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First published online October 17, 2008
Journal of Experimental Biology 211, 3512-3517 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.021949
-Opioid receptor antagonism induces NMDA receptor-dependent excitotoxicity in anoxic turtle cortex
1 Department of Pediatrics and Neuroscience, University of California San Diego,
La Jolla, CA 92093, USA
2 Department of Cellular and Systems Biology, University of Toronto, Toronto,
ON, Canada, M5S 3G5
3 Department of Ecology and Evolutionary Biology, University of Toronto,
Toronto, ON, Canada, M5S 3G5
* Author for correspondence (e-mail: buckl{at}zoo.utoronto.ca)
Accepted 15 September 2008
 |
Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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-Opioid receptor (DOR) activation is neuroprotective against
short-term anoxic insults in the mammalian brain. This protection may be
conferred by inhibition of N-methyl-D-aspartate receptors
(NMDARs), whose over-activation during anoxia otherwise leads to a deleterious
accumulation of cytosolic calcium ([Ca2+]c), severe
membrane potential (Em) depolarization and excitotoxic
cell death (ECD). Conversely, NMDAR activity is decreased by 
50% with
anoxia in the cortex of the painted turtle, and large elevations in
[Ca2+]c, severe Em depolarization
and ECD are avoided. DORs are expressed in high quantity throughout the turtle
brain relative to the mammalian brain; however, the role of DORs in anoxic
NMDAR regulation has not been investigated in turtles. We examined the effect
of DOR blockade with naltrindole (1–10 µmol l–1) on
Em, NMDAR activity and [Ca2+]c
homeostasis in turtle cortical neurons during normoxia and the transition to
anoxia. Naltrindole potentiated normoxic NMDAR currents by 78±5% and
increased [Ca2+]c by 13±4%. Anoxic neurons
treated with naltrindole were strongly depolarized, NMDAR currents were
potentiated by 70±15%, and [Ca2+]c increased
5-fold compared with anoxic controls. Following naltrindole washout,
Em remained depolarized and [Ca2+]c
became further elevated in all neurons. The naltrindole-mediated
depolarization and increased [Ca2+]c were prevented by
NMDAR antagonism or by perfusion of the Gi protein agonist
mastoparan-7, which also reversed the naltrindole-mediated potentiation of
NMDAR currents. Together, these data suggest that DORs mediate NMDAR activity
in a Gi-dependent manner and prevent deleterious NMDAR-mediated
[Ca2+]c influx during anoxic insults in the turtle
cortex.
Key words: anoxic depolarization, channel arrest, excitotoxic cell death
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Lundberg and Oscarsson,
1953). ECD and AD can be avoided and mammalian neurons can be made
relatively anoxia tolerant via an inducible neuroprotective mechanism
known as hypoxic preconditioning (HPC), whereby pretreatment with a tolerable
hypoxic insult confers neuroprotection against subsequent, otherwise
neurotoxic insults (Murry et al.,
1986; Schurr et al.,
1986). The specific mechanism of this inducible protection is
poorly understood although numerous receptors have been implicated, including
-opioid receptors (DORs).
DORs are a class of inhibitory G protein (Gi)-coupled receptor
whose activation is neuroprotective against hypoxic or ischemic insults in
mammalian neurons (Chao et al.,
2007b; Zhang et al.,
2002; Zhang et al.,
2000). Recently, DORs have also been linked to HPC-mediated
neuroprotective mechanisms. In HPC-treated animals, DOR mRNA and protein
titers are elevated concomitantly with increased neuroprotection, while a DOR
antagonist abolishes neuroprotection. In addition, DOR antagonism induces cell
death in normoxic rat cortical neurons and accelerates anoxia-induced cell
death (Zhang et al., 2002).
This finding is particularly important as it suggests that a tonic background
level of DOR activation is critical to neuronal survival in non-pathological
environments as well as during pathological events.
Despite strong evidence supporting a role for DORs in neuroprotection
against anoxic insults, the mechanism for this is unclear. Interestingly,
research into the effects of NMDAR pharmacological agents on the mechanism of
opioid dependency provided evidence for a cross-talk mechanism between DORs
and NMDARs such that activation of DORs reduces NMDAR activity and vice
versa (Cao et al., 1997;
Wang and Mokha, 1996). Since
over-activation of NMDARs is central to ECD and AD following ischemic insult,
and since DOR activation provides neuroprotection against such insults, it is
reasonable to suspect that DOR-mediated regulation of NMDAR activity is
neuroprotective.
