Departments of Physiology and Neuroscience and Neurosurgery, New York University School of Medicine, New York, New York 10016
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ABSTRACT |
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Chen, Billy T., Marat V. Avshalumov, and Margaret E. Rice. H2O2 Is a Novel, Endogenous Modulator of Synaptic Dopamine Release. J. Neurophysiol. 85: 2468-2476, 2001. Recent evidence suggests that reactive oxygen species (ROS) might act as modulators of neuronal processes, including synaptic transmission. Here we report that synaptic dopamine (DA) release can be modulated by an endogenous ROS, H2O2. Electrically stimulated DA release was monitored in guinea pig striatal slices using carbon-fiber microelectrodes with fast-scan cyclic voltammetry. Exogenously applied H2O2 reversibly inhibited evoked release in the presence of 1.5 mM Ca2+. The effectiveness of exogenous H2O2, however, was abolished or decreased by conditions that enhance Ca2+ entry, including increased extracellular Ca2+ concentration ([Ca2+]o; to 2.4 mM), brief, high-frequency stimulation, and blockade of inhibitory D2 autoreceptors. To test whether DA release could be modulated by endogenous H2O2, release was evoked in the presence of the H2O2-scavenging enzyme, catalase. In the presence of catalase, evoked [DA]o was 60% higher than after catalase washout, demonstrating that endogenously generated H2O2 can also inhibit DA release. Importantly, the Ca2+ dependence of the catalase-mediated effect was opposite to that of H2O2: catalase had a greater enhancing effect in 2.4 mM Ca2+ than in 1.5 mM, consistent with enhanced H2O2 generation in higher [Ca2+]o. Together these data suggest that H2O2 production is Ca2+ dependent and that the inhibitory mechanism can be saturated, thus preventing further effects from exogenous H2O2. These findings show for the first time that endogenous H2O2 can modulate vesicular neurotransmitter release, thus revealing an important new signaling role for ROS in synaptic transmission.
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INTRODUCTION |
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Production of reactive oxygen
species (ROS) is often considered to be a potentially harmful side
effect of cellular respiration. In particular, superoxide and
H2O2 have been linked
causally to neurodegenerative states, including Parkinson's disease,
which involves degeneration of dopaminergic pathways (Cohen et
al. 1997; Ebadi et al. 1996
; Olanow and
Tatton 1999
). Under normal physiological conditions, however,
ROS are tightly regulated and can serve as cellular messengers
(Topper et al. 1996
; Wung et al. 1999
).
Indeed, ROS have recently been implicated in a variety of redox-based signaling mechanisms that can mediate changes in neural plasticity, including activation of oxidative stress-responsive transcription factors (Suzuki et al. 1997
), modulation of LTP
induction (Auerbach and Segal 1997
; Klann et al.
1998
), and mediation of nonsynaptic communication between
neurons and glia (Atkins and Sweatt 1999
). In addition,
ROS have been implicated as modulators of synaptic transmission,
following demonstration that
H2O2 can reversibly depress
evoked population spikes in slices of guinea pig hippocampus (Pellmar 1986
, 1995
).
The site of action of H2O2
on synaptic transmission in the hippocampus appears to be presynaptic
because neither population spikes elicited by antidromic stimulation of
CA1 pyramidal cells nor EPSPs evoked by exogenous glutamate
application are altered by this ROS (Pellmar 1986,
1987
). These observations led Pellmar (1987)
to
suggest that H2O2 might
inhibit transmitter release, possibly by inactivation of presynaptic
Ca2+ channels and thus decreased
Ca2+ entry. Consistent with this hypothesis,
previous studies have shown that preincubation with
H2O2 can cause a decrease
in glutamate release from synaptosomes (Gilman et al.
1992
; Zoccarato et al. 1995
) and dopamine (DA)
release from striatal slices (Joseph et al. 1996
) after
H2O2 was washed out. In
these experiments, however, release was not evaluated in the presence
of H2O2 so that decreased release was presumably a consequence of irreversible oxidative damage
during H2O2 exposure or
washout. Reversible modulation of transmitter release by
H2O2 has not been examined previously.
Although glutamate release cannot yet be monitored in real time with
available microelectrode technology, electroactive neurotransmitters, including DA, can be detected directly using carbon-fiber
microelectrodes with fast-scan cyclic voltammetry (FCV) (Bull et
al. 1990; Rice et al. 1997
) and other
electrochemical techniques. In the present studies, we tested the
hypothesis that H2O2 can
inhibit transmitter release by determining the effect of this ROS on
stimulated release of DA in slices of guinea pig striatum. The striatum
is densely innervated with DA axon terminals arising from midbrain DA
cell body regions (Smith and Bolam 1990
); moreover
Ca2+-dependent, vesicular release of DA from
striatal synapses is well established (Dunlap et al.
