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Vol. 53, Issue 3, 564-572, March 1998
and
Inhibitory Transmitters
Departments of Ophthalmology and Visual Sciences (Q.C., P.D.L., C.R.), Psychiatry (Q.C., J.W.O., T.A.), and Anatomy and Neurobiology (P.D.L., C.R.), Washington University School of Medicine, St. Louis, Missouri 63110
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Summary |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Rapidly triggered excitotoxic cell death is widely thought to be due to
excessive influx of extracellular Ca2+, primarily through
the N-methyl-D-aspartate subtype of
glutamate receptor. By devising conditions that permit the maintenance
of isolated retina in the absence of Ca2+, it has become
technically feasible to test the dependence of excitotoxic
neurodegeneration in this intact neural system on extracellular
Ca2+. Using biochemical, Ca2+ imaging, and
electrophysiological techniques, we found that (1) rapidly triggered
excitotoxic cell death in this system occurs independently of both
extracellular Ca2+ and increases in intracellular
Ca2+; (2) this cell death is highly dependent on
extracellular Cl
; and (3) lethal Cl entry
occurs by multiple paths, but a significant fraction occurs through
pathologically activated 
-aminobutyric acid and glycine receptors.
These results emphasize the importance of Ca2+-independent
mechanisms and the role that local transmitter circuitry plays in
excitotoxic cell death.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Although
early experiments indicated that excitotoxic neurodegeneration was due
to Na+ and Cl entry into
cells on overactivation of glutamate receptors (Price et
al., 1985
; Rothman, 1985), the role of Ca2+
entry has been the focus of much of the work in this field (Choi, 1994). For example, studies by Choi (1985, 1987) with cultured embryonic rodent cortical cultures, Rothman et al. (1987)
with cultured embryonic hippocampal neurons, and Garthwaite and
Garthwaite (1986) and Garthwaite et al. (1986) with
cerebellar slices have provided evidence that in these systems,
neuronal death is triggered by the excessive entry of extracellular
Ca2+ on stimulation of ionotropic glutamate
receptors, particularly the Ca2+-permeant NMDA
receptors (McBain and Mayer, 1994). In fact, rapidly triggered neuronal
death in vitro on activation of non-NMDA receptors has been
reported to occur only in those relatively rare cultured cortical cells
expressing Ca2+-permeant AMPA receptors (Weiss et
al., 1994; Gottron et al., 1995). This is an
attractive mechanism because Ca2+ is an important
intracellular signaling molecule and plausible hypotheses explaining
cellular dysfunction on disruption of intracellular Ca2+ homeostasis have been proposed (Coyle and
Puttfarcken, 1993; Choi, 1994; Beal, 1995; Mattson et al.,
1995; Dawson and Dawson, 1996; White and Reynolds, 1996). The excessive
entry of Na+ and Cl into
neurons with overstimulation of any of the ionotropic glutamate receptors is usually described as contributing to morphological epiphenomena (cell swelling) but not to cell death (Choi, 1994).
Is this true for all neurons? In some systems, it has been difficult to
test the role of extracellular Ca2+ in
excitotoxicity because reductions in the concentration of extracellular
Ca2+ led to severe neuronal pathology even in the
absence of exogenous excitotoxins (Price et al., 1985;
Freese et al., 1990; Lehmann, 1990). This
Ca2+-omission injury has been ascribed to
membrane destabilization (Goldberg and Choi, 1993) or glial death
(Freese et al., 1990), but the mechanism remains obscure.
Several studies have indicated that extracellular
Cl may play an important role in excitotoxic
cell death in some neuronal systems. For example, Zeevalk et
al. (1989) showed that acute excitotoxic damage induced by either
NMDA or non-NMDA receptor agonists could be attenuated by removing
extracellular Cl or including the
Cl channel blockers DIDS or furosemide. Kato
et al. (1991), using cultured cerebellar granule cells,
demonstrated that KA-induced delayed LDH release was completely
prevented in the absence of extracellular Cl.
These authors also demonstrated that Ca2+
omission during the period of KA exposure did not attenuate cell death
but instead increased its rate of development.