The western painted turtle (Chrysemys picta bellii Schneider 1783)
survives months of acute anoxic exposure at 3°C and days of anoxia at
25°C without apparent detriment
(Jackson, 1968;
Musacchia, 1959). In the
anoxic turtle cortex, NMDAR activity is decreased, large increases in
[Ca2+]c are avoided and ECD and AD are not observed
(Bickler et al., 2000). With
the emerging role for DORs in mammalian HPC models, it is interesting to note
that turtle brain expresses very high endogenous levels of DORs in comparison
with rat brain and that, unlike in rat, DORs are abundantly distributed
throughout all regions of the turtle brain
(Xia and Haddad, 2001). Given
the functional connection between DORs and NMDARs in mammalian brain and the
widespread abundance of DORs in turtle brain, we hypothesized that DOR
activity may be involved in the regulation of turtle cortical NMDAR activity
during anoxia. The aim of this study was to examine the effect of DOR blockade
on turtle cortical neuron electrical activity, whole-cell NMDAR peak current
activity and [Ca2+]c homeostasis during normoxia and
during transition to anoxia with recovery.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Pamenter et al.,
2008d). All experiments were conducted at a room temperature of
22°C. Whole-cell recordings were performed using 3–6 M
electrodes containing the following (in mmol l–1): 8 NaCl,
0.0001 CaCl2, 10 NaHepes, 110 potassium gluconate, 1
MgCl2, 0.3 Na2GTP and 2 Na2ATP (adjusted to
pH 7.4). The bath reference electrode was a Ag–AgCl junction.
Cell-attached 5–20 G seals were obtained using the blind-patch
technique described elsewhere (Blanton et
al., 1989). Whole-cell patches were obtained by applying brief
suction following seal formation. Typical access resistance
(Ra) was 10–30 M. Ra was
determined prior to each measurement and patches were discarded if
Ra changed by more than 20% during the course of an
experiment. Data were collected at 2 kHz using an Axopatch-1D amplifier, a
CV-4 headstage and a digidata 1200 using Clampex 7 software (Axon Instruments,
Union City, CA, USA). The liquid junction potential was assessed as 10 mV
and all data have been corrected for this value (raw data traces are
unaltered). All chemicals were obtained from Sigma Chemical Co. (Oakville, ON,
Canada). Fluorescent probes were obtained from Molecular Probes (Eugene, OR,
USA).
Experimental design
Cortical neurons were perfused with pharmacological modifiers in the bulk
perfusate as specified in the Results section. DORs were blocked with
1–10 µmol l–1 naltrindole
(Zhang et al., 2000). NMDARs
were blocked with 2-amino-5-phosphonopentanoic acid (APV: 70 nmol
l–1). Gi proteins were stimulated with
mastoparan-7 (MP7: 0.1–1 µmol l–1). MP7 is oxygen
sensitive and was prepared under nitrogen and then injected directly into ACSF
pre-gassed with 95% N2/5% CO2. Baseline spontaneous
electrical activity, NMDAR activity and [Ca2+]c
fluorescence were measured for >10 min and cortical sheets were exposed to
anoxic ACSF or ACSF containing pharmacological modulators for a minimum of 40
min and then reperfused for 20–40 min with normoxic ACSF. Electrical
activity and [Ca2+]c were recorded throughout, while
NMDAR currents were elicited at 0 and 10 min of control perfusion and then at
20 min intervals following the switch to treatment ACSF by puffing 300 µmol
l–1 NMDA onto cortical sheets. [Ca2+]c
changes were assessed using fura-2 excited at 340 and 380 nm at 10 s intervals
to limit photo-bleaching. Fluorescence emissions above 510 nm were isolated
using an Olympus DM510 dichroic mirror and fluorescence measurements were
acquired (515–530 nm). [Ca2+]c was calculated as
described elsewhere (Buck and Bickler,
1995).
Statistical analysis
Changes in Em were assessed by comparing the average
Em from 5 min segments. For each fura-2 experiment, 25
neurons were chosen at random and the average change in fluorescence 5 min
after each perfusion switch was used for statistical comparison. Significance
was determined using a one-way repeated ANOVA followed by Tukey's
post-hoc test. NMDAR current data were normalized and analyzed using
two-way ANOVA with a Student–Neuman–Keuls post-hoc test.