1995
; Moghaddam and Bunney 1989
). Together,
these characteristics make the striatal slice an ideal preparation in
which to investigate modulation of synaptic transmitter release. Using
an experimental protocol of
H2O2 application similar to
that used previously (e.g., Pellmar 1995
), we first
tested the hypothesis that exogenous
H2O2 inhibits synaptic
transmission by inhibiting transmitter release. Whether DA release was
modulated by endogenous
H2O2 was then addressed by
examining the effect of the specific degradative enzyme for
H2O2, catalase (EC
1.11.1.6). Because vesicular transmitter release (Dodge and
Rahamimoff 1967
; Llinás et al. 1982
), as
well as mitochondrial respiration and subsequent production of ROS
(Dykens 1994
; Jung et al. 2000
;
Kojima et al. 1994
), are dependent on extracellular
Ca2+ concentration
([Ca2+]o) and
Ca2+ entry, we further examined the
Ca2+ dependence of the effects of exogenous and
endogenous H2O2 on DA release.
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METHODS |
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Slice preparation and solutions
Male Hartley guinea pigs (150-250 g) were deeply anesthetized
with 40 mg/kg pentobarbital (ip) and decapitated. All animal handling
procedures were in accordance with National Institutes of Health
guidelines and were approved by the New York University School of
Medicine Animal Care and Use Committee. Neostriatal slices, 400-µM
thick, were prepared as previously described (Rice et al.
1997). In most experiments, slices were cut in ice-cold artificial cerebrospinal fluid (ACSF), which contained (in mM): 124 NaCl, 3.7 KCl, 26 NaHCO3, 1.5 CaCl2, 1.3 MgSO4, 1.3 KH2PO4, and 10 glucose
saturated with 95% O2-5%
CO2. Slices were allowed to recover in this
normal ACSF for
1 h at room temperature before transfer to a
submersion recording chamber (Warner Instrument, Hamden, CT). In some
experiments, slices were cut and allowed to recover in HEPES-buffered
ACSF, containing (in mM): 120 NaCl, 5 KCl, 20 NaHCO3, 6.7 HEPES acid, 3.3 HEPES salt, 2 CaCl2, 2 MgSO4, and 10 glucose saturated with 95% O2-5%
CO2, which appeared to improve slice quality.
Once in the recording chamber, slices were equilibrated with normal
ACSF at 32°C for an additional 30 min before experimentation;
superfusion media was ACSF with 1.5 or 2.4 mM
Ca2+ as indicated, at a flow rate of 1.2 mL/min.
To examine the effects of endogenous
H2O2, slices were
preincubated for 30 min with 250 IU/ml of catalase in normal ACSF (1.5 mM Ca2+) before transfer to the recording
chamber; catalase was also included during the equilibration period in
the chamber in 1.5 or 2.4 mM Ca2+ as indicated.
Microelectrodes and voltammetric instrumentation
Carbon-fiber electrodes were made from 7- to 8-µm carbon
fibers (type HM, unsized, Courtaulds) that were spark-etched to a 2- to
4-µm tip (MPB Electrodes; Queen Mary and Westfield College, Miles End
Road; London, UK). The voltammetric method used for all experiments was
FCV. Electrodes were calibrated in the appropriate ACSF in the
recording chamber at 32°C (Chen and Rice 1999;
Kume-Kick and Rice 1998
). Measurements of
[DA]o in the presence of
H2O2, sulpiride or catalase
were calculated from calibration factors determined in the presence of
these agents; only catalase consistently altered electrode sensitivity,
with a decrease of <20%. Data were obtained using a Millar
Voltammeter (PD Systems International, West Molesey, Surrey, UK). Data
acquisition was controlled by Clampex 7.0 software (Axon Instruments,
Foster City, CA), which imported voltammograms to a 333-MHz Pentium II
PC via a DigiData 1200B D/A board (Axon Instruments). Scan rate for FCV
was 800 V/s, with a sampling interval of 100 ms controlled by an
external timing circuit. Scan rage was
0.7 to +1.3 V (vs. Ag/AgCl).
Voltammograms were obtained in two-electrode mode, with an Ag/AgCl wire
in the recording chamber as the reference electrode.
Electrical stimulation
Teflon-coated platinum wire bipolar stimulating electrodes
(50-µm bare, 75-µm-coated diameter) with tip separation of 50 µm were placed on slice surface as previously described with the carbon-fiber microelectrode positioned between the electrical poles and
inserted 50-100 µm below the slice surface (Rice et al.
1997). Two stimulation paradigms were used: normal stimulation with 50 pulses delivered at 10 Hz (Rice et al. 1997
) and
high-frequency stimulation with 10 pulses, delivered at 100 Hz
(Cragg and Greenfield 1997
; Singer 1988
).
In both paradigms, pulse duration was 100 µs with amplitudes of
0.4-0.8 mA.
Experimental design
Whether experiments were conducted using normal or
high-frequency stimulation, the interval between stimulation trains was 10 min. Only slices with at least three consistent evoked increases in
extracellular DA concentrations ([DA]o) under
control conditions were tested further. For experiments with exogenous
H2O2, after consistent DA
release was confirmed, H2O2
was superfused for 15 min, followed by a 30-min washout period.