Although these studies suggested that there may be
Ca2+-independent mechanisms of excitotoxic cell
death in which extracellular Cl may play an
important role, they have not been definitive. For example,
Cl channel-blocking agents are notoriously
nonspecific drugs (Cabantchik and Greger, 1992), and some have been
shown to directly block glutamate receptors (Lerma and Martin del Rio,
1992). Similarly, impermeant anions used as Cl
substitutes in Cl removal experiments also may
be glutamate receptor blockers. Replacement anions are necessarily used
at very high concentrations (>100 mM), so they need not be
very potent antagonists to provide substantial receptor blockade.
Ca2+-omission experiments have not used prolonged
periods of Ca2+ removal because of the toxicity
this causes, and whether manipulations in extracellular
Ca2+ can affect intracellular free
Ca2+ levels has not been addressed in any of
these studies.
We reexamined the ionic basis of excitotoxicity in an isolated, intact
retina preparation. In addition to using biochemical and histological
measures of excitotoxicity, we used whole-cell patch-clamping in a
retinal slice preparation to assess receptor activation and blockade
and Fura-2 Ca2+ imaging to examine intracellular
free Ca2+ during the experimental manipulations.
We conclude that excitotoxicity in this preparation is independent of
extracellular Ca2+ and dependent on extracellular
Cl and that excitotoxicity depends on
Cl-gating inhibitory transmitters present in
retinal circuits.
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Materials and Methods |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Chick embryo retina preparation.
The chick embryo retina
preparation protocols have been described previously (Romano et
al., 1995). Briefly, eyes were isolated from chick embryos
(15 ± 1 day old), placed in standard BSS at 0-4°, enucleated,
and cut into thirds. The retina segments then were isolated from the
rest of the ocular tissues and transferred to a 7-ml glass vial
containing 1 ml of the standard BSS saturated with 95%
O2/5% CO2. The standard
BSS contained 124 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM
MgCl2, 1.25 mM
KH2PO4, 22 mM
NaHCO3, 20 mM glucose, and 30 µM phenol red, pH 7.4. For experiments in the absence of
Ca2+, the BSS was modified so 2 mM
MgCl2 was substituted for
CaCl2 and 0.1 mM EGTA was added.
22 hr. An aliquot of the medium was taken at the end of the 22-hr
incubation for measuring delayed toxicity. For the measurement of
residual LDH remaining in the retinal tissue (as part of the total LDH
in a retinal segment), the retina was treated with Triton X-100 (0.2%)
and lysed by freezing and thawing. The lysate was centrifuged, and an
aliquot of the supernatant was taken for LDH assay.
LDH assay. The cocktail for LDH assay contained 0.1 mg/ml NADH, 2 mM sodium pyruvate, 0.1 M potassium phosphate, pH 7.4, and an aliquot of the BSS medium containing LDH released from the retina. LDH activities were measured spectrophotometrically by the rate of NADH disappearance. The assay has a >20-fold linear range. Levels of LDH in the aliquots taken for the acute and delayed releases are expressed as a percentage of the total LDH in a retinal segment, which is the sum of the LDH from the aliquots of the two releases and the retinal lysate. All the data in this report reflect total LDH release as a fraction of total retinal LDH content.
Within each experiment, each condition was tested on triplicate retinal segments, and all experiments were repeated at least three times. All reported differences are statistically significant (Student's t test or analysis of variance and multiple-comparison Tukey's test) but values for p are shown only in cases in which the differences were not apparent by simple inspection.Ca2+ imaging.
Intracellular
Ca2+ concentrations in the retina were measured
with digital fluorescence microscopy (Wong, 1995b). The retinal dissection was performed as described above. After the retina was
isolated, a segment of the retina was cut and mounted (ganglion cell
side up) onto a filter (HABP 045; Millipore, Bedford, MA). For whole
mounts, the retinal segment was transferred to a chamber containing the
incubation medium (BSS), described above, with the addition of Fura-2
AM (2 µM) and pluronic acid (0.001%), and incubated for
1 hr at 30-35°. For retinal slices, the retinal segment on a piece
of Millipore filter was sliced with a tissue chopper at 150-µm
intervals before Fura-2 incubation. The tissue then was washed and
transferred to a temperature-controlled recording chamber on the stage
of a fluorescence microscope. Imaging was performed at 25° to mimic
the standard conditions of the toxicity experiments.