Significance was determined at P<0.05, and all data are expressed
as the mean ± s.e.m. (standard error of the mean).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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8 mV that was reversed following reoxygenation
(Fig. 1B;
Fig. 2; N=15). The
anoxic changes in turtle NMDAR activity and [Ca2+]c
homeostasis are well documented but these experiments were repeated here for
statistical comparisons (Bickler et al.,
2000; Pamenter et al.,
2008d; Shin and Buck,
2003). During 90 min of control recordings, normoxic NMDAR peak
currents did not change significantly, ranging from 986±120 to
1087±73 pA, and [Ca2+]c was determined to be
142±8 nmol l–1
(Fig. 1A; Figs
3 and
4; N=5 for each).
Anoxic perfusion induced a 50±7% and 53±9% reduction in NMDAR
activity at 20 and 40 min of anoxia, respectively
(Fig. 1B;
Fig. 3; N=8). Also,
[Ca2+]c increased with anoxia by 40% to
199±15 nmol l–1
(Fig. 1B;
Fig 4; N=5). Following
normoxic reperfusion, Em, NMDAR currents and
[Ca2+]c returned to control levels.
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Naltrindole perfusion during normoxia did not lead to significant sustained Em depolarization; however, large discrete depolarizing events were observed at a frequency of 0.06±0.01 Hz (Fig. 1C, inset; Fig. 2; N=20). APV perfusion abolished these depolarizing currents, suggesting they were mediated by NMDARs. Naltrindole potentiated normoxic NMDAR peak currents by 49±6% and 78±4% following 20 and 40 min treatment, respectively (Fig. 1C; Fig. 3; N=6), and increased [Ca2+]c by 13±4% (Fig. 1C; Fig. 4; N=6). Both these changes were reversed by washout of naltrindole. APV also abolished the naltrindole-mediated increase in [Ca2+]c in normoxic controls, indicating the increase was due to NMDAR-mediated calcium influx (Fig. 4; N=5).
During anoxia, naltrindole resulted in hyper-excitability, extended
depolarization and, in 7 out of 23 experiments (31%), terminal loss of
Em (Fig.
1D; Fig. 2;
N=23). In all 23 neurons, the extended depolarization was not
reversed by reoxygenation, suggesting impaired neuronal viability following
anoxia. The depolarization may have been due to increased NMDAR activity
because naltrindole prevented the anoxic decrease in NMDAR peak currents. In
fact, naltrindole potentiated NMDAR currents by 44±19% and
70±15% following 20 and 40 min of anoxic perfusion
(Fig. 1D;
Fig. 3; N=6). This
increase resembles the potentiation of turtle NMDAR currents by naltrindole
during normoxia but is striking compared with the 50% decrease in NMDAR
activity normally observed in anoxic cortical neurons. Concomitant with the
increase in NMDAR current magnitude and enhanced anoxic depolarization,
naltrindole treatment during anoxia resulted in significantly greater
increases in [Ca2+]c compared with anoxic controls. In
these neurons [Ca2+]c increased 212±16% to
302±22 nmol l–1; and unlike in control anoxic
recordings, [Ca2+]c continued to increase following
washout with normoxic naltrindole-free saline to 544±56 nmol
l–1, a 382% increase over control
[Ca2+]c and 350% greater than
[Ca2+]c in control anoxic experiments following
reoxygenation (Fig. 1D;
Fig. 4; N=5).
We hypothesized that the electrical hyper-excitability and loss of Em observed in most anoxic turtle cortical neurons treated with naltrindole may be due to over-activation of NMDARs and subsequent NMDAR-mediated excitotoxic elevations in [Ca2+]c. Blockade of NMDARs with APV prevented whole-cell NMDA-evoked currents (Fig. 1E), and had no effect on [Ca2+]c or Em (data not shown). Perfusion of APV during anoxia prevented naltrindole-mediated electrical hyper-excitability and extended depolarization (Fig. 1E; Fig. 2; N=7). The electrical response of anoxic neurons treated with naltrindole and APV resembled that of the response to anoxia alone and not that of anoxic plus naltrindole-treated neurons. Perhaps more importantly, in anoxic plus naltrindole-treated neurons already undergoing electrical hyper-excitability, APV perfusion both abolished the excitation and enhanced Em depolarization (Fig. 5). APV also prevented [Ca2+]c increases beyond changes observed in anoxic controls and [Ca2+]c decreased significantly following reoxygenation (Fig. 1E; Fig. 4; N=4).