Illustrated data were obtained 5 min before
H2O2 application (control);
after 15 min H2O2; and
after 30 min washout of
H2O2, unless otherwise
indicated. To evaluate effects of endogenous
H2O2 on evoked
[DA]o, slices were preincubated with catalase
(Auerbach and Segal 1997), then transferred to the
recording chamber. Three consistent evoked [DA]o records were obtained in the presence of
catalase, then the enzyme was washed out for 40 min. The three evoked
[DA]o records obtained in catalase were
averaged and compared with the average of three consistent records
taken during washout.
HPLC analysis of DA content in neostriatal slices and in solution
In separate experiments, DA content in striatal tissue was
analyzed following 32°C incubation of slices in normal ACSF or ACSF
containing H2O2. Incubation
chambers were bubbled continuously with 95%
O2-5% CO2. Slices were
prepared as usual, then incubated for 15 min at 32°C in ACSF or in
ACSF plus H2O2. One
hemisphere was used as control while the contralateral hemisphere was
incubated with H2O2.
Following incubation, excess ACSF was carefully removed from the
slices, and a sample of striatal tissue (7-10 mg) was taken, weighted,
frozen on dry ice, then stored at 80°C until HPLC analysis. On the
day of analysis, samples were diluted 1:10 with ice-cold, deoxygenated
mobile phase, sonicated and spun for 2 min at 14,000 g, and
the supernatant was injected directly into the HPLC system. The
analytical column was a 10-cm C18 reversed-phase catecholamine column (HRA-80, ESA, Wiggens, MA) preceded by a 7-µm
guard column (1.5 cm, ODS, BAS, West Lafayette, IN); the detector was a
glassy carbon electrode set at +0.7 V versus Ag/AgCl. The mobile phase
was 50 mM NaH2PO4, with 3 mg/l sodium octylsulfate, 23.2 mg/L heptanesulfonic acid, 8 mg/L EDTA,
and 10% methanol, pH 3 (Witkovsky et al. 1993
).
The mobile phase was deoxygenated with argon, filtered, then maintained
under an argon atmosphere throughout the analysis. Flow rate was 1.2 mL/min. In separate experiments to test the stability of DA in ACSF in
the presence of H2O2,
nominally 1 µM DA was superfused through the recording chamber in 1.5 and 2.4 mM Ca2+-containing ACSF at 32°C, with
and without H2O2. Samples
of DA from the chamber were collected after 2 min of superfusion.
Immediately after collection, samples were diluted 1:10 in ice-cold
deoxygenated eluent and the concentration of DA determined using HPLC.
Lipid peroxidation assay
To address whether H2O2 might cause lipid peroxidation under the conditions tested, we determined total levels of the lipid peroxide byproducts, malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE), using a spectrophotometric assay (Bioxytech LPO-586 kit; Oxis International, Portland, OR). Slices were incubated for 30 min in normal ACSF (1.5 mM Ca2+), exposed to 1.5 mM H2O2 for 15 min, then H2O2 washed out for 30 min; control slices were maintained at 32°C for an equivalent incubation period of 75 min in normal ACSF. After incubation, excess solution was removed from each slice, three slices were pooled, weighed, and frozen on dry ice until analysis; for these pooled measurements, n was the number of samples rather than the number of slices.
Drugs and chemicals
ACS grade H2O2 (30%
w/w), sulpiride, and all components of ACSF and HEPES-ASCF were
obtained from Sigma (St. Louis, MO). At 30%,
H2O2 is 8.8 M; the
concentration of H2O2 used
in all experiments was 1.5 mM (0.005%). The stability of
H2O2 solutions in some
experiments was determined spectrophotometrically using a commercially
available kit (Oxis International); after 15 min superfusion,
H2O2 concentration in the
recording chamber remained within 5% of the initial concentration. Catalase (from bovine liver) was obtained from Calbiochem (San Diego,
CA), a preparation that has been shown to have pure catalase activity
(Esch et al. 1998); enzyme concentration was 250 IU/mL, which was mid-range for effective levels reported previously
(Auerbach and Segal 1997
; Desagher et al.
1997
). All solutions were freshly made immediately before use.
Statistical analysis
All data are expressed as means ± SE, where n is the number of slices unless otherwise indicated. The controls in each experiment with H2O2 were the stimulated [DA]o increases obtained before H2O2 exposure; the maximum evoked [DA]o under these control condition was considered to be 100%. Two different statistical analyses were used to assess the effects of H2O2. First, the average maximal evoked [DA]o during stimulation was compared using paired Student's t-test, then [DA]o throughout the stimulus was compared using one-way ANOVA followed by Tukey post hoc analysis. The same statistical assessments were used to evaluate the effect of catalase except that instead of comparing single stimulation records, as was necessary for the H2O2 experiments, three catalase and three washout records were averaged from each slice. Significance of differences in striatal DA content, media DA concentrations, or tissue lipid peroxidation products between control and H2O2-exposed samples were determined using unpaired t-tests.
