all elicited [Ca2+]i rises. The pattern of these responses consisted of an initial rapid rise to peak within 5 to 10 s of agonist application, followed by a slow decline to a plateau level higher than the prestimulus level of [Ca2+]i (Fig. 2, A-D). Rarely did the fluorescence signal decline to prestimulus levels in the presence of agonist, but on removal of agonist it promptly returned to resting levels. In many instances (25 of 44 cells), oscillatory changes in [Ca2+]i were observed when slices were perfused with adenosine (Fig. 2D). Brief (15 s) applications of 2 MeSATP elicited repeated [Ca2+]i transients of reduced amplitude, which remained constant over a number of trials (Fig. 2E). Similar results were obtained in 35 separate slices (140 cells), and the results are compiled in Table 1. These purinergic receptor-mediated [Ca2+]i signals were most likely due to direct stimulation of Müller cells as the responses were unaffected by blocking neuronal stimulation and action potential propagation by pretreatment of slices with TTX (1 µM, 4 slices).
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While all the cells responded to ATP or 2 MeSATP, very few of the cells
responded to norepinephrine or glutamate. Norepinephrine (500 µM)-induced Ca2+ responses were observed only
in two of six retinal slices, while all cells in the field of view in
the same six slices showed Ca2+ rises in response
to ATP (10 µM) and 2 MeSATP (1 µM). In contrast to findings in
freshly isolated Müller cells (Keirstead and Miller 1995, 1997
), application of glutamate (up to 1 mM) had little effect on Müller cells in situ in retinal slices
(15 cells in 3 slices).
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Müller cell [Ca2+]i signals are due to Ca2+ release from cellular stores
To test whether the presence of extracellular Ca2+ is required for purinergic receptor-evoked [Ca2+]i signals, we replaced normal ACSF with medium containing zero Ca2+ and 100 µM EGTA. We found that removal of extracellular Ca2+ over short periods of time (up to 10 min of perfusion) did not significantly alter either the amplitude or the pattern of 2 MeSATP-evoked responses (Fig. 3A). Prolonged incubation in Ca2+-free medium with repeated agonist stimulations, however, resulted in the loss of response, probably due to complete depletion of the intracellular Ca2+ stores (Fig. 3A).
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In another set of experiments, intracellular
Ca2+ stores were depleted with thapsigargin,
which inhibits the sarcoendoplasmic reticulum
Ca2+ ATPase and thus depletes the endoplasmic
reticulum Ca2+ stores in cells (Thastrup
1990). A 15-min perfusion with thapsigargin (5 µM) abolished
all [Ca2+]i responses to
ATP or 2 MeSATP (Fig. 3B). Taken together, these results
show that the Müller cell
[Ca2+]i responses to ATP
and 2 MeSATP are due to Ca2+ release from
intracellular stores rather than from an influx of extracellular
Ca2+. Newman and Zahs (1997)
obtained similar results in their experiments on retinal whole-mount preparations.
Pharmacology of purinergic receptor-mediated [Ca2+]i signals in Müller cells
Purinergic receptors have been classified into two major
types. P1 adenosine receptors (subtypes A1 and A2) are
G-protein-coupled receptors and act via phospholipase-C
(Burnstock et al. 1983; Cusack and Hourani
1982
). P2 purinergic receptors have two subtypes, ionotropic
P2X receptors and metabotropic P2Y receptors, which like P1 receptors
act via phospholipase-C. Seven different subtypes of P2Y receptors have
been described based on the rank order of potency of different
nucleotide agonists (see Burnstock 1997
). Since the
Müller cell [Ca2+]i
responses to ATP, 2 MeSATP, and adenosine were not abolished in the
absence of [Ca2+]o, but
were abolished by treatment with thapsigargin, we conclude that these
responses are mediated by P2Y and P1 types of metabotropic purinoceptors.
-Methylene-ATP (
meATP, 1 µM), the known
agonist for P2X subtype of receptors, produced small increases
in [Ca2+]i in <10% of
Müller cells examined.