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Blockade of DORs with naltrindole prevents DOR-mediated Gi cascades. MP7 is a direct stimulator of Gi proteins and perfusion of MP7 should relieve the naltrindole-mediated DOR blockade since it acts downstream of DORs and thus of naltrindole. Therefore, we co-perfused cortical sheets with MP7 and naltrindole during anoxia. MP7 prevented the naltrindole-mediated electrical hyper-excitability and severe depolarization was not observed (Fig. 1F; Fig. 2; N=6). Furthermore, the anoxic decrease in NMDAR activity was restored and NMDAR activity decreased (Fig. 1F; Fig. 3; N=6). Finally, large increases in [Ca2+]c were not observed and cells behaved like control anoxic cells: [Ca2+]c increased 46±3% with anoxia to 208±25 nmol l–1 and this change was reversed by reoxygenation (Fig. 1F; Fig. 4; N=5).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Bickler et al.,
2000; Pamenter and Buck,
2008; Pamenter et al.,
2008d). These deleterious events are likely to be dependent on
NMDAR-mediated Ca2+ influx since naltrindole-mediated electrical
hyper-excitability, severe prolonged depolarization and enhanced
Ca2+ influx were abrogated in cortical slices pre-treated with the
NMDAR antagonist APV. Furthermore, in cells exhibiting seizure-like events
during anoxic naltrindole perfusion, APV application abolished
hyper-excitability and Em recovered following
reoxygenation.
Turtle neurons employ numerous mechanisms to limit glutamatergic
hyper-excitability during anoxia, including: depressed NMDAR activity and
receptor abundance, reduced glutamate release, and decreased AMPA receptor
activity (Bickler et al., 2000;
Milton et al., 2002;
Pamenter et al., 2008c). As a
result, NMDAR-mediated damage due to toxic [Ca2+]c
accumulation and deleterious nitric oxide production are avoided in the anoxic
turtle cortex (Bickler, 1992;
Pamenter et al., 2008a). In
previous studies examining turtle cortical NMDAR activity, neurons were
voltage clamped and perfused with tetrodotoxin to prevent action potentials.
As a result, the true importance of NMDAR regulation in anoxic neuronal
survival could not be determined. In the present study, we directly correlate
enhanced NMDAR activity with excessive [Ca2+]c
accumulation and terminal depolarization in cortical neurons, highlighting the
critical role of NMDAR depression in the turtle's anoxia tolerance. Indeed
turtle neurons exposed to naltrindole during anoxia responded in a fashion
similar to mammalian neurons exposed to glutamate toxicity or ischemia. In
such neurons, excessive NMDAR-mediated Ca2+ influx is observed
along with severe depolarization (Coulter
et al., 1992; Garthwaite and
Garthwaite, 1986; Garthwaite
et al., 1986). Furthermore, upon reoxygenation, mammalian
Em remains depolarized and, as in our experiments, these
effects can be prevented by perfusion of an NMDAR antagonist prior to the
onset of severe depolarization (Limbrick
et al., 2003).
The mechanism by which DORs and NMDARs interact is poorly understood, but
as DORs are coupled to Gi proteins, the pathway probably involves
Gi activation. There is some indirect evidence to support a role
for G protein-mediated responses in the turtle's anoxia tolerance. Whole-brain
cAMP concentration decreases significantly in the anoxic cortex, and since
cAMP is directly mediated by Gi activity these data suggest
anoxia-induced changes in Gi signaling occur in turtle brain
(Pamenter et al., 2007).
Furthermore, we have recently shown that the anoxic depression in turtle NMDAR
activity is blocked by pertussis toxin, a Gi inhibitor
(Pamenter et al., 2008b).
