1Division of Neurology, Department of Pediatrics, University of Pennsylvania School of Medicine, the Pediatric Regional Epilepsy Program of the Children's Hospital of Philadelphia, and the Joseph Stokes Jr. Research Institute of Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318; and 2Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0599
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ABSTRACT |
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Lin, Dean D.,
Akiva S. Cohen, and
Douglas
A. Coulter.
Zinc-Induced Augmentation of Excitatory Synaptic Currents and
Glutamate Receptor Responses in Hippocampal CA3 Neurons.
J. Neurophysiol. 85: 1185-1196, 2001.
Zinc is
found throughout the CNS at synapses co-localized with glutamate in
presynaptic terminals. In particular, dentate granule cells' (DGC)
mossy fiber (MF) axons contain especially high concentrations of zinc
co-localized with glutamate within vesicles. To study possible
physiological roles of zinc, visualized slice-patch techniques were
used to voltage-clamp rat CA3 pyramidal neurons, and miniature
excitatory postsynaptic currents (mEPSCs) were isolated. Bath-applied
zinc (200 µM) enhanced median mEPSC peak amplitudes to 153.0% of
controls, without affecting mEPSC kinetics. To characterize this
augmentation further, rapid agonist application was performed on
perisomatic outside-out patches to coapply zinc with glutamate
extremely rapidly for brief (1 ms) durations, thereby emulating release
kinetics of these substances at excitatory synapses. When zinc was
coapplied with glutamate, zinc augmented peak glutamate currents
(mean ± SE, 116.6 ± 2.8% and 143.8 ± 9.8%
of controls at 50 and 200 µM zinc, respectively). This zinc-induced
potentiation was concentration dependent, and pharmacological isolation
of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptor-mediated currents (AMPAR currents) gave results similar to
those observed with glutamate application (mean, 115.0 ± 5.4%
and 132.5 ± 9.1% of controls at 50 and 200 µM zinc,
respectively). Inclusion of the AMPAR desensitization blocker
cyclothiazide in the control solution, however, abolished zinc-induced
augmentation of glutamate-evoked currents, suggesting that zinc may
potentiate AMPAR currents by inhibiting AMPAR desensitization. Based on
the results of the present study, we hypothesize that zinc is a
powerful modulator of both excitatory synaptic transmission and
glutamate-evoked currents at physiologically relevant concentrations.
This modulatory role played by zinc may be a significant factor in
enhancing excitatory neurotransmission and could significantly regulate
function at the mossy fiber-CA3 synapse.
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INTRODUCTION |
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Zinc, a heavy-metal, divalent
cation, has long been implicated in a myriad of cellular processes,
ranging from interactions with zinc-finger proteins and protein kinases
to modulation of ion channels (as reviewed in Smart et al.
1994). In the CNS, zinc localizes to numerous regions,
including the cerebrocortical and corticostriatal pathways
(Perez-Clausell and Danscher 1985
, 1986
). Most notably, zinc has been shown to localize within the axon terminals
of dentate granule cells. These mossy fibers synapse on the proximal
dendrites of pyramidal neurons in region CA3 (Crawford and
Connor 1972
; Haug et al. 1971
). It is at these
axon terminals that zinc colocalizes with glutamate in synaptic
vesicles and may be released into the synaptic cleft together with
glutamate (Assaf and Chung 1984
; Cole et al.
1999
; Howell et al. 1984
; Wenzel et al.
1997
). Despite continuing studies for decades, however, precise
determination of zinc's physiological role at these mossy fiber
synapses and in the CNS in general remains elusive. This study was
conducted in an attempt to determine how zinc co-release may impact
excitatory neurotransmission and glutamate receptor function in CA3
neurons during both normal and pathological states.
Zinc affects multiple voltage-gated and ligand-gated ion channels. Most
noteworthy are the GABAA, the
N-methyl-D-aspartate (NMDA), and the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
(AMPAR)-gated channels (for reviews, see Harrison and Gibbons
1994
; Smart et al. 1994
). Although zinc's
inhibitory effects on both GABAA and NMDA
receptor-mediated currents have been extensively studied in the past
two decades (as reviewed in Choi and Koh 1998
;
Smart et al. 1994
), relatively few studies have been
conducted examining zinc's effects on the AMPA subtype of glutamate
receptors, perhaps because of the difficulties involved in assessing
the peak of AMPAR currents. Zinc's role in modulating function at this
glutamate receptor subtype is especially crucial in the pyramidal
neurons of region CA3 because of the relative dearth of NMDA receptors
at mossy fiber-CA3 (MF-CA3) synapses (Benke et al.
1993
; Monaghan et al. 1983
; but see Vogt
et al. 2000
). In this brain region, long-term potentiation
(LTP) induction is known to be an NMDA-independent phenomenon
(Williams and Johnston 1989
; Zalutsky and Nicoll
1990
), unlike in many other hippocampal regions, in which LTP
has been shown to be dependent on both the AMPA- and NMDA-subtype of
glutamate receptors (Bliss and Collingridge 1993
). Given
that zinc is co-released with glutamate on high-frequency stimulation
of the MF axon terminals (Assaf and Chung 1984
;
Howell et al. 1984
), this heavy metal's presence in
these excitatory synapses may be of great physiological import in the
modulation of function at MF-CA3 synapses.