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RESULTS |
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Inhibition of evoked DA release by exogenous H2O2
Reproducible, TTX-sensitive DA release in striatal slices could be elicited using a 10-Hz, 50-pulse stimulation train at 10-min intervals for at least 2 h under control conditions. As described in METHODS, two different aspects of the effect of H2O2 on stimulated DA release were examined: maximum evoked [DA]o during stimulation and [DA]o throughout the pulse train.
In normal ACSF containing 1.5 mM Ca2+, the average maximum evoked [DA]o was 0.66 ± 0.07 µM (n = 10; Fig. 1A). Maximum evoked [DA]o was decreased by 56% to 0.29 ± 0.05 µM after 15-min exposure to H2O2 (P < 0.001, n = 10). Moreover, throughout the stimulus, evoked increases in [DA]o were depressed in the presence of H2O2, when compared with control stimulation (P < 0.001). Both peak [DA]o (0.58 ± 0.08 µM, n = 11) and DA release throughout the stimulus recovered to control levels after 30-min washout of H2O2 (P > 0.05; Fig. 1B).
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Because decreased DA release in the presence of H2O2 might reflect oxidative loss of DA during H2O2 exposure, we determined the DA content of striatal tissue immediately after incubation with H2O2. Striatal DA content was not altered significantly (P > 0.05) by H2O2 exposure, with 62 ± 5 nmol/g tissue wet weight (n = 24) in control slices and 56 ± 5 nmol/g (n = 24) in slices incubated for 15 min with H2O2. Similarly, media concentrations of DA were relatively stable in the presence of H2O2 after superfusion through the recording chamber for 2 min at 32°C; this was substantially longer than the 5-s period of [DA]o elevation in the extracellular space during electrical stimulation. In ACSF with 1.5 mM Ca2+, the measured concentration of a nominally 1 µM DA was 0.98 ± 0.02 µM (n = 6) and 0.93 ± 0.02 µM (n = 6) in the same medium plus H2O2. In ACSF with 2.4 mM Ca2+, DA concentration was 0.97 ± 0.02 µM (n = 6) and 0.91 ± 0.01 µM (n = 6) with H2O2. This 5-6% decrease in DA concentration was statistically significant in 2.4 mM Ca2+ (P < 0.05) but not in 1.5 mM Ca2+. In addition, total MDA + 4-HNE levels, reflecting lipid peroxide formation, were indistinguishable between control and H2O2-exposed slices, with 1.6 ± 0.2 nmol/g tissue wet weight (n = 6) in control slices and 1.4 ± 0.2 nmol/g (n = 8) in slices sampled after 30-min washout of H2O2. Levels of MDA + 4-HNE were not determined in slices sampled during H2O2 exposure because of the potential for interference of this oxidant under assay conditions.
Ca2+ dependence of inhibition of DA release by exogenous H2O2
To investigate the potential involvement of Ca2+ entry on the effect of H2O2 on DA release, the Ca2+ dependence of H2O2 inhibition was examined in ACSF containing normal (1.5 mM) [Ca2+]o or 2.4 mM [Ca2+]o. As expected, evoked [DA]o was enhanced in 2.4 mM [Ca2+]o compared with that in 1.5 mM (2.16 ± 0.38 µM, n = 8). Under these conditions, however, H2O2 had no significant effect on either peak [DA]o or overall release levels (P > 0.05). Moreover, concentrations of exogenous H2O2 of up to 10 mM did not alter evoked [DA]o when [Ca2+]o was 2.4 mM (data not illustrated).
To determine whether the inhibition of DA release seen in 1.5 mM Ca2+ could be reversed as well as prevented by raising [Ca2+]o to 2.4 mM, we first determined the effect of H2O2 on stimulated DA release in 1.5 mM [Ca2+]o, then switched to 2.4 mM [Ca2+]o in the continued presence of H2O2 (Fig. 2). In 1.5 mM [Ca2+]o, maximum evoked [DA]o decreased from 0.69 ± 0.16 to 0.37 ± 0.10 µM (P < 0.001, n = 5) in the presence of H2O2 (Fig. 2A), with lower release levels throughout stimulation (P < 0.001). When [Ca2+]o was increased to 2.4 mM in H2O2 for an additional 10 min, evoked [DA]o increased significantly, with a peak level of 2.27 ± 0.63 µM (n = 5), which was 320% of control in 1.5 mM [Ca2+]o in the absence of H2O2 (P < 0.001; Fig. 2B). Evoked DA release throughout the stimulus was also elevated (P < 0.001). Maximum (2.26 ± 0.65 µM, n = 5) and overall evoked [DA]o in 2.4 mM [Ca2+]o were the same as release levels in the presence of H2O2 (P > 0.05) or after H2O2 washout (Fig. 2B).