, 1995b).
Electrophysiology.
Membrane currents were measured by
whole-cell patch recording of ganglion cells in the salamander retinal
slice. The procedures for preparation and recording of the retinal
slices have been described in detail previously (Lukasiewicz and
Werblin, 1994). Briefly, a small square was cut from isolated retina,
placed photoreceptor side down on a piece of Millipore filter, and
sliced with a tissue chopper at 150-µm intervals. The sliced
retina/filter complex was transferred to the recording chamber and
immobilized by embedding the ends of the filter into two rails of
vacuum grease that had been laid down in the chamber. The retinal slice
was viewed with a Nikon Optiphot 2 microscope modified to have a fixed
stage. A Nikon 40× long working-distance water-immersion objective
with Hoffman Modulation contrast (Modulation Optics, Greenvale, NY) was
used for visualization of cells on the surface of the slice.
.
Recordings were obtained with a Dagan 3900A (Minneapolis, MN)
patch-clamp amplifier. The data were digitized and stored with a 33-MHz
386 PC using a Labmaster DMA data acquisition board (Scientific Solutions, Solon, OH). Responses were filtered at 2 kHz with the four-pole Bessel low-pass filter and sampled at 0.7-2 kHz. Electrodes of <5 M
resistance were pulled from borosilicate glass
(TW150F-4; World Precision Instruments, Sarasota, FL) with a
Sachs-Flaming puller (Sutter Instruments, Novato, CA). The measured
series resistances were typically 15-25 M. The
magnitude of the series resistance compensation, read from the Dagan
3900A compensation counting dial, was 5-10 M. Patchit
software (Geo. Grant, Sommerville, MA) was used to generate voltage
command outputs, acquire data, gate the drug perfusion valves, and
trigger the Picospritzer. Data were analyzed using Tack (Geo. Grant,
Sommerville, MA) and expressed as mean ± standard deviation.
Fertilized chick eggs were purchased from SPAFAS (Roanoke, IL). KA and
MK-801 were obtained from Research Biochemicals (Natick, MA). GYKI
53655 [1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine] was kindly provided by Eli Lilly and Co. (Indianapolis, IN). All other
reagents were obtained from Sigma Chemical (St. Louis, MO).
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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KA-induced excitotoxicity in isolated retina is independent of extracellular Ca2+. If extracellular Ca2+ is important for excitotoxic neurodegeneration, the application of an excitotoxic agonist in the absence of extracellular Ca2+ should eliminate, or attenuate, the damage. However, as has been observed in many other neuronal preparations, the incubation of E14 chick retinas in the absence of Ca2+ leads to massive neuronal degeneration (Fig. 1B), precluding the experimental use of this simple manipulation. Two different strategies were used to overcome this limitation.
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), a selective antagonist of AMPA-preferring
glutamate receptors, was ineffective against
Ca2+-omission toxicity.
Because KA-induced toxicity is not blocked by MK-801, the effect of
Ca2+ omission on KA-induced excitotoxicity could
be examined by including MK-801 in the incubation medium. The omission
of extracellular Ca2+ did not attenuate
KA-induced damage to E14 chick retinas; as with the E11 retinas, the
toxicity instead was exacerbated (Fig. 1C). Even in the absence of
Ca2+, KA-induced toxicity could be prevented
completely by GYKI 53655, indicating that the same receptor mechanisms
are operative despite the altered ionic conditions. These data indicate
that KA-induced excitotoxicity is not dependent on the entry of
extracellular Ca2+.
These experiments demonstrated clearly that AMPA/KA receptor-mediated
cell damage can occur in the presence or absence of extracellular
Ca2+ and, in fact, is more severe in the absence
of Ca2+. However, increases in intracellular
Ca2+ by mobilization of intracellular stores
during KA exposure may occur in the absence of
Ca2+ influx, and this could contribute to the
mechanism of toxicity. To explore this possibility, intracellular free
Ca2+ concentrations in ganglion cells in whole
mounted retinas (Fig. 2) or in amacrine
and bipolar cells in retinal slices (not shown) were monitored during
and after KA treatment, using the ratiometric fluorescent
Ca2+ indicator Fura-2. As shown in Fig. 2, in the
presence of 2 mM extracellular Ca2+,
KA caused a substantial and prolonged rise in intracellular Ca2+ in these cells. This increase was nearly
abolished when Ca2+ was removed from the
extracellular medium. This was not correlated with a decrease in
KA-induced toxicity but with the exacerbation demonstrated above.