The majority of electrophysiological studies investigating zinc's
effects on AMPARs have been conducted employing bath application of
zinc onto transfected oocytes, HEK cells, or hippocampal cultures during whole cell patch-clamp recordings (for review, see Smart et al. 1994). In these studies, zinc has been demonstrated to have concentration-dependent effects on AMPAR currents. Mayer et
al. (1989)
demonstrated that, at concentrations <300 µM,
zinc potentiated AMPAR currents, whereas at higher concentrations
(>500 µM), zinc inhibited AMPAR currents. AMPARs, however, are
characterized by ultrarapid desensitization (cf. Zorumski and
Thio 1992
), and it is impossible to change solutions rapidly
enough using whole cell patch techniques to determine the peak of the
AMPA receptor currents. In addition, AMPARs on these cultured neurons
and transfected cells would not be exposed to zinc under physiological
conditions, where extracellular zinc concentrations may reach 100-300
µM during normal functioning of the MF-CA3 synapse
(Frederickson et al. 1983
). Although a recent study
describes effects of synaptically released zinc on postsynaptic NMDA
receptors at MF synapses (Vogt et al. 2000
), to date
there have been no published reports that assess zinc's effects at
physiologically relevant concentrations on synaptic miniature
excitatory postsynaptic currents (mEPSCs) or on native perisomatic
AMPAR currents in CA3 neurons. Therefore to accomplish these goals, in
the present study, whole cell slice patch techniques examining zinc's
effects on excitatory synaptic currents were combined with ultrafast
solution switching techniques to investigate zinc's effects on
glutamatergic synapses and glutamate receptors in perisomatic
outside-out patches of CA3 neurons.
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METHODS |
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Slice preparation
Experiments were performed on 15- to 21-day-old male
Sprague-Dawley rats. Rats were anesthetized and decapitated, and their brains were rapidly removed and placed into cold (4°C), oxygenated (95% O2-5% CO2)
sucrose-based artificial cerebrospinal fluid (SACSF) containing (in mM)
200 sucrose, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 10 glucose, 2 MgCl2, and 2 CaCl2. The
brains were chilled for 3 min before being cut into slices with a
vibratome (Lancer 1000, St. Louis, MO). For synaptic studies, 300-µm
hippocampal-entorhinal-cortical (HEC) slices were cut as described
previously (Rafiq et al. 1995), to maintain the maximal
number of intact dentate granule cell-CA3 axons and synapses. The
presence of intact axons has been shown to impact the frequency of
mEPSCs (Staley and Mody 1991
). Brains were hemisected
and each side was glued, rostral-side up, onto a 12° agar ramp. HEC
slices were then isolated by trimming away the cortical and thalamic
tissue with a scalpel. For outside-out patch studies, 250-µm
transverse slices were cut. All slices were immediately transferred to
oxygenated artifical cerebrospinal fluid (ACSF) in a holding chamber
containing (in mM) 130 NaCl, 3 KCl, 26 NaHCO3,
1.25 NaH2PO4, 10 glucose,
1.0 MgCl2, and 2.0 CaCl2.
Slices were incubated at 32-33°C for >1 h prior to initiating experiments.
Visualization
Brain slices were placed in a recording chamber at room temperature and continually perfused with oxygenated ACSF at a rate of 5 ml/min. CA3 pyramidal neurons were visualized using video-enhanced infrared differential interference contrast microscopy with an Olympus BH-2 upright microscope (Olympus America, Melville, NY) fitted with Nomarski optics, an infrared filter, and a ×40 long working distance water-immersion lens.
Electrodes and intracellular solutions
Patch electrodes were pulled from thick-walled borosilicate
capillary glass (WPI, Sarasota, FL) and filled with an intracellular pipette solution, comprised of either (in mM) 145 K-gluconate, 10 HEPES, 10 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA), and 5 MgATP (K-gluconate internal solution) or 145 CsCl, 10 HEPES, 10 BAPTA, and 5 MgATP (CsCl internal solution). Results using either intracellular solution were similar. Osmolarity was adjusted to
290 mOsm with sucrose, and pH was adjusted to 7.2 with CsOH or KOH. A
Narishige PP-83 microelectrode two-stage puller was used to pull 4-6
M electrodes for whole cell recordings and 8-12 M
electrodes for
outside-out patch studies.
Rapid application
Fast application of agonist was performed as described by Jonas
and colleagues (Jonas 1995). Application
pipettes were fashioned by pulling theta glass (2 mm OD, 0.3 mm wall
thickness, 0.1167 mm septum, Hilgenberg GmbH, Malsfeld, Germany) on a
Narishige PP-83 electrode puller. A diamond scribe was then used to
score the theta glass such that each barrel had a sharp, clean edge and
a diameter ranging between 100 and 150 µm. The theta glass was
mounted on a piezoelectric transducer (Burleigh LSS-3100, Burleigh
Instruments, Fisher, NY) and power supply (Burleigh PZ-150 amplifier/driver). Either Clampex 6.0 or 7.0 (Axon Instruments, Foster
City, CA) waveform protocols were used to deliver command potentials
resulting in applications of 1-ms pulses of agonists to the excised
patches. Agonists and antagonists were dissolved in a control Ringer
solution containing (in mM) 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH adjusted to 7.2 with NaOH, 325 mOsm (Spruston et al.
1995
). This prevented pH changes that would occur in normal
HCO
On excision of the outside-out patch, the tip of the patch electrode was positioned in the control stream, approximately 20 µm from the interface separating the control and drug streams. Typically, the patches yielded AMPAR currents between 10 and 500 pA and NMDA receptor-mediated currents between 5 and 70 pA. After completion of patch recording, patches were expelled, and the exchange time was measured by recording the tip potential across an interface between control solution and a 80% control/20% distilled H2O solution. The 20-80% exchange times typically ranged between 100 and 175 µs.