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Loss of exogenous H2O2-mediated inhibition under conditions of enhanced excitation
The dependence of the effect of H2O2 on [Ca2+]o suggested that other conditions that increase Ca2+ entry and subsequent DA release might also decrease the effectiveness of exogenous H2O2 on inhibiting release. We tested this hypothesis using two additional experimental paradigms, both conducted in 1.5 mM [Ca2+]o. First, we assessed the effect of enhanced stimulus frequency by using brief, high-frequency stimulation (10 pulses at 100 Hz) to evoke DA release. Because of the short duration of this stimulus paradigm, only mean peak evoked [DA]o was compared statistically in these experiments. With this protocol, maximum evoked [DA]o was significantly higher than with 10-Hz stimulation (167% of control, n = 5; P < 0.05). Moreover, DA release evoked by high-frequency stimulation in 1.5 mM [Ca2+]o was unaffected by H2O2 (P > 0.05 for both H2O2 and washout compared with control; Fig. 3A). Representative voltammograms of the maximum [DA]o obtained under each conditions are also illustrated (Fig. 3B).
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Second, we assessed the effect of blocking D2
autoreceptors (Fig. 4). Under normal
conditions, D2 receptor activation should hyperpolarize presynaptic DA terminals (Lacey et al. 1987,
1988
; Liu et al. 1994
). In addition,
D2 receptor activation can have a direct effect
on Ca2+ entry, and hence DA release, by
inhibiting voltage-dependent N- and P/Q-type Ca2+
channels (Kuzhikandthil and Oxford 1999
; Yan et
al. 1997
). Consistent with these actions, a supramaximal
concentration of the D2-receptor antagonist
sulpiride (1 µM) (Cragg and Greenfield 1997
;
Lacey et al. 1987
) caused a significant increase in peak
evoked [DA]o during 10-Hz, 50-pulse
stimulation, compared with evoked levels in the absence of
D2-receptor blockade (P < 0.01)
with an average peak [DA]o of 1.76 ± 0.40 µM (n = 5), which was 275% of control. In the
presence of sulpiride, exogenous
H2O2 again caused a
significant decrease in peak evoked [DA]o (to
1.36 ± 0.36 µM, n = 5, P < 0.01) and a decrease in overall release (P < 0.01;
Fig. 4). However, the decrease was attenuated from that in control
conditions, with an inhibition of only 20%, compared with the roughly
60% decrease in the absence of D2-receptor
blockade (compare Figs. 1A and 4). Unlike control
conditions, maximum evoked [DA]o did not return to pre-H2O2 levels after
H2O2 was washed out in the
presence of sulpiride (not shown). By contrast, maximum evoked
[DA]o during high-frequency stimulation (10 pulses at 100 Hz) was unaffected by sulpiride (P > 0.05, n = 5), as shown previously (Cragg and Greenfield 1997
). Neither exposure to
H2O2 nor subsequent washout in the continued presence of sulpiride altered
[DA]o evoked by high-frequency stimulation
(data not illustrated).
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Catalase increased evoked [DA]o
To determine whether DA release was modulated by endogenously produced H2O2 during stimulation, we tested the effect of catalase on evoked [DA]o during 10-Hz, 50-pulse trains and compared this to evoked [DA]o recorded after catalase was washed out (Fig. 5). Following three consistent recordings in the presence of catalase, washout was initiated; evoked [DA]o typically began to decrease after washout for 10 min. A new, lower level was seen by 20-min washout, which remained stable until the end of recording (up to 1 h of washout). In the presence of catalase, average peak [DA]o in 1.5 mM Ca2+ was significantly higher than in paired control stimulations after catalase washout (165% of control, n = 5; P < 0.01) as was release throughout the stimulus (P < 0.001 vs. washout; Fig. 5A). In contrast to exogenously applied H2O2, catalase was not only effective in the presence of 2.4 mM [Ca2+]o, but its effect was enhanced compared with that in 1.5 mM [Ca2+]o (Fig. 5B). Peak [DA]o in catalase was 181% higher than after washout in 2.4 mM [Ca2+]o (n = 7; P < 0.001 for peak [DA]o and for [DA]o throughout the train vs. washout). The greater effect of catalase in higher [Ca2+]o can also be seen in the normalized DA voltammograms shown in Fig. 5. To ascertain that the enzymatic action of catalase was responsible for these effects, catalase was inactivated in some experiments by boiling; when active catalase was washed out with inactivated enzyme (in either 1.5 or 2.4 mM [Ca2+]o), a decrease in evoked [DA]o was observed that was indistinguishable from that seen with ACSF washout alone.
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In studies with active catalase, the shape of evoked [DA]o responses differed somewhat from that in control slices never exposed to catalase (compare pre- or post H2O2 records in Figs. 1 and 2 with catalase washout records in Fig. 5). During stimulation under control conditions, evoked [DA]o reached a maximum after 200-400 ms, then declined to plateau levels that were roughly 40-50% of the peak concentration (Figs. 1A and 2B). By contrast, after catalase incubation and washout, [DA]o fell to 10% of peak concentration by the end of stimulation in both 1.5 and 2.4 mM [Ca2+]o.