Combined with our present report that the Gi activator MP7
depressed NMDAR currents and prevented extended depolarization and large
accumulations of [Ca2+]c following anoxic/naltrindole
treatment, there is now mounting evidence that Gi signaling is
critical to the regulation of NMDARs in the anoxic cortex. G proteins are well
suited to act as anoxic messengers because they mediate numerous downstream
mechanisms in neurons. Of particular interest are mitochondrial ATP-sensitive
K+ channels (mKATP), whose activity can be directly
regulated by binding of G protein subunits to the sulfonylurea subunit of the
mKATP channel (Wada et al.,
2000). Recently we demonstrated that mKATP channels
mediate the anoxic decrease in turtle NMDAR activity, such that activation of
mKATP during anoxia partially uncouples mitochondria, decreasing
the mitochondrial Ca2+-buffering capacity and subsequently
elevating [Ca2+]c, which regulates NMDARs via a
phosphatase-dependent process (Bickler et
al., 2000; Pamenter et al.,
2008d; Shin et al.,
2005). Although mKATP activation is critical to anoxic
decreases in NMDAR activity, these channels are not oxygen sensitive and are
thus unlikely to be the primary detector of low oxygen that initiates
downstream NMDAR depression. Therefore, the pathway upstream of
mKATP activation in turtle cortical NMDAR depression remains of
interest.
Interestingly, just as anoxic turtle neurons treated with naltrindole
resemble ischemic mammalian neurons, mammalian mechanisms of inducible
neuroprotection may parallel the endogenous mechanism of NMDAR regulation in
the anoxic turtle cortex. In mammals, DOR and mKATP activation are
both critical to HPC-induced neuroprotection and agonists of either receptor
are neuroprotective in a range of ischemia-tolerance models, including HPC
(Chao et al., 2007a;
Kis et al., 2003;
Yoshida et al., 2004;
Zhang et al., 2000). Since
targeted blockade of either receptor prevents HPC-mediated neuroprotection in
mammals and NMDAR depression in turtles, it is logical that the two receptors
must function at separate but critical points in the same pathway. Indeed,
there is evidence supporting this hypothesis: blockade of KATP
channels abolishes DOR agonist-induced neuroprotection in ischemic mammalian
heart and brain (Lim et al.,
2004; Patel et al.,
2002). The mechanism of DOR-/mKATP-induced
neuroprotection in mammalian brain remains unresolved; however, regulation of
NMDAR activity as occurs in the turtle is an attractive possibility as this
regulation is critical to preventing ECD in mammalian neurons
(Arundine and Tymianski, 2003;
Pamenter et al., 2008d). This
hypothesis is supported indirectly by the inverse relationship between NMDAR
activity and DOR activation observed in mammalian brain whereby increases in
DOR activity suppress NMDAR activity (Cao
et al., 1997; Wang and Mokha,
1996). Therefore, the final mediator of neuroprotection in
DOR-mediated HPC models may be a reduction in NMDAR current.
DOR protein expression in the turtle brain is high relative to that in rat
brain tissue (Xia and Haddad,
2001), and this difference in receptor expression could partly
explain the turtle's remarkable innate anoxia tolerance relative to the
mammalian brain, despite their apparent reliance on similar neuroprotective
mechanisms. Greater receptor expression may confer a higher sensitivity to
opioid-mediated signaling, allowing the turtle brain to rapidly decrease NMDAR
activity in response to decreasing environmental oxygen while long-term
mechanisms of metabolic depression are being activated [e.g. removal of
channels from neuronal membranes (Bickler
et al., 2000; Perez-Pinzon et
al., 1992)]. Conversely, mammalian brain requires HPC
pre-treatment to upregulate DOR protein expression and `prime' the brain
against subsequent low-oxygen insults. Thus turtle brain is better able to
respond quickly to low-oxygen insults than murine brain. Conversely, the role
of DOR-mediated signaling in long-term anoxia tolerance in the turtle remains
undetermined; however, NMDAR activity remains depressed during at least the
first 6 weeks of anoxia in turtle brain and DOR-mediated pathways may regulate
this depression (Bickler,
1998). DORs are activated endogenously by enkephalins and
determination of anoxia-induced changes in turtle brain enkephalin
concentration would be an informative next step in the elucidation of this
pathway.
