Departments of Physiology and Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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Lei, Saobo and John F. MacDonald. Gadolinium Reduces AMPA Receptor Desensitization and Deactivation in Hippocampal Neurons. J. Neurophysiol. 86: 173-182, 2001. The actions of the trivalent cation Gd3+ on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd3+ (0.1-10 µM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 µM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd3+ also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd3+ reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd3+ inhibited both peak and steady-state currents, indicating that Gd3+ both reduces desensitization and inhibits these currents. Gd3+ reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd3+. The Gd3+-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd3+ reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd3+ potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.
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INTRODUCTION |
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Glutamate activates three
different families of ion channels including
N-methyl-D-aspartate (NMDA),
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate
receptors. One particular property of glutamate receptors is that they
are modulated by divalent cations such as Mg2+
(Ault et al. 1980
; Mayer et al. 1984
;
Nowak et al. 1984
). The Mg2+ block
of NMDA receptors is voltage dependent and plays an important physiological role. Currents mediated by these receptors are similarly blocked by other members of group IIB metal cations including cadmium,
cobalt, nickel, and manganese (Mayer and Westbrook 1987
; Mayer et al. 1989
). Nevertheless, millimolar
concentrations of Mg2+ also increase NMDA-evoked
currents by interacting with the glycine-binding site of the receptor
(Paoletti et al. 1995
; Wang and MacDonald 1995
). The effects of polyvalent cations on AMPA and kainate
receptor-mediated currents have also been investigated, although to a
lesser extent than for NMDA receptors. At micromolar concentrations the
trivalent cations, La3+ and
Gd3+ inhibit kainate receptor function in both
neurons and cell lines expressing the GluR6 subunit (Huettner et
al. 1998
). In contrast, Zn2+ and
Cd2+ enhance AMPA receptor function at low
concentrations and inhibit at higher concentrations (Mayer et
al. 1989
; Rassendren et al. 1990
).
Zn2+ also reduces AMPA receptor desensitization
in cultured superior colliculus neurons (Bresink et al.
1996
).
We recently demonstrated that some positive modulators of AMPA
receptors, such as cyclothiazide (CTZ), act to reduce a proton-induced enhancement of AMPA receptor desensitization (Lei et al.
2001). In the present paper, we examine the effects of
Gd3+ on AMPA receptor function.
Gd3+ is a highly effective blocker of a variety
of ion channels likely because of its potent ability to reduce negative
surface charges on the membrane (Xiong et al. 1997
). Our
results demonstrate that Gd3+ reduces AMPA
receptor desensitization and deactivation while it also blocks AMPA receptors.
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METHODS |
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Preparation of acutely isolated hippocampal CA1 neurons
The animals used in this work were handled under the regulations
of the Canadian Institutes of Health Research. CA1 hippocampal pyramidal neurons were acutely isolated using modified procedures of
Wang and MacDonald (1995) except that protease (type
XIV, Sigma) instead of papain was used. Briefly, Wistar rats of 2- to
3-wk old were decapitated under halothane anesthesia using a
guillotine. Hippocampi were quickly removed and placed in a dish
containing cold oxygenated external solution consisting of (mM) 140 NaCl, 1.3 CaCl2, 5.4 KCl, 25 HEPES, 33 glucose, 1 MgCl2, and 0.0003 tetrodotoxin (TTX; pH, 7.4, osmolarity, 320-335 mosmol/l). The hippocampi were cut into 300- to
500-µm-thick slices by hand with a razor blade. The hippocampal
slices were digested at room temperature (20-22°C) in the preceding
solution containing 1.5 mg/ml protease. The incubation medium was
stirred with pure oxygen blown in at the bottom of the vessel. After 40 min of enzymatic digestion, slices were rinsed three times with
enzyme-free solution. The slices were maintained in the external
solution, bubbled with oxygen, and used for up to 8-10 h. The CA1
region was dissected out with a scalpel under a phase-contrast
microscope and then triturated with a fire-polished glass pipette. Data
were obtained from the pyramidal cells that had a bright color and
clear outline.