Electrophysiology
In the whole cell configuration, mEPSCs were isolated by the
addition of 400 nM tetrodotoxin (TTX) and 10 µM bicuculline
methiodide (BMI) to the ACSF. Recordings in both whole cell and
outside-out patch configurations were performed with an Axopatch-1D
(Axon Instruments, Foster City, CA) and filtered at 1-3 kHz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). In the
whole cell configuration, series resistances were <20 M and checked
frequently to ensure that they did not deviate. Cells in which series
resistance was >20 M
or in which series resistance fluctuated were
discarded. A series resistance compensation level of 80% was utilized
in all experiments. Data were digitized at 44 kHz with a PCM device
(Neurodata Instruments, New York, NY) before being stored on videotape.
For excised patch experiments, currents were concurrently digitized at
10-20 kHz and recorded onto disk with pClamp software and a Digidata
1200 A/D board (Axon Instruments).
Data analysis
Whole cell voltage-clamp data were played back into Dempster (Strathclyde, Glasgow, UK) software with a sampling interval of 50 µs. Ten to 20-min periods of activity were reaquired using Dempster software in both control and zinc-exposed conditions (number of events typically 500-2,000 per epoch), and kinetic analyses performed using either Dempster or Mini analysis software (Synaptosoft, Leonia, NJ). For rapid application studies, Clampfit 6.0 (Axon Instruments) was used to average five or more consecutive traces to ascertain peak amplitude and trace kinetics. Curve fits were performed with SigmaPlot 2.0 (Jandel Scientific, San Rafael, CA), and statistical analyses were performed using SigmaStat 2.0 (Jandel Scientific), and values expressed in the text are means ± SE. The statistical significance was determined at a P < 0.05 value using the Student's paired t-test for zinc effects, and Student's unpaired t-test for group comparisons.
Reagents
All solutions were prepared fresh from stocks each day. ZnCl2 was diluted from a pH 7.2 10-mM stock solution, which was prepared every 3-4 days. Buffering the pH of the ZnCl2 stock solution enhanced stability. D-AP5 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from RBI (Natick, MA). TTX was obtained from Calbiochem (La Jolla, CA). All other chemicals and reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted.
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RESULTS |
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CA3 mEPSCs are augmented by zinc
Spontaneously occurring mEPSCs were recorded from 25 CA3 neurons
using TTX to block action potentials and BMI to block
GABAAergic neurotransmission. Similar to other
reports (Capogna et al. 1996; McBain and
Dingledine 1992
), the frequency of these events was extremely
low. In only seven of these neurons was the mEPSC frequency high enough
for adequate analysis. Previous investigators have determined that the
fast peak component of CA3 mEPSCs is mediated by the AMPA subtype of
glutamate receptors rather than the NMDA and kainate subtypes
(Henze et al. 1997
; Jonas et al. 1993
;
McBain and Dingledine 1992
). The median, putatively
AMPAR-mediated peak amplitude of these CA3 mEPSCs was 18.1 ± 1.0 (SE) pA, similar to the results of other investigators (Henze et
al. 1997
; Jonas et al. 1993
). Mean rise and
decay times were 0.54 ± 0.1 ms and 1.6 ± 0.3 ms,
respectively. The mEPSC frequency of these seven cells with
sufficiently high frequency ranged from 0.13 to 3.6 Hz (mean, 1.5 ± 0.4 Hz).
On bath application of 200 µM ZnCl2, mEPSC amplitude increased in six of seven CA3 neurons recorded (Fig. 1, A and B). The median peak amplitude of the synaptic events, presumably mediated by AMPAR activation, increased significantly to 31.9 ± 3.2 pA during zinc application (P < 0.05, Fig. 2A), while the mean mEPSC frequency was not significantly increased (frequency, 2.3 ± 0.7, P > 0.05, Fig. 2D). No significant changes in mEPSC rise and decay kinetics were observed during zinc application (rise time, 0.58 ± 0.16 ms; decay time, 2.5 ± 0.5 ms; Fig. 2C) compared with controls (Fig. 2, B and C). During zinc application, the amplitude and frequency of the mEPSCs varied largely from minute to minute without a consistent increase in mEPSC amplitude or frequency during the first few minutes of zinc application. Rather, occasional bursts of transient, high-frequency mEPSC activity (20-40 Hz) were observed; these bursts typically lasted <0.1 min and occurred at random intervals (data not shown). During burst periods, large-amplitude mEPSCs (>75 pA) were often observed. Because of the rapid kinetics of mEPSCs (Fig. 1), overlapping events were extremely rare and therefore did not confound analyses even during burst periods. Excluding these periods of bursting activity from the analysis did not alter the finding that zinc significantly augmented mEPSC amplitudes in CA3 neurons.
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Glutamate-evoked currents are potentiated by zinc
Zinc colocalizes with glutamate in synaptic vesicles and is
co-released with glutamate on high-frequency stimulation of nerve terminals (Cole et al. 1999; Howell et al.
1984
; Wenzel et al. 1997
). Since zinc presumably
augments the AMPAR component of excitatory synaptic events in region
CA3, rapid agonist application experiments on perisomatic CA3 glutamate
receptors were performed to characterize further zinc's effects on
glutamate receptors expressed in these neurons. With this technique,
concentrations of agonists and modulators are easily regulated,
allowing for more controlled experimental conditions that approximate
synaptic release of glutamate. A saturating glutamate concentration of
3 mM (Min et al. 1998
; Spruston et al.
1995
), as well as 10 µM glycine, was chosen because previous studies have shown that glutamate may achieve millimolar concentrations within the synaptic cleft of hippocampal neurons
(Clements 1996
; Clements
et al. 1992
; Henze et al. 1997
).