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DISCUSSION |
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The data presented here demonstrate for the first time that
neurotransmitter release can be modulated by
H2O2. In these experiments, both endogenous and exogenous
H2O2 were shown to inhibit
DA release in striatum. These findings, together with previous reports
that exogenous H2O2 can
inhibit glutamatergic transmission in the hippocampus (Avshalumov et al. 2000; Pellmar 1986
,
1987
, 1995
), suggest that inhibition of
transmitter release by H2O2
might represent a general synaptic response to ROS.
Ca2+ dependence of H2O2-mediated inhibition of DA release
In the present studies, exogenous H2O2 had no effect on evoked DA release in the presence of 2.4 mM [Ca2+]o or during high-frequency stimulation. Both of these conditions would increase presynaptic Ca2+ entry. At face value, this result suggests that increased intracellular Ca2+ might act competitively with H2O2. However, even when H2O2 concentration was increased to 10 mM, there was still no effect on evoked release in 2.4 mM [Ca2+]o, as noted in RESULTS, indicating that direct competition between Ca2+ and H2O2 was unlikely. Furthermore unaltered evoked [DA]o in the presence of exogenous H2O2 under these conditions argued against a direct chemical interaction between H2O2 and DA to decrease [DA]o. This was verified by our studies of DA stability in H2O2-containing ACSF.
In contrast to the lack of effect of exogenous
H2O2 in 2.4 mM
[Ca2+]o, the effect of
catalase was somewhat greater in 2.4 than in 1.5 mM
[Ca2+]o. This suggests
that a Ca2+-dependent increase in endogenous
H2O2 levels generated
during stimulation caused a maximal inhibitory effect that occluded any further response to exogenous
H2O2. In support of this
hypothesis, previous studies have shown that mitochondrial respiration
(the primary source of endogenous
H2O2, see following text)
and consequent ROS production are Ca2+ dependent.
Increases in intracellular Ca2+ concentration
([Ca2+]i) are accompanied
by enhanced mitochondrial O2 consumption
(Jung et al. 2000; Kojuma et al. 1994
).
In addition, in presynaptic terminals, elevated
[Ca2+]i serves as a
trigger for mitochondrial uptake of Ca2+
(Giovannucci et al. 1999
) and for active clearance of
[Ca2+]i by plasma
membrane transporters (Zenisek and Matthews 2000
). Both
of these processes are ATP-dependent processes and thus require increased oxidative metabolism. Moreover, Dykens (1994)
has shown directly that increases in [Ca2+]
cause an increase in ROS production by isolated brain mitochondria.
The effect of H2O2 on DA
release was also minimal or absent when release was elicited in 1.5 mM
[Ca2+]o under conditions
that enhance Ca2+ entry. The first paradigm
tested was brief, high-frequency stimulation (Fig. 3). Physiologically,
both terminal excitability and DA release are dependent on stimulation
frequency, in part reflecting frequency-dependent Ca2+ entry (Chergui et al. 1994;
Garris et al. 1997
). In vivo, DA cells exhibit two main
activity patterns: single spike firing at 0.5-8 Hz and burst firing of
three to eight spikes at frequencies of 6-15 Hz (Grace and
Bunney 1984
), with the ability to follow stimulation
frequencies up to 100 Hz (Tepper et al. 1991
).
Increasing stimulus frequency from 10 to 100 Hz in the present
experiments caused an increase in evoked [DA]o
as expected. Consistent with a concomitant increase in
H2O2 generation under these
conditions, exogenous H2O2
had no effect on evoked [DA]o during
high-frequency stimulation, again suggesting that the inhibitory
mechanism was already fully activated by endogenous
H2O2.
The second approach was to manipulate DA terminal excitability using an
antagonist of D2 autoreceptors, which should also increase Ca2+ entry. Normally,
D2-receptor activation hyperpolarizes the
presynaptic membrane of DA terminals, which decreases excitability and
thus decreases DA release (Cragg and Greenfield 1997;
Lacey et al. 1987
, 1988
). In addition,
D2-receptor activation can decrease Ca2+ currents through direct links to N- and
P/Q-type Ca2+ channels (Kuzhikandathil and
Oxford 1999
; Yan et al. 1997
), which are
required for synaptic release of DA (Turner et al.
1993
). Consistent with these actions, the presence of a
D2-receptor antagonist markedly enhances evoked
[DA]o (Cragg and Greenfield
1997
) (Fig. 4). In the present studies, the
D2-receptor blockade with sulpiride also
decreased the effect of exogenous
H2O2 on DA release (Fig. 4).