Whole cell recording
Whole cell recordings were performed with an Axopatch-1B
amplifier (Axon Instruments) in voltage-clamp mode. Recording
electrodes with resistances of 3-5 M were constructed from
thin-walled borosilicate glass (1.5-mm diam, WPI) using a two-stage
puller (PP83, Narishige). Data were digitized, filtered (5kHz) and
acquired on-line using the program pClamp6 (Axon Instruments). The
standard internal solution for recording electrodes consisted of the
following (in mM) 140 CsF, 35 CsOH, 10 HEPES, 2 MgCl2, 2 tetraethylammonium, 11 EGTA, and 4 Na2ATP (pH 7.3, osmolarity, 300 mosmol/l). The time course of whole cell AMPA currents was similar when CsF was replaced with CsCl2 (Lei et al.
2000
; Wang and MacDonald 1995
). The standard
external solution was the same as that previously described except that
Mg2+ (3 mM) and AP5 (50 µM) were included to
block N-methyl-D-aspartate (NMDA) receptors.
After formation of the whole cell configuration, the recorded cells
were voltage-clamped at
60 mV and lifted into the stream of solution.
Saturating concentrations of glutamate (3-10 mM) were applied to the
neurons for 2 s to evoke responses. Under these recording
conditions, the currents evoked by glutamate were completely blocked by
GYKI 53655, confirming their identity as AMPA receptor responses.
Nucleated or outside-out patch recording and ultra-fast perfusion
Outside-out patch recordings were carried out as previously
described (Bai et al. 1999), and glutamate was applied
using theta tubing connected to a piezoelectric translator (PZS-100,
driven by PZ-150, Burleigh, Fishers). For solution exchange,
is
less than 200 µs as determined by measuring the open-tip junction
potentials. Currents were filtered at 5 kHz and digitized at 50 kHz.
Nonstationary variance analysis
Nonstationary variance analysis was used to estimate
conductance and the open probability of the channels at the peak of the response (Sigworth 1980). Generally, 60 responses evoked
by applications of 10 mM glutamate for 100 ms at 5-s intervals were
recorded for each outside-out patch. During an epoch of 60 responses,
an average of 10% run-down was observed. Data from patches showing
>20% run-down were not included for analysis. Responses from each
patch were divided into 10-12 groups (5 for each group). After
aligning each of the five responses to the peak, local means of each
group were calculated to minimize the distortion originating from
run-down. Each individual response was subtracted from the local mean
of the group to compute the variance. Current responses 90 ms from the
peak were selected for analysis. The mean current was divided into 100 equally sized bins, and the corresponding variances were pooled. The
binned variance versus the mean current was plotted and fit with the
equation:
2 = iI
I2/N +
base, where
2 is the variance, I is
the mean current, N is the number of channels activated
at the peak, i is the single channel current, and
base is the background variance.
Open probability at the peak, PO,Peak was calculated by PO,Peak = Ipeak/(iN), where
Ipeak is the peak current; then, the
single channel conductance was measured from
= i/(E
Erev), where E is the
holding potential, Erev is the reversal potential (0 mV under our recording conditions).
Miniature excitatory postsynaptic currents
Miniature excitatory postsynaptic currents (mEPSCs) mediated by
AMPA receptors were recorded from mouse hippocampal neurons taken from
wild-type or GluR2 null-mutant mice (Jia et al. 1996) cultured for 14-17 days as previously described (Lei et al.
2000
). The extracellular solution was supplemented with TTX
(0.5 µM), AP5 (50 µM), strychnine (1 µM), and bicuculline
methiodide (20 µM). mEPSCs were filtered at 2 kHz. The
synaptic events were acquired and analyzed using the SCAN program
(Strathclyde Software). For detection, the trigger level was set
approximately three times higher than the baseline noise. False events
were eliminated by subsequent inspection of the raw data.
Gd3+ was dissolved in the extracellular solution
and applied to the cells.
Data analysis
The time course of the onset of desensitization in outside-out patches was determined by fitting the decay of the response (beginning 10 points after the peak) with a single-exponential function. The decay of deactivation was fit with single-exponential function from 10 to 40% of the peak to exclude the influence of desensitization. Data were expressed as means ± SE. The values in parentheses refer to the number of cells used for the statistical analysis. Concentration-response curves were fit by Hill equation: I = Imax × {1/[1 + (EC50/[ligand])n]}, where Imax is the maximum response, EC50 is the concentration of ligand producing a half-maximal response, and n is the Hill coefficient. Statistical analyses were performed using Student's t-test or by one-way ANOVA when appropriate. P values less than 0.05 were taken as an indication of a significant difference.