We systematically tested a number of agonists to determine the major
purinoceptor subtype involved in the evoked
[Ca2+]i response in
Müller cells. In these experiments, ATP was applied at the
beginning and the end of each experiment, and only cells with
comparable responses at the beginning and end were included in the
analysis. Agonists were tested from low to high concentrations with a
15-min wash in between. Dose-response relationships were constructed
for ATP, 2 MeSATP, 2 MeSADP (Fig. 4A), and ADP (Fig. 4B). The threshold concentrations were 1.0, 0.01, and 0.1 µM for ATP, 2 MeSATP, and ADP, respectively. The
EC50 for the different nucleotides were (in
µM), ATP, 7.0 ± 0.67; 2 MeSATP, 0.12 ± 0.06; 2 MeSADP,
0.22 ± 0.05; and ADP, 0.93 ± 0.03, showing that ADP, 2 MeSATP, and 2 MeSADP have 10-fold higher affinity for the receptor than
ATP. These results are in general agreement with other reports (Burnstock et al. 1983; Cusack and Hourani
1982
; Dixon 2000
) and support the conclusion
that the Müller cells in rat retina express P2Y purinoceptors and
their stimulation results in a robust
[Ca2+]i response.
Furthermore, in separate trials, the P2Y antagonists suramin (200 µM)
and reactive blue (100 µM) both blocked the
[Ca2+]i responses in
Müller cells evoked by ATP and 2 MeSATP (Fig. 5, A-C).
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In addition, we also tested MeATP, UTP, and uridine diphosphate
(UDP) and found that the P2Y agonists, 2 MeSATP, 2 MeSADP, ADP, and ATP were by far the most potent and stimulated most
Müller cells (Table 1). Only 36 and 8% of Müller cells
responded to the P2 agonists UTP and UDP, respectively (Table 1). The
rank order of potency for the purine and pyrimidine nucleotides was 2 MeSATP = 2 MeSADP > ADP > ATP
meATP = UTP > UDP. These results suggest that P2Y1
subtype of receptors mediate the
[Ca2+]i signals evoked by
the purinoceptor agonists in Müller cells. Adenosine was as
potent as 2 MeSATP in eliciting Müller cell [Ca2+]i responses (Table
1, Figs. 2D and 5D), suggesting that Müller cells also express P1 purinoceptors. To determine the adenosine receptor subtype (A1 or A2) involved, in separate experiments, we
tested the selective A1 antagonists
1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PAPCX) or
8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or the selective A2 antagonist 3,7-dimethyl-1-propargylxanthine
(DMPX). Slices were stimulated with adenosine (10 µM) in the presence
or absense of antagonists. We found that while PAPCX and DPCPX were
totally ineffective in antagonizing the adenosine evoked
[Ca2+]i responses (5 trials, data not shown), DMPX (100 µM) completely abolished the
responses in 10 separate trials (Fig. 5D). Taken together,
these data suggest that in the retina, both intercellular communication
and intracellular Ca2+ waves through the
thickness of the retina within Müller cells could occur by a
purinergic receptor-mediated mechanism.
Ca2+ wave propagation in individual Müller cells
To investigate the spatiotemporal characteristics of the Ca2+ signals in Müller cell processes, we acquired images rapidly (every 100 ms). Movies of image sets showing Ca2+ responses revealed that the signals always initiated in one region of Müller cells and propagated as waves through the processes. Figure 6 shows a montage of a series of images showing Ca2+ responses in a retinal slice stimulated with 2 MeSATP (10 µM). This representative image sequence shows a Ca2+ wave originating at the end-feet (top of picture) and propagating to the cell body (bright spots at the middle of the image) and then toward the photoreceptor layer at the bottom of the picture. In addition, bi-directional propagation of waves from a single initiation site was often observed (see Fig. 8), suggesting that diffusion of agonist across the slice does not determine the direction of the wave. Propagation of Ca2+ waves from one Müller cell to others can also be seen in the bottom half of the montage. Waves between cells were often observed, and the direction of propagation of these intercellular waves did not depend on the direction of fluid flow. We analyzed wave propagation through the cell body and the thin processes using digital image processing techniques (see Figs. 7 and 8).