Similar to reports by others, 1-ms pulses of 3 mM glutamate to
outside-out patches elicited glutamate-evoked currents that consisted
of fast and slow components (Colquhoun et al. 1992; Livsey et al. 1993
; Spruston et al.
1995
). An initial rapid, presumably AMPAR-mediated rise and
decay comprised the first component and was followed by a much slower,
presumably NMDA receptor-mediated decay phase. Only patch currents
with rise times <1.0 ms were accepted. Decay times (10-90%) ranged
between 3.6 and 17.0 ms and were best fit with the sum of two exponentials.
To mimic zinc's concurrent release with glutamate at presynaptic
terminals, zinc was included only in the drug barrel with glutamate. As
noted previously, at lower concentrations (<300 µM), zinc has been
shown to augment non-NMDA glutamate receptor-mediated currents while
antagonizing NMDA receptor-mediated currents (Bresink et al.
1996; Mayer et al. 1989
; Rassendren et
al. 1990
; Xie et al. 1993
). As seen in Fig.
3, the addition of zinc (200 µM)
together with glutamate reversibly potentiated the peak currents
elicited by 1-ms pulses of 3 mM glutamate alone in five of eight
excised patches. In the five patches that demonstrated zinc
sensitivity, zinc (200 µM) significantly augmented glutamate-evoked
current amplitudes to 143.8 ± 9.8% of control levels evoked by
glutamate alone (P < 0.05). The remaining three
patches showed no zinc sensitivity, i.e., were neither potentiated nor
depressed by zinc coapplication. This existence of dual glutamate
receptor populations, one sensitive and one insensitive to zinc
potentiation, has previously been reported by other investigators
(Mayer et al. 1989
; Rassendren et al.
1990
); it is possible that zinc-insensitive patches may contain
more glutamate receptor subunit heteromers with zinc-insensitive subunit combinations (Dreixler and Leonard 1994
,
1997
). Figure 3B illustrates the averages of
five consecutive agonist applications on a faster time scale. Zinc had
no effect on rise and decay time constants of the glutamate response,
as illustrated in the superimposed and normalized current traces in
Fig. 3C.
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Zinc specifically augments the AMPAR currents in a concentration-dependent manner
Application of glutamate to somatic patches has been shown
to activate both AMPA and NMDA receptors simultaneously (Jonas and Sakmann 1992; Spruston et al. 1995
). Similar
to mEPSCs, previous research has determined that the fast peak and fast
decay of the glutamate-evoked current is mediated primarily by the
AMPAR, whereas the NMDA receptor is the principle mediator of the
slower decay component (Colquhoun et al. 1992
;
Spruston et al. 1995
). To experimentally differentiate
zinc's effects on these receptors, antagonists were employed to
isolate specific receptor subtypes prior to zinc exposure.
Since neither the kainate nor the NMDA receptor mediate the peak of the
glutamate-evoked currents, we chose to initially investigate the
effects of zinc on AMPAR currents. To block the NMDA receptor contribution to the glutamate-evoked currents, 30 µM
D-AP5 was added to both theta glass barrels, thereby
isolating the AMPAR and kainate receptor-mediated currents. Since the
kainate receptor does not contribute significantly to the fast peak of
glutamate-evoked currents, primarily effects on the AMPAR contribution
to the peak are assumed to be considered in this study. Figure
4 depicts the results of this experiment.
Isolated AMPAR currents closely resembled glutamate-evoked currents
and, similar to the glutamate-evoked currents, were significantly
potentiated by 200 µM zinc (Fig. 4, A and B).
As was the case with glutamate application, not all patches were zinc
sensitive. In 7 of 14 patches, zinc significantly augmented the AMPAR
current amplitude to 132.5 ± 9.1% of control AMPAR amplitudes
(P < 0.05); zinc had no effect on the remaining seven
patches. This augmentation level was not significantly different from
zinc augmentation of glutamate currents (P > 0.05).
Figure 4C depicts normalized traces of the AMPAR currents
with and without zinc; similar to zinc's effects on glutamate-evoked
currents, no consistent change in rise or decay kinetics was observed
with zinc coapplication. Therefore it is likely that the zinc-mediated augmentation of the glutamate-evoked responses are solely a consequence of augmented AMPAR currents. This may have physiological relevance since zinc can be released into the MF-CA3 synapses at concentrations up to 100-300 µM (Frederickson et al. 1992;
Xie and Smart 1991
), and excitatory
neurotransmission and long-term potentiation at the MF-CA3 synapses on
apical dendrites have been shown to be AMPAR and not NMDA receptor
dependent (Williams and Johnston 1989
; Zalutsky
and Nicoll 1990
).
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To explore the concentration dependence of zinc effects on glutamate receptor responses, additional experiments were conducted using lower concentrations of zinc. With no antagonists present and 50 µM zinc in only the agonist barrel, glutamate-evoked current amplitudes were significantly augmented in five of eight patches (mean, 116.6 ± 2.8%, P < 0.05) compared with control glutamate-elicited responses (data not shown). In additional experiments using lower zinc concentrations, 30 µM D-AP5 was added to the perfusing solutions to isolate the AMPAR component of the glutamate-evoked currents. The results of these experiments are illustrated in Fig. 5. Figure 5, A and B, depict the results of 1-ms pulses of 50 µM zinc coapplied with glutamate on AMPAR responses. In four of seven patches, zinc augmented AMPAR current amplitudes significantly (mean, 115.0 ± 5.4% of control glutamate-evoked currents, P < 0.05), which was not statistically different from the 50 µM zinc-induced augmentation of raw glutamate currents (P > 0.05). Zinc (50 µM) had no effects on the rise and decay kinetics of the AMPAR current decay as illustrated in Fig. 5C. When these experiments were repeated with 100 µM zinc, the AMPAR currents were significantly potentiated to 121.8 ± 2.4% in three of six patches (P < 0.05, data not shown). In addition, the AMPAR current rise and decay kinetics during 50-, 100-, and 200-µM zinc coapplication were similar, i.e., there was no significant changes in the rise and decay kinetics compared with control responses. Therefore zinc-induced augmentation of both raw glutamate and isolated AMPAR current amplitudes was concentration dependent and occurred without significant effects on response kinetics.