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KA-induced excitotoxicity in isolated retina is dependent on extracellular Na+. To clarify further the mechanisms for KA-induced toxicity, we focused our remaining investigations on Ca2+-independent mechanisms. One such mechanism, excessive Na+ influx, was studied in Na+-substitution experiments. Choline or N-methyl-D-glucamine was substituted for Na+ in the incubation medium. Unlike Ca2+ omission (Fig. 1B), Na+ omission alone did not cause any cell damage (Fig. 3A). Na+ omission during exposure to a low concentration of KA (32 µM) blocked toxicity but was ineffective against a higher concentration of KA (320 µM, Fig. 3B). One potential explanation for the failure of Na+ omission to protect against KA toxicity at high concentrations is that high concentrations of KA activate some Ca2+-permeable channels and that the excessive Ca2+ influx through these channels might cause the toxicity. This did not prove to be the case because simultaneous omission of Na+ and Ca2+ did not protect against KA toxicity at 320 µM (Fig. 3B).
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KA-induced excitotoxicity in isolated retina is dependent on
extracellular Cl.
Effective protection against
KA-induced toxicity in the retina came from Cl
substitution (Fig. 4). With 75% of the
extracellular Cl replaced by methylsulfate,
toxicity of KA at either of the two concentrations (32 and 320 µM) was nearly blocked. Similar neuroprotection was
observed using the impermeant Cl substitutes
isethionate and gluconate but not the permeant anion bromide (data not
shown).
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substitution compared with Na+ substitution was
surprising. To verify that the protection afforded by this manipulation
was not due to a direct inhibitory action of the
Cl substitute methylsulfate on the receptor,
the effect of methylsulfate on KA-elicited currents was examined using
whole-cell patch-clamp recordings of salamander retinal ganglion cells
in an intact retinal slice preparation. We chose to use salamander
retina because it provides a robust preparation that survives for long
periods of time and is technically amenable to stable, long-duration,
whole-cell patch-clamping. Importantly, most of the major findings that
have come out of studies of this preparation have been confirmed in other species, including birds and mammals. For example, the
transmitters utilized by distinct subtypes of retinal neurons are
similar across species (e.g., photoreceptors and bipolar cells are
glutamatergic, amacrine cells are either glycinergic or GABAergic), and
the retinal circuitry of salamander is similar to chick as well as to
other vertebrate retinas. Fig. 5A shows
the response of a ganglion cell to KA in the presence or absence of
methylsulfate. With membrane holding potential of 
75 mV, substitution
of methylsulfate for Cl did not inhibit
KA-elicited currents. Instead, it enhanced the currents in all 14 cells
recorded. This is strong evidence that methylsulfate does not block the
receptor mediating the action of KA.
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37 mV and that replacement of 75%
of the Cl by methylsulfate shifted the reversal
potential to 12 mV (Fig. 5B). These results indicate that KA
activates a Cl conductance in these ganglion
cells. The enhancement of KA-elicited currents by the substitution of
methylsulfate for Cl can be readily explained
in terms of an increase in Cl efflux because
the equilibrium potential for Cl becomes more
positive with the reduction of extracellular
Cl. Because there is no evidence that
endogenous retinal ionotropic glutamate receptors are permeable to
Cl, it seems likely that activation of the
Cl current must be indirect. One potential
explanation for the increased Cl conductance in
ganglion cells in this slice preparation would be that KA stimulates
GABAergic and/or glycinergic amacrine cells that synaptically activate
GABA and/or glycine receptors on ganglion cells, leading to the
increased Cl conductance. Consistent with this
explanation, the combined application of bicuculline and strychnine,
GABA and glycine receptor antagonists, respectively, blocked the
Cl current and shifted the reversal potential
of KA-elicited currents to near 0 mV, which is expected for the typical
cation current gated by AMPA/KA receptors (Fig. 5C).
These results suggest that KA treatment of isolated retina, an intact
neural network that contains a diverse population of neurons, leads to
pathological release of inhibitory as well as excitatory transmitters.