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Ca2+ wave propagation in situ was analyzed using
paradigms developed in our laboratory to investigate propagation in
cultured glial cells (Fig. 7) (Simpson et al. 1997;
Yagodin et al. 1994
). Cell images were sectioned into
serial slices, and the average fluorescence intensity over time within
each of these regions was plotted with an offset along the
Y-axis (Fig. 7B). The offset plot shows that in
this Müller cell, the very first Ca2+ wave
initiated close to the end-foot and another one a little later near the
photoreceptor layer and both waves propagated toward the cell body. To
obtain quantitative estimates of wave speed and local
Ca2+ release kinetics, the cell was sectioned
into 0.85-µm-thick slices, and fluorescence intensity data were
extracted from each slice. Peak amplitude, rate-of-rise, as well as the
time at which 50% peak was reached were extracted from each trace and
were plotted against cell length (Fig. 8) (see Simpson et al.
1997
; Yagodin et al. 1994
for methods). For
reasons of clarity only data from the end foot to the cell body region
is shown in Fig. 8. This analysis revealed that the peak amplitude and
rate of rise of the Ca2+ response were nonuniform
through the cell, with some regions of the cell showing higher
Ca2+ release rates than found in surrounding
regions (arrows). At these sites, the local signal amplitude was higher
(Fig. 8), and the rate of rise of the response was steeper (not shown).
Thus the kinetic profile of these regions appeared similar to the
specializations (wave amplification sites) previously observed in
cultured glial cells (Simpson and Russell 1996
;
Yagodin et al. 1994
).
A plot of the delay times to 50% peak of the signal against cell
length localizes wave initiation sites in the cell. At these sites, the
wave reaches 50% of maximum amplitude sooner than the surrounding
regions and appears as minima in this plot (Fig. 8, ). In this cell,
the wave initiates at approximately 18 µm from the end-foot
(*). Wave speed was calculated from the slope of the line
through the data points (Fig. 8). While the average wave speed was
approximately 18 µm/s, waves slowed and sped up during propagation
through different regions of the cell. Wave speed varied between 10 and
40 µm/s along processes. On average, waves took about 4-10 s to
travel from end-feet to the cell body. From the initiation sites, waves
propagated in both directions (see Fig. 8). In most of our experiments,
waves initiated in the end-foot region of Müller cells and
propagated toward the cell body. Of the 56 separate cells analyzed,
55% of waves initiated at the end-feet, 39% in the process region,
and only in 5% of cells, waves initiated at the cell soma.
The peaks in Ca2+ release amplitude or the wave
speed observed along Müller cell processes did not correspond to
the peaks in the fluorescence intensity profile of the process (data
not shown). This observation suggests that irregular process shape and
thus variations in dye volume along processes do not contribute to the
differences in Ca2+ release kinetics we measured.
Such differences in fluorescence intensity along processes were
corrected by the F/F0
normalization procedure. Furthermore, transient dynamic changes in
shape were never observed during purinergic stimulation, and plots of
F/F0 in unstimulated
processes did not show any peaks. Taken together, these data indicate
that, like in cultured glial cells, Ca2+ wave
propagation in Müller cells in situ is achieved through a series
of wave amplification sites where the local Ca2+
release function is specialized.
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DISCUSSION |
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In this study, we analyzed agonist-evoked Ca2+ wave propagation within Müller cell processes in slices of adult rat retinae. Using a novel indicator loading technique, we have discretely filled Müller cells with calcium green and measured Ca2+ transients evoked by bath-applied agonists. Immunocytochemical identification of cells using anti-GFAP antibodies clearly showed that the cells loaded with Ca2+ indicator were indeed Müller glial cells (Fig. 1). We found that purinergic agonists were very potent agents for eliciting Ca2+ responses in Müller cells. These Ca2+ responses initiated in specific regions of the Müller cell and propagated bi-directionally at nonuniform rates through cellular processes. There appeared to be specific cellular locations at which Ca2+ release rate and amplitude were significantly higher suggesting wave amplification sites.
Newman and Zahs (1997) have recently shown that in whole
flat mount preparations of rat retina, exogenous addition of agonists as well as electrical stimulation can evoke Ca2+
waves that propagate between Müller cell end-feet. In addition, they showed that these Ca2+ waves modulate
ganglion cell excitability (Newman and Zahs 1998
). We
were interested to know whether these Ca2+ waves
impinge on signaling within the retina, specifically, do Ca2+ waves travel from the end-feet through the
entire length of Müller cells to the photoreceptor layer of the
retina. Our present study shows that Ca2+ waves
propagate along Müller cell processes and this propagation occurs
by regenerative Ca2+ release at specialized
release sites along the processes. These results suggest that
Müller cells may indeed act to modulate signaling throughout the
thickness of the retina.