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RESULTS |
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Modulation of whole cell AMPA receptor currents by Gd3+
We first studied the effect of Gd3+ on glutamate-evoked AMPA receptor-mediated whole cell currents in isolated rat hippocampal CA1 neurons. Applications of Gd3+ (5 µM) both in the control and glutamate barrels had little effect of the peak (Ip) of glutamate-evoked currents (Fig. 1, A and B). However, the steady-state currents (Iss) were enhanced to 200 ± 25% of the control (n = 10, P < 0.01, Fig. 1B). The ratio of the Iss/Ip was, therefore increased to 201 ± 25% of the control (n = 10, P < 0.01, Fig. 1B) suggesting that the extent of desensitization was decreased by Gd3+.
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We then constructed concentration-response relationships for
Gd3+ using glutamate as an agonist. At
concentrations up to 10 µM, Gd3+ potentiated
Iss of glutamate-evoked currents in a
concentration-dependent manner with a threshold of about 0.03 µM and
an EC50 value of 1.0 ± 0.2 µM
(n = 7; Fig. 1, C and D). In
contrast, higher concentrations (up to 30 µM or higher) of
Gd3+ depressed
Iss (Fig. 1D). This
resembled the effects of Zn2+ on AMPA receptor
function (Rassendren et al. 1990).
AMPA receptor-mediated currents can also be evoked with applications of kainate that demonstrate much less complete desensitization than with glutamate. Similar to steady-state glutamate-evoked currents, the steady state of kainate responses was enhanced by low concentrations of Gd3+ (0.1-10 µM; Fig. 1E) and inhibited by higher concentrations (Fig. 1, E and F). The EC50 value for Gd3+ was 0.63 ± 0.06 µM (n = 8) with a Hill coefficient of 1.60 ± 0.07 (n = 8).
Gd3+ increases the potency of glutamate
We next examined whether or not low concentrations of Gd3+ would enhance glutamate potency. Gd3+ (10 µM) shifted the peak current versus glutamate concentration curve to the left (control, EC50 = 0.15 ± 0.03 µM; Gd3+, 0.09 ± 0.02, n = 9, P < 0.01, Fig. 2, A and B) without changing that for steady-state currents (control, EC50 = 0.06 ± 0.01 µM; Gd3+, 0.05 ± 0.01, n = 9, P > 0.05, Fig. 2C).
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To determine if the enhancement induced by Gd3+ was voltage dependent, we examined its effect on AMPA receptors activated by three different agonists, glutamate, AMPA, and kainate, at different holding potentials. Gd3+ (5 µM) increased the steady-state currents evoked by all three agonists at each of the holding potentials tested, suggesting that the effect of Gd3+ was unlikely to be voltage dependent (Fig. 3, A-C). The lesser degree of enhancement at depolarized potentials likely reflects the voltage dependence of desensitization itself.
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Gd3+ both inhibits AMPA receptor-mediated currents and reduces desensitization
Our results suggest that Gd3+ may have reduced AMPA receptor desensitization, and therefore we examined the rate of onset of desensitization of AMPA currents in recordings from outside-out patches using an ultra-fast perfusion system. The decay of the current evoked by the application of glutamate (10 mM, 100 ms, the onset of desensitization) was well fit by single exponential function. Gd3+ (5 µM) increased the apparent time constant of desensitization (control, 8.2 ± 1.5 ms; Gd3+, 12.5 ± 2.0 ms, n = 9, P < 0.01, Fig. 4, A and B), suggesting that Gd3+ does indeed reduce AMPA receptor desensitization. The peak resolved by our ultra-fast perfusion likely represents the activity of a majority of nondesensitized receptors. In this respect, Gd3+ inhibited these peak currents by 14.6 ± 2.5% (Fig. 4, Ad and B, n = 9, P < 0.01), suggesting that Gd3+ may also block AMPA currents at low concentrations in addition to reducing desensitization. To confirm this possibility, we used cyclothiazide (CTZ) to block AMPA receptor desensitization and anticipated that a blockade would be revealed even at relatively low concentrations of Gd3+. For example, a low concentration of Gd3+ enhanced kainate-evoked currents (Fig. 4C, left) in the absence of CTZ. As anticipated, applications of CTZ to the same cells dramatically increased the kainate-evoked current by reducing desensitization (Fig. 4C, right). However, in the presence of CTZ applications of Gd3+ consistently inhibited currents by 6.4 ± 0.6% (n = 5, P < 0.01, Fig. 4, C and D), confirming the inhibition of AMPA receptor currents.