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Inhibition of desensitization reversibly occludes zinc augmentation
Zinc's precise mechanism of action on AMPAR currents remains
unknown. A review by Smart and colleagues (Smart et al.
1994) hypothesized that zinc may potentiate AMPAR currents by
alleviating desensitization. More recent reports have provided evidence
in support of this hypothesis using whole cell recording techniques (Bresink et al. 1996
). To test this hypothesis using
fast solution-switching techniques, allowing recording of the peak and
full decay of AMPAR currents, a set of experiments were undertaken
examining the effects of blockade of AMPAR desensitization using high
concentrations of cyclothiazide (CTZ) (cf. Patneau et al.
1993
; for review, see Yamada 1998
) on zinc
modulation of AMPAR currents. In these experiments, isolated AMPAR
currents were elicited with 1-ms applications of 3 mM glutamate
followed by coapplication of glutamate with zinc (100 µM). Once the
zinc sensitivity of the patch was assayed, CTZ, at a concentration
shown to inhibit AMPAR desensitization and slow AMPAR deactivation (100 µM), was added to the control solution, and then the zinc sensitivity
of the patch was reassessed.
Figure 6 illustrates the results of these experiments. In three of six patches, zinc reversibly potentiated AMPAR currents by 121.8 ± 2.4% of control responses as depicted in Fig. 6, A and D. Because of the slow time-dependent AMPAR current wash out, patch currents were corrected for rundown and normalized as described (see Fig. 6 legend); the normalized current amplitude percentages, plotted against time, appear in Fig. 6D. Once it had been ascertained that the patch demonstrated zinc sensitivity (Fig. 6, A and D), 100 µM CTZ was added to the control solution, and the patches were allowed to equilibrate in CTZ for 1 min prior to repeating drug applications. As illustrated in Fig. 6B, CTZ augmented the glutamate-evoked currents such that they were 111.3 ± 1.9% of the rundown-subtracted current amplitudes. In addition, consistent with its mechanism of action, CTZ significantly prolonged the current decay of AMPAR currents as depicted in Fig. 6B (316.7 ± 57.8% prolongation of decay). More importantly, CTZ occluded zinc-induced augmentation of AMPAR currents as evidenced by the reduced zinc-induced augmentation of AMPAR currents during CTZ exposure (Fig. 6, B and D). After a 10-min CTZ wash out period in normal control solution, the reversibility of CTZ's effects was tested by reassessing AMPAR zinc sensitivity (Fig. 6C). By this time, the basal pre-CTZ AMPAR properties had been reestablished: the current deactivation rates returned to faster baseline states, and zinc once again reversibly augmented glutamate-evoked currents (Fig. 6, C and D). These data support the previously established hypothesis that reductions in desensitization may account for a significant proportion of zinc's effects in augmenting peak AMPAR-mediated currents, but also suggest that additional mechanisms may also play a role, since CTZ-induced occlusion of zinc's effects was not complete (Fig. 6).
|
Jones and Westbrook (1995) have shown that deactivation
of GABAA receptor-mediated currents is mediated
by the rapid transition into and out of desensitized states. Previous
reports studying glutamate receptors, however, have shown that
AMPAR desensitization, following brief (1 ms) saturating pulses of
glutamate, does not play a major role in shaping the rate of
deactivation in CA3 patches (Colquhoun et al. 1992
).
Given the extremely rapid desensitization of the AMPAR, however, a
portion of the AMPARs may desensitize during the 1-ms agonist
applications (Colquhoun et al. 1992
). Since our results
using CTZ suggest that zinc may be inhibiting AMPAR desensitization,
paired-pulse applications with interpulse intervals ranging from 10 to
500 ms were conducted on isolated AMPARs with 3 mM glutamate alone and
glutamate with 200 µM zinc to examine zinc's effects on
desensitization using alternative methods. The first of the pair of
applications was defined to occur at t = 0 ms. As
demonstrated in Fig. 7A,
AMPARs were significantly desensitized at t = 10 ms
following the initial glutamate application such that glutamate-evoked
current amplitudes were only 47.2 ± 3.3% of the initial pulse
amplitude (n = 10 patches), indicating that
approximately 50% of AMPARs were desensitized by a 1-ms application of
glutamate. With increasing interpulse intervals, however, the currents
eventually recovered and returned to baseline amplitudes. The average
percent recovery of the patch currents is plotted with respect to the
interpulse intervals in Fig. 7C. Like results reported by
previous investigators (Colquhoun et al. 1992
), the curve fitted to the recovery from desensitization could be well-fitted by a single exponential curve. Zinc coapplication with glutamate exhibited a similar time course of recovery as illustrated in Fig.
7B (n = 4-5 patches). The initial pulse
current amplitudes are normalized in Fig. 7, A and
B, to facilitate desensitization time course recovery
comparisons since zinc potentiated the AMPAR currents. Like glutamate,
recovery from desensitization during zinc application with glutamate
could be well-fitted by a single exponential curve, with a time
constant indistinguishable from that seen in glutamate alone (Fig.