It was important to test whether the Müller cell
Ca2+ responses are a direct consequence of
stimulation of purinergic receptors on Müller cells or if the
waves are due to indirect stimulation of Müller cells by synaptic
release of transmitter resulting from purinergic stimulation of neural
networks. We provide evidence that the responses are due to direct
stimulation of Müller cells. Removal of
Ca2+ from the bathing medium that blocks
vesicular transmitter release (Llinas 1982) did not
abolish the Ca2+ waves. Furthermore, blockade of
action potentials with tetrodotoxin (TTX, 1.0 µM) did not inhibit the
agonist-evoked Ca2+ signals. While it can be
argued that under conditions of extracellular Ca2+ removal and TTX blockade, cellular signaling
can occur through generation of graded potentials using surface charge
effects; the more direct interpretation of our results is that
purinergic agonists act directly on Müller cells. Finally, a
graded dose-response relationship was obtained with increasing agonist
concentrations, which further suggests that the primary
Ca2+ response occur in Müller cells.
The fact that complete removal of Ca2+ from the
bathing medium did not abolish Ca2+ waves, while
depleting intracellular stores of Ca2+ did
abolish waves, suggests that the ATP- and adenosine-evoked Müller
cell Ca2+ waves were due to stimulation of
metabotropic purinergic receptors, which include P2Y and P1 subtypes.
Previous studies have resulted in classification of the P2Y
purinoceptor subtypes using rank order of agonist potency (see for
reviews Burnstock 1997; Dixon 2000
;
North and Barnard 1997
). The rank order of potency in
Müller cells in our experiments was 2 MeSATP = 2MeSADP > ADP > ATP
meATP = UTP > UDP,
suggesting that Müller cells in rat retina express
P2Y1 subtype of purinergic receptors.
Furthermore, since Ca2+ waves were also evoked by
the P1 agonist, adenosine, and these waves were blocked by DMPX and not
by PAPCX or DPCPX, we conclude that adenosine is acting via stimulation
of P1A2 purinergic receptors in Müller
cells. The fact that the P2X-specific purinergic agonist (
meATP)
failed to elicit [Ca2+]i
in Müller cells in thapsigargin-treated and untreated retinal slices suggests that Müller cells may not express this subtype of
purinoceptors. This result, however, should be interpreted with
caution, since the P2X receptor channels are known to undergo fast
inactivation, and measurements of membrane currents by patch-clamp methods are required to verify this possibility. While retinal neurons
have been shown to express P2X2 purinoceptors (Greenwood et al.
1997
; Neal and Cunningham 1994
), recent
studies have shown that Müller cells in adult rat retinae also
express P2X purinergic receptors (Neal et al.
1998
).
Ca2+ waves evoked by purinergic receptors
propagate bidirectionally from initiation sites within Müller
cell processes. Analysis of Ca2+ waves revealed
the presence of discrete Ca2+ release sites where
the magnitude of Ca2+ release was significantly
higher than in surrounding regions of the cell. Nonuniform distribution
of purinergic receptors on Müller cells processes alone could not
explain the observation of specialized release sites, but could
contribute to wave initiation. Inositol 1,4,5-trisphosphate
(InsP3) generated during receptor activation
diffuses extremely rapidly in cytoplasm (D 180 µm2/s) (Allbritton et al.
1992
), and any gradient in InsP3 levels will be expected to dissipate rapidly. Wave propagation on the other
hand occurs at rates of up to 40 µm/s. In theory, localized patches
of purinergic receptors on Müller cell processes together with
focal release of agonists could elicit localized wave initiation. Whether such an arrangement exists in the retina needs to be
investigated. The regenerative nature of wave propagation that we
observed, however, suggests specialized Ca2+
release sites along Müller cell processes where
Ca2+ release function is enhanced.
The phenomenon of specialized Ca2+ release sites
is remarkably similar to observations in cultured glial cells of the
astrocytic and oligodendrocyte lineages (Simpson et al.