Sources of endogenous H2O2
Oxidative metabolism in brain tissue occurs in the mitochondria as
in all cells. During the process of oxidative phosphorylation, a
significant amount of O2 consumed is diverted to
form superoxide (O). The amount of
H2O2 produced by brain
mitochondria is up to 5% of the amount of O2
consumed (Arnaiz et al. 1999
). Given that the rate of
O2 consumption in gray matter is 2-5 µmol/g
tissue wet weight per minute (Hagerdal et al. 1975
;
McIlwain and Bachelard 1985
), or 2-5 mM (assuming 1 g = 1 mL), this would mean that concentrations of up to 250 µM
H2O2 could be generated
every minute within brain neuropil. Because the rate of
O2 consumption is roughly 10-fold higher in neurons than in glia (Siesjö 1980
), however, this
H2O2 would be produced
predominantly in the neuronal compartment, which could lead to higher,
intra-neuronal concentrations. Moreover, the presence of mitochondria
within 250 nm of the synapse in DA terminals (Delle Donne et al.
1997
; Nirenberg et al. 1997
) suggests that even
higher levels could be reached in the restricted, intracellular
compartment of a synaptic terminal. It is relevant to note, however,
that such local increases are likely to be transient because
H2O2 is membrane permeable
and thus can readily leave the compartment in which it is produced.
In addition to mitochondrial sources,
H2O2 will also be produced
in DA terminals by monoamine oxidase (MAO), which is a metabolizing enzyme for DA (Cohen et al. 1997; Sandri et al.
1990
). Importantly, MAO is localized on the outer membrane of
mitochondria, which would further enhance
H2O2 concentrations near DA
synapses. Given that increased DA metabolism accompanies enhanced DA
release, generated H2O2
might provide additional feedback inhibition to decrease transmitter
release and thus augment the effect of
D2-autoreceptor-mediated inhibition.
Actual concentration of
H2O2 at a given location
will depend not only on the activity of sources of
H2O2 and the size of the compartment it enters, but also on the activity of the local
antioxidant network. In brain tissue,
H2O2 levels are regulated
largely by the intracellular enzyme, glutathione (GSH) peroxidase, and
by endogenous catalase in peroxisomes (Cohen 1994).
Importantly, the present results with exogenous catalase indicate that
cellular antioxidant regulation does not completely remove endogenously generated H2O2. Rather
these processes appear to permit levels of
H2O2 that are sufficient to
exert modulatory actions. The lack of effect of up to 10 mM exogenous
H2O2 in elevated
[Ca2+]o, however,
indicates that
H2O2-mediated effects are
saturable; this is also consistent with the calculation that competing
levels of endogenous H2O2
might be millimolar. Indeed, the concentration of exogenous
H2O2 used in the present
studies (1.5 mM) is within the range (1-3 mM) found to have biological
effects in many other studies in the literature (e.g., Frantseva
et al. 1998
; Krippeit-Drews et al. 1999
;
Pellmar 1987
, 1995
; Seutin et al. 1995
).
One caveat in these arguments is that the activity of antioxidant
enzymes and other cellular antioxidants could result in levels of
exogenously applied and endogenously generated
H2O2 that are much lower
than this. Available analytical methods cannot yet address this
question quantitatively. Regardless of absolute concentrations,
however, the fact that catalase enhanced evoked
[DA]o by roughly the same percentage that
exogenous H2O2 decreased
peak [DA]o suggests that similar effective
levels were achieved with both sources.
Possible mechanisms of H2O2-mediated modulation of DA release
It has been previously suggested that inhibition of synaptic
transmission in the hippocampus by
H2O2 might be the result of decreased presynaptic Ca2+ entry and consequent
loss of transmitter release (Pellmar 1987). The present
results confirm that H2O2
can indeed inhibit transmitter release. In addition, in separate
studies in hippocampal slices, we have monitored presynaptic
Ca2+ entry indirectly using ion-selective
microelectrodes to monitor decreases in
[Ca2+]o during
pulse-train stimulation (Avshalumov et al. 2000
). Evoked [Ca2+]o decreases were
unaffected by H2O2,
suggesting that its action was downstream from
Ca2+ entry. Inhibition of evoked population
spikes in the hippocampus by
H2O2 in those studies was
further shown to be mediated by hydroxyl radical
(
OH) formation, which can be catalyzed by
metal ions (Avshalumov et al. 2000
). It is likely that
OH is also involved in modulating DA release;
however, we were not able to test this in the present studies because
the metal ion chelator typically used to prevent
OH generation, deferoxamine, is electroactive
and thus interfered with voltammetric detection of DA.