In addition to limiting glutamatergic excitability, the turtle brain
exhibits a wide range of neuroprotective responses to anoxia. In general,
mechanisms that are electrically inhibitory (e.g. GABAergic and adenosinergic)
are elevated during anoxia, while excitatory mechanisms (e.g. glutamatergic)
are depressed (Nilsson and Lutz,
1992; Nilsson et al.,
1990). Experimentally, blocking NMDAR depression (with
naltrindole), GABAergic activation (M.E.P. and L.T.B., unpublished
observations) or adenosinergic signaling
(Milton et al., 2007) all
induce cell death in anoxic turtle neurons. Therefore, each of these
mechanisms appears to be individually critical to anoxic survival. The
existence of multiple neuroprotective responses to anoxia may be due to the
activation of numerous oxygen-sensitive mechanisms that are initiated at
sequentially lower PO2 during a normoxic to anoxic
transition. For example, adenosine receptors regulate NMDAR activity under
hypoxic (5% O2) but not anoxic (0% O2) conditions
(Pamenter et al., 2008b). The
occurrence of overlapping oxygen-sensitive systems in the turtle brain is
logical as this organism repeatedly experiences rapid changes in O2
availability in its natural environment
(Ultsch, 2006). Multiple
oxygen sensors calibrated to detect changes in small ranges of
PO2 and subsequently initiate specific cellular
responses to that PO2 would be evolutionarily
advantageous to this oxygen conformer and confer a greater degree of
sensitivity in terms of cellular responses to changes in oxygen availability.
Furthermore, the individual importance of each of these mechanisms suggests
the metabolic state of the anoxic turtle brain is very tightly regulated and
that any significant deviation from this state that increases electrical
activity may quickly overcome the turtle neurons' ability to match its
metabolic depression to decreased energy availability during anoxia. This may
explain why naltrindole is neurotoxic in turtles during anoxia but not
normoxia, despite potentiating NMDAR currents in both experiments. During
normoxia, the Na+/Ca2+ exchanger and the associated
Na+/K+-ATPases that maintain the Na+ gradient
counter excessive NMDAR-mediated Ca2+ influx. However, during
anoxia, turtle ATPase activity is decreased to match decreased ATP production
(Buck and Hochachka, 1993;
Buck and Pamenter, 2006;
Hylland et al., 1997). Thus an
increase of Na+/Ca2+ exchanger activity to counter
NMDAR-mediated Ca2+ influx would deplete the Na+
gradient more rapidly than the ATPase can match in its reduced activity state,
leading to rundown of Em and deleterious
[Ca2+]c accumulation.
In conclusion, we have shown that blockade of DORs potentiates normoxic
NMDAR currents and not only prevents but also reverses the anoxic decrease in
NMDAR activity, leading to excitotoxic events, increased Ca2+
influx and terminal depolarization in a significant percentage of anoxic
neurons. Our results offer a strong hypothesis to researchers examining
mammalian models of HPC-mediated neuroprotection against ischemic insults. DOR
activation is neuroprotective in mammalian models of stroke, and in both the
turtle and mammals, DORs regulate NMDAR activity. As in turtle neurons,
depression of NMDAR activity is critical to ischemic survival in mammalian
neurons since blockade of NMDARs reduces [Ca2+]c
accumulation and prevents ECD following ischemic insult. However, direct
blockade of NMDARs results in sedative or psychomimetic side-effects
(Ikonomidou and Turski, 2002).
DOR-mediated inhibition of NMDARs, as occurs in turtle cortex, may offer an
indirect mechanism of NMDAR depression that is independent of direct
pharmacological NMDAR blockade.
|
Acknowledgments |
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[Ca2+]c)
correspond to the same treatment regime, as indicated by the solid black bars.
The whole-cell NMDAR currents (bottom trace) were recorded at the time points
indicated by a, b or c on the Em trace within the same
panel. All experiments were conducted on individual cortical sheets exposed to
(A) normoxia, (B) anoxia, (C) normoxia plus naltrindole, (D) anoxia plus
naltrindole, (E) anoxia plus naltrindole in the presence of the NMDAR
antagonist 2-amino-5-phosphonopentanoic acid (APV), or (F) anoxia plus
naltrindole in the presence of the Gi agonist mastoparan-7
(MP7).
data significantly different from anoxic
controls. Significance was assessed at P<0.05. The bar indicates
the duration of the treatment indicated in the key.
data significantly different from anoxic neurons
treated with naltrindole. Significance was assessed at P<0.05. The
bar indicates the duration of the treatment indicated in the key.