7C). Since zinc appears to inhibit desensitization but does
not alter the recovery from desensitization with paired applications,
this suggests that zinc may specifically retard the transition of
AMPARs into a desensitized state, but has little effect on the recovery
from desensitization. Zinc does not prolong the decay of AMPAR currents
(Figs. 3-5), but densensitization has been demonstrated to make only a
minimal contribution to deactivation of AMPARs in rapid application
experiments (Colquhoun et al. 1992
). It appears that
zinc only inhibits desensitization as channels are opening during the
rising phase of the glutamate-evoked currents.
|
Pulse applications of zinc have no effect on NMDA receptor-mediated currents
It is well established that tonic applications of zinc block NMDA
receptor-mediated currents using both whole cell and fast application
techniques (cf. Spruston et al. 1995; Traynelis
et al. 1998
; Westbrook and Mayer 1987
).
Similar to results previously reported (Spruston et al.
1995
), tonically applied zinc reduced NMDA receptor-mediated
currents on patches excised from CA3 neurons to 30.0 ± 4.45% (50 µM, n = 3) and 22.0 ± 9.7% (200 µM,
n = 3) of control applications (data not shown). Under
physiological conditions, however, subsynaptic NMDA receptors are not
continuously exposed to zinc. Rather, since glutamate and zinc are
colocalized within the same synaptic vesicles and co-released
simultaneously, and both glutamate and zinc are rapidly taken up, NMDA
receptors are only briefly exposed to zinc when glutamate is present.
In contrast to zinc's strong block of NMDA receptors when tonically applied, prior reports have demonstrated that even high concentrations of zinc (1 mM), when included only in the agonist solution, only blocks
NMDA receptor-mediated currents to a very small degree and slightly
slows the rise time (Spruston et al. 1995
). However, in
other experiments, in addition to potentiating AMPAR currents, zinc has
also been shown to augment NMDA receptor-mediated currents when
certain splice variants of the NMDA receptor are present (Hollmann et al. 1993
). To determine whether or not the
AMPAR was the only glutamate receptor subtype on CA3 pyramidal neurons modulated by pulsatile application of physiological concentrations of
zinc, NMDA receptor-mediated currents were isolated with 5 µM CNQX,
and 3 mM glutamate was applied. Since the voltage-dependent magnesium
block of the NMDA receptor pore is very effective at a holding
potential of
60 mV, magnesium was omitted from the perfusion
solutions to elicit larger, more easily analyzed currents. Even without
magnesium, NMDA receptor-mediated currents were much smaller and
reached peak current amplitudes much more slowly than AMPAR currents.
Under these conditions, brief (1 ms) pulses of zinc (50 and 200 µM),
coapplied with glutamate demonstrated no significant effect on NMDA
receptor-mediated currents (P > 0.05 for both 50 and
200 µM, n = 4 for 50 µM, n = 7 for
200 µM; Fig. 8). Furthermore, zinc had
no effect on the rise time or the decay time of the NMDA
receptor-mediated currents. Thus pulsatile application of zinc
appeared to potentiate glutamate-evoked currents by specifically enhancing AMPAR currents, without impacting NMDA receptors at all.
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DISCUSSION |
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To date, there is no consensus regarding zinc's physiological
role in the CNS. One reason for this uncertainty is that zinc has an
extremely wide spectrum of involvement; zinc plays a role in hundreds
of processes ranging from DNA transcription to modulation of ion
channels (for reviews, see Choi and Koh 1998;
Smart et al. 1994
). To exacerbate the confusion, these
effects are often seemingly contradictory; for example, zinc has been
shown to block apoptosis (Sunderman 1995
), and yet it is
a potent inducer of excitotoxicity (Choi and Koh 1998
).
Zinc's presence in certain specific telencephalic excitatory pathways
(Perez-Clausell and Danscher 1985
, 1986
)
and not others indicates that it most likely subserves a specific
purpose at select synapses. Previous studies have demonstrated that CNS
zinc concentrations of up to 300 µM are achievable on repetitive
stimulation of nerve terminals (Assaf and Chung 1984
;
Howell et al. 1984
). These concentrations are relevant
because they easily exceed those necessary to impact heavily on
excitatory neurotransmission (Mayer and Vyklicky 1989
; Rassendren et al. 1990
). Therefore since zinc is
colocalized exclusively in glutamatergic pathways, modulation of
excitatory neurotransmission is probably one of zinc's chief roles. It
is this potential role that is addressed in this study. Prior reports
have demonstrated zinc-induced potentiation of MF-CA3 field potentials
with tetanic stimulation (Budde et al. 1997
) as well as
zinc-induced augmentation of whole cell AMPAR currents (Mayer
and Vyklicky 1989
; Xie et al. 1993
). In a recent
study, it has been shown that, at MF-CA3 synapses, zinc tonically
occupies the high-affinity binding site of NMDA receptors, and will
also occupy the lower affinity binding site during action
potential-dependent zinc release. Both of these effects result in zinc
shaping MF synaptic responses, but do not appear to alter LTP in
control animals compared with transgenic or mutant strains lacking
vesicular zinc (Vogt et al. 2000
). Unlike previously
published results, the present study examines zinc's effects on
excitatory neurotransmission at both a single-cell and isolated
receptor level in slice. By using whole cell slice patch techniques and
ultrafast solution changing techniques, we demonstrate zinc-induced
augmentation of mEPSCs and a concentration-dependent potentiation of
AMPAR currents.