A number of presynaptic mechanisms that are downstream from
Ca2+ entry could be involved in mediating the
actions of H2O2 on DA release. For example, it was recently shown that
H2O2 caused a decrease in
ATP production, which interfered with stimulus secretion in pancreatic
cells (Krippeit-Drews et al. 1999). Another possible mechanism might be through oxidative modification of one or more intracellular proteins involved in vesicular release, including those
involved in Ca2+-dependent SNARE binding
interactions (Bezprozvanny et al. 1995
; Rettig et
al. 1997
; Sheng et al. 1996
; Südhof
1997
). Such a process would involve oxidation of
redox-sensitive amino acids like cysteine. Alternatively, because ROS
shift the balance between phosphatases and kinases (Klann and
Thiels 1999
; Klann et al. 1998
), the effects of
H2O2 could involve
increased phosphorylation of Ca2+ binding
proteins or other synaptic proteins. Tying these latter two mechanisms
together is the fact that cysteine residues on protein tyrosine
phosphatases have been shown to be selective targets of
H2O2-induced oxidation
(Denu and Tanner 1998
).
In addition to these possible mechanisms, there is evidence in the
literature to suggest that
H2O2 application can cause
membrane hyperpolarization mediated by an increased
K+ in some cell types, including CA1 pyramidal
neurons (Krippeit-Drews et al. 1999; Seutin et
al. 1995
), which could lead to a decrease in transmitter
release. This is not seen in all cells, however. For example, although
H2O2 was also found to
cause changes in membrane properties of some cells in thalamocortical
slices, the net effect of
H2O2 application was
excitatory, apparently by selective loss of inhibitory transmission
(Frantseva et al. 1998
). How changes in membrane
properties might contribute to inhibition of release of DA from
synaptic terminals in striatum has not been examined.
The slightly altered shape of evoked
[DA]o records after prolonged catalase
incubation suggests that
H2O2 might contribute to DA
regulation by mechanisms in addition to inhibition of release. The
shape and amplitude of [DA]o records during
normal stimulation is a consequence of many factors, including specific
inhibitory effects of D2 autoreceptors and DA uptake via
the dopamine transporter (DAT) as well as general slice quality. The
influence of D2 receptors can be seen in Fig. 4, in which
the increase in [DA]o was enhanced and
prolonged when inhibitory autoreceptors were blocked by sulpiride compared with control records (e.g., Fig. 1). Whether sulpiride was
present or not, however, DA release curves typically fell to 40-50%
of peak value by the end of the pulse train. After incubation in
catalase, however, the fall in evoked [DA]o was
exaggerated, with a decay to only 10% of peak by the end of the pulse
train (compare Figs. 1, 2, and Fig. 5). One possible explanation is that prolonged depletion of tonic levels of
H2O2 could decrease protein
kinase C-dependent phosphorylation of the DAT, which would increase DA
uptake activity (Bauman et al. 2000; Vaughan et
al. 1997
). In addition, there is evidence in the literature to
suggest that decreased phosphorylation of D2 receptors
might increase agonist sensitivity (Elazar and Fuchs
1991
). Either effect could contribute to the faster return of
evoked [DA]o to baseline after catalase
incubation. On the other hand, direct oxidation of the DAT under
conditions of oxidative stress can inhibit DAT activity (Berman
et al. 1996
) such that added catalase might protect the DAT by
decreasing oxidative stress. It should be noted, however, that possible
oxidative inhibition of the DAT by
H2O2 during exogenous application would increase rather than decrease
evoked [DA]o. Moreover, the similarity of
evoked [DA]o in the presence and absence of
H2O2 in 2.4 mM
[Ca2+]o (e.g., Fig.
2B) suggests that DAT activity is not altered during 15-min
H2O2 application.
Independent of the possible mechanism(s) by which
H2O2 may act, the finding
that H2O2 can inhibit
transmitter release reveals a novel process by which synaptic
transmission might be modulated physiologically. In addition, these
data point to a normal process that could lead to neuropathology if
regulation were disrupted. As noted earlier, Parkinson's disease,
which involves degeneration of DA cells and terminals, has been linked
to oxidative stress (Cohen et al. 1997; Ebadi et
al. 1996
; Olanow and Tatton 1999
). An imbalance
in ROS production and regulation could contribute to this process.
Moreover, excess H2O2 can
damage mitochondria (Hoyt et al. 1997
), such that
disregulation could initiate a cycle of increased
[Ca2+]i, increased ROS
production, cytotoxicity in striatum (Brouillet et al.
1995
), and degeneration of DA neurons in midbrain
(Sonsalla et al. 1997
). The present findings, therefore
not only provide a new perspective on modulation of synaptic
transmission by ROS but also suggest normal regulatory processes that
could, over time, contribute to the pathology of neurodegenerative disorders.
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ACKNOWLEDGMENTS |
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We are grateful to M. Chesler and S. J. Cragg for insightful comments on the manuscript, to M. L. Chao for HPLC analysis of dopamine, and to K. A. Helmin for lipid peroxide determination.
These studies were supported by National Institute of Neurological Disorders and Stroke Grant NS-36362.
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FOOTNOTES |
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Address for reprint requests: M. E. Rice, Dept. of Physiology and Neuroscience, NYU School of Medicine, 550 First Ave., New York, NY 10016 (E-mail: margaret.rice{at}nyu.edu).
Received 31 August 2000; accepted in final form 17 January 2001.
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REFERENCES |
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