1997; Yagodin et al. 1994
). The specialized
Ca2+ release sites serve as wave amplification
sites, and mathematical models have provided evidence that they might
behave as partially coupled cellular oscillators being coupled by the
diffusing Ca2+ ions (Li and Rinzel
1994
; Roth et al. 1995
). We and others have shown that wave amplification sites are endowed with a number of
structural specializations, including high-density patches of
IP3Rs, sarcoendoplasmic reticulum
Ca2+ pumps (SERCA), beadlike concentration of
intraluminal calreticulin, and close apposition with one or more
mitochondria (Rizzuto et al. 1998
; Simpson and
Russell 1996
). Indeed, fluorescence-based immunocytochemistry
of IP3Rs in Müller cells in retinal slices revealed beadlike staining along processes (data not shown). Thus it is
likely that cellular specializations similar to what has been shown in
cultured glial cells (Simpson et al. 1997
) may support the enhanced local Ca2+ release observed in
Müller cells in situ.
We found that a majority of Ca2+ waves started at
the end-feet, or at the apical end of Müller cells and propagated
toward cell bodies. This observation is different from results obtained in isolated dispersed tiger salamander Müller cells, where wave initiation sites were most often observed in the apical region (Keirstead and Miller 1997). This may be due to
differences in the analysis paradigm, species differences, or possible
damage to cells during the dissociation procedure (Newman
1993
). We observed many sites of wave initiation in any given
cell, where a Ca2+ response begins before the
arrival of a wave propagated from other areas of the cell. Multiple
wave initiation may be explained by patchy distribution of agonist
receptors on the cell membrane, the amount of second messenger formed
locally in relation to the internal cell volume, high-density local
patches of IP3Rs, or a lower threshold for
activation of IP3Rs in local regions of the cell.
Without adequate receptor distribution studies, it is difficult to
differentiate these and other possibilities.
There is strong experimental evidence in both the peripheral and
central nervous systems that suggests that ATP released from synaptic
terminals participates in modulation of synaptic plasticity (Burnstock 1990; Edwards et al. 1992
;
Illes and Norenberg 1993
; Sawynok et al.
1993
). Similarly, ATP released from glial cells has also been
shown to elicit glial Ca2+ signals that propagate
both within cells and between cells in vitro (Cotrina et al.
2000
; Guthrie et al. 1999
). ATP is one of the
several different neurotransmitters present in the retina, and indirect
evidence suggests that ATP could be co-secreted with acetylcholine in
rabbit retina (Neal and Cunningham 1994
). It is not
clear what role such purinergic activation of Müller cells plays
in modulating activity of retinal neural networks. Furthermore, the
primary source of ATP release is not clearly understood. Our results
show that ATP is an important signaling molecule in the retinal
Müller cells, while adrenergic and glutamatergic agonists were
much less efficacious. In a recent study, Newman and Zahs (1997)
showed that initiation of Ca2+
waves in Müller cells by mechanical stimulation alters
excitability of ganglion cells in the vicinity of the wave. This
observation, taken together with studies in other brain regions
(Duffy and MacVicar 1995
; Grosche et al.
1999
; Porter and McCarthy 1996
), strongly
suggests that Ca2+-based excitability in glial
cells may have important implications for excitability of neighboring
neuronal circuits. Our present study clearly demonstrates that retinal
Müller cells possess Ca2+-based
excitability similar to glial cells in other brain regions. In
addition, we show that cellular mechanisms supporting regenerative wave
propagation are remarkably similar in Müller cells in situ and in
cultured glial cells and suggest a role for Müller cells in
modulating signaling through the retina.
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ACKNOWLEDGMENTS |
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We thank Dr. Laurel Haak for critical reading of the manuscript and Dr. Michael Yvonne for discussions on the retinal preparation.
Present address of Y. Li: Dept. of Pharmacology, Uniformed Services University of Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20014.
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FOOTNOTES |
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Address for reprint requests: J. T. Russell, Laboratory of Cellular and Molecular Neurophysiology, NICHD, NIH, Bldg. 49, Rm. 5A-78, Bethesda, MD 20892 (E-mail: james{at}helix.nih.gov).
Received 8 May 2000; accepted in final form 11 October 2000.
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REFERENCES |
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