Mechanism of zinc-induced augmentation of AMPARs
It has been hypothesized that zinc may enhance AMPAR currents by
reducing AMPAR desensitization (Bresink et al. 1996;
Smart et al. 1994
). In this study, evidence for such a
mechanism is provided in experiments illustrated in Fig. 6, which
demonstrates that CTZ, a potent AMPAR desensitization blocker, occludes
zinc's ability to augment AMPAR currents. There are three possible
explanations for this loss of zinc sensitivity. First, CTZ and zinc may
be competing for an allosteric modulatory site, and the tonic presence of CTZ is antagonizing zinc's ability to bind to this site. Another possibility is that CTZ is maximally relieving AMPAR desensitization. If zinc augments AMPAR currents by inhibiting desensitization, then
CTZ's competing mechanism of action would occlude zinc's effects.
Given the vastly different molecular structures of the thiazides and
zinc, it is unlikely that the two compounds would be competing for the
same modulatory site. In addition, to date there have been no prior
reports of zinc binding to the thiazide modulatory site. A third
possibility is that CTZ may be combining with zinc and chelating it,
reducing zinc effects through a receptor-independent mechanism.
Although the first and third mechanisms cannot be completely ruled out,
the second mechanism appears more likely, given that it is supported
both by this study and by binding and whole cell electrophysiological
studies (Bresink et al. 1996
). Zinc may have additional
mechanisms contributing to its AMPAR actions. Zinc effects on AMPAR
peaks were larger than CTZ's, and the occlusive effects of CTZ were
not complete. The close apposition of zinc binding sites in the S1/S2
region of AMPARs to AMPA binding sites suggest that zinc could
potentially modulate agonist binding to the receptor (Armstrong
and Gouaux 2000
).
Possible mediators of zinc-sensitive and zinc-insensitive AMPA responses
One consistent finding throughout all of the above-described patch
experiments is that only 60% of the patches were zinc sensitive. This
was not true for AMPAR-mediated synaptic responses, where the majority
of cells (6/7) had mEPSCs that were augmented by zinc (Figs. 1 and 2).
One potential factor that could mediate this bimodal responsiveness of
AMPARs in patches could be differences in the subunit composition of
extrasynaptic, but not synaptic, receptors. A recently published study
has characterized five putative zinc-binding histidine residues in the
S1/S2 region of AMPARs and found that some of these histidines are not
conserved in all subunits, in particular His-412, which is present in
GluR2-4, but absent in GluR1 (Armstrong and Gouaux
2000). This agrees well with Xenopus oocyte data
that demonstrated that AMPARs composed of homomeric GluR3 subunits were
zinc sensitive, while homomeric GluR1 AMPARs were not (Dreixler
and Leonard 1994
, 1997
). There may be additional
complexity in AMPAR subunit determinants of zinc sensitivity: flip
AMPAR splice variants may be more zinc sensitive than flop variants
(Shen and Yang 1999
). These data, combined with our
patch data, suggest that, in CA3 neurons, zinc-sensitive synaptic
AMPARs may be primarily comprised of GluR2-4 subunits and/or flip
splice variants, while extrasynaptic AMPARs may have a higher
contribution of GluR1-containing receptors, and/or a higher
contribution of flop splice variants, conferring zinc insensitivity to
40-50% of patches.
Data from in situ hybridization, immunohistochemical, and single cell
expression profiling studies have provided extensive information
concerning the AMPAR subunits expressed by 15- to 20-day-old rat CA3
neurons. Using mRNA expression profiles in individual CA3 pyramidal
neurons from 12- to 17-day-old rats, Geiger et al.
(1995) demonstrated that these neurons expressed mostly GluR1
and GluR2, lower levels of GluR3, and no GluR4 mRNA, which agrees
relatively well with in situ hybridization studies (Keinänen et al. 1990
;
Pellegrini-Giampietro et al. 1991
). Individual CA3
neurons also expressed primarily flip splice variants, with lower
levels of expression of flop spice variants (Geiger et al. 1995
). This is also in relatively good agreement with in situ hybridization studies, which demonstrated that GluR1 and 2 flip mRNAs
are present early in development, while flop mRNAs gradually increase
expression over the first 2-3 postnatal weeks (Monyer et al.
1991
). Given that flop splice variants may be differentially insensitive to zinc modulation (Shen and Yang 1999
),
this developmental enhancement in flop expression in 2- to 3-wk-old
animals might contribute to zinc insensitivity in some patches pulled
from 2- to 3-wk-old CA3 neurons.
Immunohistochemical studies in adult rat CA3 region have demonstrated
that GluR1 staining is associated with pyramidal neurons and is
particularly strong in CA3 dendritic regions, while immunostaining for
GluR 2/3 is primarily localized in the somatic layer, with weaker (but
still noticeable) staining in dendritic regions (Leranth et al.
1996). This suggests that both GluR1 and GluR2/3 subunits may
combine to form AMPARs in perisomatic membranes, which in turn would
constitute the receptors studied in pulled patch experiments. Immunogold studies in area CA3 of adult rats examining subcellular distribution of AMPAR subunits demonstrated strong localization of
GluR2/3 and 4 subunits and weaker localization of GluR1 subunits at
mossy fiber synapses, suggesting that synaptically released glutamate
may preferentially activate GluR2-4 subunit-enriched AMPARs
(Baude et al. 1995
). In contrast, cell bodies in region CA3 labeled for both GluR1 and GluR2/3 subunits (Baude et al. 1995
; Leranth et al. 1996
). Given that
GluR1-containing AMPARs may be relatively zinc insensitive
(Dreixler and Leonard 1994
, 1997
), this
suggests that AMPARs in perisomatic patches may have a higher
proportional expression of GluR1 subunits than AMPARs activated during
mEPSCs. This in turn could contribute to the finding that 40% of
perisomatic AMPAR responses were zinc insensitive, while only 14% of
CA3 neurons had zinc-insensitive synaptic responses.