These transmitters then activate their receptor-associated
Cl channels, thus contributing to excessive
Cl influx in the neurons. Because the
ion-substitution experiments described above indicated that
extracellular Cl is essential for excitotoxic
cell death, block of these receptors should be protective against
excitotoxicity. Consistent with this proposal, when both GABA and
glycine receptors were blocked by combined application of picrotoxin
(which blocks both GABAA and the retina-selective
GABAC receptors; Lukasiewicz, 1996) and
strychnine, the toxicity of 320 µM KA was attenuated
partially (Fig. 5D). Interestingly, individual blockade of GABA or
glycine receptors did not attenuate KA toxicity.
Extracellular Cl also is important for excitotoxic
neurodegeneration caused by NMDA receptor overactivation.
Are the
ionic mechanisms mediating KA-induced toxicity also operative in NMDA
receptor-mediated forms of excitotoxic neurodegeneration in the chick
retina? When retinas were incubated with NMDA (500 µM) or
in the absence of Ca2+ for 60 min (which as shown
results in NMDA receptor-mediated toxicity), substantial
neurodegeneration occurred (Fig. 6, A and B). The toxicity induced by NMDA treatment was almost entirely eliminated when either Na+ or
Cl was removed during the NMDA exposure, as was
toxicity caused by Ca2+ omission. The combination
of bicuculline and strychnine also provided significant protection
(Fig. 6, C and D), indicating a role for GABA and glycine in both of
these forms of toxicity. Bicuculline alone provided complete protection
against the toxicity resulting from 1 hr of Ca2+
omission, whereas strychnine was without effect (Fig. 6D).
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The evidence presented here indicates that rapidly triggered
excitotoxic cell death is a Ca2+-independent
process in some neurons and that it shows a remarkable dependence on
extracellular Cl. Some of the lethal
Cl entry occurs through neurotransmitter-gated
Cl channels activated by pathologically
released inhibitory transmitters, pointing to a role for GABA and
glycine, as well as glutamate, in excitotoxicity. The differences among
neuronal populations in the mechanisms of excitotoxic cell death
therefore depend on both the intrinsic properties of the neurons (i.e.,
which receptors, channels, and so on they express) and the nature of
the network in which they are embedded (i.e., the complement of
transmitters and other pathological environmental elements they will
encounter during an excitotoxic insult).
Ca2+ independence of excitotoxicity and
Ca2+ omission-induced damage.
In a previous study
providing evidence for Ca2+-independent,
Cl-dependent excitotoxic cell death (Kato et
al., 1991), Ca2+ was absent from the
incubation medium only during the period of excitotoxin exposure. It
remained possible that Na+ loading during the
agonist exposure led to a later marked increase in intracellular
Ca2+ due to greatly enhanced
Na+/Ca2+ exchange. By using
conditions permitting incubation in the absence of
Ca2+ for the duration of the 24-hr experimental
period, the current study does not have this limitation. In addition,
Ca2+ imaging experiments documented that there
was minimal or no increase in free intracellular
Ca2+ during the agonist exposure, or the period
immediately after, when the retinas were maintained in the
Ca2+-free medium (Fig. 2); nevertheless, the
cells died.
). Second, if the
integrity of the cell membrane were breached, the rapid increase of
intracellular Ca2+ to extracellular levels would
have been immediately apparent during the imaging experiments. This was
never observed. It is likely that there are roles for cell swelling and
intracellular osmotic/ionic imbalances in cell death, but the precise
mechanism involved is neither trivial nor apparent and is not likely to be rapid mechanical failure of the cell membrane.
Ca2+ omission triggered excitotoxicity in the E14
retina dependent on NMDA receptor activation (Figs. 1B and 6C). The E11
retina is not susceptible to Ca2+-omission injury
(Fig. 1A) or NMDA-mediated excitotoxicity (data not shown), which is
consistent with the requirement for NMDA receptor overactivation in
Ca2+-omission injury. Similar observations
pertaining to Ca2+-omission injury in other types
of neurons have been made by other investigators (McCaslin and Smith,
1990; Eimerl and Schramm, 1991). The precise mechanism of this
Ca2+ omission-induced excitotoxicity is unknown
and was not the explicit subject of this study. Nevertheless, the
mechanism presumably involves the induction of hypersensitization of
NMDA receptors, a pathological release of glutamate, or both, upon
lowering extracellular Ca2+. Evidence for both
these possibilities has been gathered in other systems (Clark et
al., 1990; McCaslin and Smith, 1990; Legendre et
al., 1993; Rosenmund et al., 1995). The ability
of bicuculline to prevent Ca2+ omission-induced
(Fig. 6D) damage indicates that overactivation of GABA receptors plays
an important role in this form of toxicity.
Ca2+ omission increased KA-induced delayed LDH
release (Fig. 1C). This probably was due to the increased
Na+ conductance of KA-activated channels in the
absence of Ca2+ because extracellular
Ca2+ modulates KA-activated currents by impeding
the permeation of monovalent cations (Gu and Huang, 1991).
Because NMDA receptor antagonists must be present to completely block
Ca2+-omission injury and they also provide
complete or near-complete blockade of NMDA receptor-mediated
excitotoxicity, we could not use this method to investigate the
Ca2+ dependence of this NMDA receptor-mediated
injury. However, the marked neuroprotection provided by
Cl omission against NMDA treatment (Fig. 6A)
and the fact that Ca2+ omission injury itself is
NMDA receptor mediated are not easily explained by postulating a
Ca2+-dependent mechanism for NMDA
receptor-dependent toxicity in this system.
Another potential means for examining the Ca2+
dependence or independence of cell death is by use of the
membrane-permeant "prochelator" BAPTA (Tymianski et al.,
1993). On hydrolysis by intracellular esterases, the acid is formed,
which (1) cannot leave the cell and (2) is a Ca2+
chelator. Therefore, extracellular application of this substance should
lead to a large increase in the Ca2+-buffering
capacity of the cytoplasm and theoretically should protect against
Ca2+-dependent, but not
Ca2+-independent, forms of excitotoxicity. We
have found that BAPTA acetoxymethyl ester (20-100 µM)
does not protect against KA toxicity, which is consistent with our
other findings documenting a Ca2+-independent
mechanism for KA-induced cell death (Q. Chen and C. Romano,
unpublished). However, because several investigators have reported that
BAPTA acetoxymethyl ester is not a very effective neuroprotectant
against agonist-induced excitotoxicity (Dubinsky, 1993; Abdel-Hamid and
Tymianski, 1997), even in systems in which cell death is known to be
Ca2+ dependent, this evidence, although
consistent, is not compelling.
Na+, Cl, and inhibitory transmitters in
excitotoxic cell death.
The mechanism for excessive
Na+ influx in excitotoxicity is understood
readily in terms of the conductance of
Na+-permeable receptor channels activated by all
excitotoxins. The mechanisms for excessive Cl
influx triggered by excitotoxins are less direct. Rothman and Olney
(1987) proposed a general mechanism by which excessive
Na+ influx would draw Cl
into cells to restore ionic balance. However, they did not identify specific ion channels, transporter systems, or receptors that might
mediate the influx of Cl. Zeevalk et
al. (1989) reported that the
Cl/bicarbonate anion channel blocker DIDS
protected the chick retina from acute excitotoxicity. We have observed
that DIDS also reduced KA-induced delayed LDH release in our retinal
preparation (data not shown). However, the effect of DIDS against
excitotoxicity is difficult to interpret because we found that DIDS
completely inhibited KA-induced currents in ganglion cells of
salamander retina, suggesting it is a receptor blocker (data not
shown). The blockade of GABA release by DIDS observed by Zeevalk
et al. 1989) also indicates that DIDS may be operating as a
receptor blocker. The poor specificity of other
Cl antagonists has been described (Cabantchik
and Greger, 1992; Lerma and Martin del Rio, 1992).
influx in ganglion cells
during brief exposure to KA (i.e., puffs). The straightforward
implication of this is that KA treatment induced the release of GABA
and glycine receptor agonists from nearby neurons. It has been
established that KA treatment of intact chick retina (Zeevalk et
al., 1989) or chick retinal cells in culture (Ferreira et
al., 1994) leads to GABA release. Much of this release is
independent of extracellular Ca2+ (Ferreira et
al., 1994) and presumably is mediated by the reversed operation of the voltage-dependent GABA transporter. Such
Ca2+-independent release of GABA from retinal
neurons has been well documented for several classes of retinal neurons
(Schwartz, 1987; O'Malley et al., 1992). Taurine also is
released on KA from intact chick retina (Zeevalk et al.,
1989), and this amino acid is well known to be an agonist at some
subtypes of inhibitory (Cl gating) glycine
receptors (Schmieden et al., 1992).