Possible roles of zinc in the CNS
Although spontaneous zinc release has been reported
(Charton et al. 1985), repetitive activation of
excitatory pathways appears to be the principle stimulus for zinc
release in the CNS (Assaf and Chung 1984
; Budde
et al. 1997
; Howell et al. 1984
). Given this
finding and the fact that tonically applied zinc inhibits NMDA
receptor-mediated currents, some investigators initially proposed that
zinc may serve to inhibit excitotoxicity (Kida and Matyja
1990
; Peters et al. 1987
; Weiss et al.
1993
). The present study, as well as others (Spruston et
al. 1995
), has shown that, rather than inhibiting neuronal
excitability, zinc enhances neuronal excitability (Mayer and
Vyklicky 1989
) and strongly potentiates excitotoxicity (for
review, see Choi and Koh 1998
). Although the precise
mechanisms by which zinc mediates excitotoxicity remains unknown,
previous studies have demonstrated that zinc has numerous effects on
ion channels and neurotransmitter release that tend to potentiate
excitability (Choi and Koh 1998
). For instance, at
concentrations of 300 µM, it is possible that zinc diffuses from
glutamatergic synapses to sites containing zinc-sensitive GABAA receptors, thereby decreasing regional
inhibitory tone (Buhl et al. 1996
; Gibbs et al.
1997
; Shumate et al. 1998
). In addition, zinc suppresses glutamate uptake by inhibiting the glutamate
transporter EAAT1 (Spiridon et al. 1998
;
Vandenberg et al. 1998
), which may lead to higher basal
levels of glutamate at excitatory nerve terminals. Thus under
physiological release conditions, zinc's depression of
GABAA receptor function, coupled with an
augmentation of AMPAR currents and decreased glutamate uptake, overall
may predispose the limbic system to enhanced excitatory neurotransmission.
A zinc-mediated increase in MF-CA3 synaptic strength may serve to
facilitate LTP induction. Given that zinc may act similarly to the
thiazide family of AMPAR modulators (Fig. 6), this could enhance
synaptic plasticity. Thiazides have been shown to facilitate LTP
induction in other hippocampal regions (as reviewed in Yamada 1998), most likely by augmenting EPSC amplitudes and prolonging EPSC decays. Since zinc appears to have a mechanism of action similar
to these compounds, zinc could potentially serve as an endogenous AMPAR
modulator (Harrison and Gibbons 1994
; Huang
1997
) capable of lowering the stimulus threshold required for
LTP induction (however, see Vogt et al. 2000
; Xie
and Smart 1994
).
This study was undertaken to determine zinc's effects in situ on
native receptors that normally receive zinc-containing synaptic innervation. The results demonstrate that zinc augments
glutamate-evoked currents in both synaptic and extra-synaptic receptors
in a concentration-dependent manner. Preliminary investigation of
zinc's mechanism of action suggests that a relief of AMPAR
desensitization may contribute substantially to its modulatory actions
(Bresink et al. 1996). The potentiation of AMPAR
currents, coupled with reports that zinc can affect protein kinase C
(PKC) (Baba et al. 1991
), a protein known to play a role
in the maintenance phase of LTP (Kuba and Kumamoto 1990
;
Linden and Routtenberg 1989
), suggest the hypothesis that one of zinc's physiological roles may be to modulate function of
glutamatergic synapses. Because LTP may be a synaptic modulatory mechanism important in learning and memory (Bliss and
Collingridge 1993
), zinc may play a pivotal role in these
higher cognitive functions as well. The studies of Vogt et al.
(2000)
using ZnT-3 knockout animals lacking vesicular zinc
argue against a role for zinc modulation of LTP. However, recent
reports using the same ZnT-3 knockout transgenic strain have suggested
that not all physiologically relevant zinc may be localized within
ZnT3-decorated vesicles, since seizure-induced zinc-mediated
excitotoxic mechanisms in hippocampus persist unabated in animals
lacking vesicular zinc (Lee et al. 2000
). In addition to
a physiological role, zinc may be a major factor in certain
pathological states such as epilepsy, in which zinc is aberrantly
released from sprouted mossy fibers (Babb et al. 1991
;
Houser et al. 1990
; Sutula et al. 1989
;
Tauck and Nadler 1985
). In this environment, zinc's
inhibition of GABAA receptors, coupled with the
zinc-induced augmentation of AMPAR currents, may have catastrophic
ramifications on limbic excitability (Brooks-Kayal et
al. 1998
; Buhl et al. 1996
;
Gibbs et al. 1997
; Shumate et al. 1998
).
Further research is required for a more definitive characterization of
zinc's possible roles in these pathological conditions.
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ACKNOWLEDGMENTS |
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We thank Drs. Annette M. L. McClellan and Stefano Vicini for generous time and assistance in assembling the solution-exchange system.
This research was supported by National Institute of Neurological Disorders and Stroke Grant RO1-NS-32403 to D. A. Coulter and by the Sophie and Nathan Gumenick Neuroscience and Alzheimer's Research Fund. D. D. Lin was supported by the MD/PhD program of the Medical College of Virginia, Virginia Commonwealth University.
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FOOTNOTES |
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Address for reprint requests: D. A. Coulter, 3516 Civic Center Blvd., Abramson Research Center, Rm. 707, Philadelphia, PA 19104-4318 (E-mail: coulterd{at}emailchop.edu).
Received 19 September 2000; accepted in final form 8 December 2000.
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REFERENCES |
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