Consistent with the hypothesis that excitotoxic cell death may depend
on overactivation of these ligand-gated Cl
channels, block of GABA and glycine receptors by picrotoxin and strychnine during KA exposure provided a partial protection against excitotoxicity in the chick retina (Fig. 5D). The fact that protection afforded by the inhibitors was less than that provided by
Cl omission suggests involvement of other modes
of Cl entry. The exposure to KA was prolonged
(30 min) during the toxicity experiments and very brief during the
electrophysiological experiments. Perhaps Cl
currents (other than the ligand-gated ones we observed) were not seen
in the electrophysiological experiments because they develop slowly.
NMDA receptor-mediated toxicity was also Cl
dependent and blocked by bicuculline and strychnine (Fig. 6),
suggesting a more general involvement of GABA and glycine in
excitotoxic injury.
We propose the model in Fig. 7 to
illustrate mechanisms mediating the Cl influx
triggered by excitotoxins. Persistent activation of ionotropic glutamate receptors, in addition to causing Na+
influx, could persistently activate GABA/glycine-gated
Cl channels (via excess GABA/glycine release),
causing excessive Cl entry into the neurons
already subjected to excessive Na+ entry. Other
pathways for Cl entry would be activated,
perhaps with slower time courses. This would create osmotic conditions
conducive to obligatory water entry and cell swelling. Prolonged and
massive NaCl influx and water uptake may eventually lead to cell death.
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) are also responsible for
excitotoxin-induced neurodegeneration defined biochemically in terms of
the delayed LDH release. These findings are in contrast to observations
in cultured cerebrocortical and hippocampal neurons in which NMDA receptors mediate a Ca2+-dependent delayed
toxicity (Choi, 1987; Rothman et al., 1987). These apparent
differences cannot be readily explained in terms of differences in
methodological approach and more likely reflect some intrinsic
differences among neurons or neural networks in their ability to cope
with a specific ionic overload produced during excitotoxin exposure.
These intrinsic differences may be essential for our understanding of
multiple mechanisms for excitotoxicity.
The results presented here demonstrate that Cl
influx plays an essential role in excitotoxic degeneration of the chick
retina, but it remains to be determined how the
Cl ions contribute to the toxic process.
Cl transmembrane movement is important for
membrane excitability, intracellular pH regulation, and cell volume
control. We can only speculate, at present, that prolonged disruption
in any of these functions may be detrimental to the neuron. If
excessive Cl influx proves to be a mechanism
that is generally operative in excitotoxicity, it may be possible to
develop therapeutic approaches to the management of neurological
disorders based on this principle.
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Acknowledgments |
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We thank Dr. R. O. Wong (Department of Anatomy and Neurobiology, Washington University School of Medicine) for generously providing us with her expertise and device for the Ca2+ imaging experiments.
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Footnotes |
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Received August 4, 1997; Accepted November 26, 1997
This work was supported by Grants EY08089, EY08922, EY09370, EY02687, and DA07261 and an unrestricted grant from Research to Prevent Blindness, Inc.
Send reprint requests to: Carmelo Romano, Ph.D., Department of Ophthalmology & Visual Sciences, Campus Box 8096, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: romano{at}am.seer.wustl.edu
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Abbreviations |
|---|
NMDA, N-methyl-D-aspartate;
GABA, -aminobutyric
acid;
KA, kainic acid;
EGTA, ethylene glycol bis(
-aminoethyl
ether)-N,N,N
,N-tetraacetic
acid;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
AMPA, 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
DIDS, 4,4-diisothiocyanatostilbene-2,2-disulfonate;
LDH, lactate
dehydrogenase;
BSS, balanced salt solution;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic
acid.
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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subunits: identification of amino acid residues involved in taurine activation.
EMBO J
11:
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