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One possible explanation for the enhancement of steady-state currents was that Gd3+ increased the open probability of AMPA channels even though it can potentially block open channels. Therefore we examined the effect of Gd3+ on the open probability and the single-channel conductance of AMPA channels at the peak of the response using outside-out patches and nonstationary variance analysis. Gd3+ reduced the open probability at the peak (control, 0.69 ± 0.04; Gd3+, 0.45 ± 0.09, n = 5, P < 0.05) without changing the single-channel conductance (control, 13.6 ± 4.7 pS; Gd3+, 10.2 ± 2.8 pS, n = 5, P > 0.05; Fig. 5). The reduction in the open probability may have resulted from a Gd3+-induced inhibition of channel gating or a slowing of activation kinetics (see following text).
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Another possibility is that Gd3+ enhances
steady-state currents by increasing the rate of recovery from
desensitization. Therefore we examined recovery in outside-out patches
using a paired-pulse paradigm in which the first application of
glutamate (10 mM for 100 ms) was followed at variable intervals
ranging, from 20 to 420 ms, by a second application of glutamate (10 mM, 20 ms; Fig. 6A). The ratio
of the amplitude of the second to the first response (P2/P1) was
plotted against the interpulse interval thus providing the time course
of recovery from desensitization (Fig. 6B). The time
constant of recovery, estimated by a single exponential fit, was
reduced by Gd3+ (control, = 47.8 ± 4.9 ms; Gd3+,
= 30.4 ± 3.7 ms,
n = 5, P < 0.05) demonstrating that
Gd3+ likely enhances the rate of recovery from
desensitization.
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Gd3+ reduces both the activation and deactivation of AMPA receptors
Several modulators of AMPA receptor desensitization, such as
cyclothiazide, aniracetam and thiocyanate, also modulate AMPA receptor
deactivation (Partin et al. 1996). Therefore we next examined the effect of Gd3+ on AMPA receptor
deactivation using the ultra-fast perfusion system. In the presence of
Gd3+, the deactivation time constant was enhanced
by 18.0 ± 4.3% (control,
= 2.7 ± 0.2 ms;
Gd3+,
= 3.2 ± 0.2 ms,
n = 7, P < 0.01), indicating the
deactivation process was slowed by Gd3+ (Fig.
7, A and B). We
also observed an increase in the 10-90% rise time in the presence of
Gd3+, suggesting that activation kinetics were
also slowed (Fig. 7). Therefore Gd3+ may slow
both the binding and unbinding of agonist to the receptor.
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Several important functions of AMPA receptors such as the
determination of Ca2+ permeability, sensitivity
to a blockade by polyamines and relative rates of desensitization are
dependent on the presence or absence of the GluR2 subunit in the AMPA
receptor (Dingledine et al. 1999). Most AMPA receptors
in CA1 neurons contain the GluR2 subunit and this subunit determines
the permeability of the channels to Ca2+.
Therefore we considered the possibility that the
Gd3+-induced modulation of desensitization
required the presence of this subunit. However,
Gd2+ enhanced glutamate-evoked currents in the
hippocampal CA1 neurons isolated from GluR2-deficient mice (Jia
et al. 1996
) (Fig. 8). Neither
was the potency of Gd3+ changed (Fig. 8,
A and B).
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The kinetics of excitatory postsynaptic currents in individual cells
are determined by a combination of receptor deactivation, desensitization, and the rate of recovery from desensitized state (Edmonds et al. 1995; Trussell and Otis
1996
). Since Gd3+ slowed both
deactivation and desensitization kinetics and enhanced the rate of
recovery from desensitization, we next examined whether or not
Gd3+ modulates the kinetics of mEPSCs recorded in
cultured hippocampal neurons. Bath application of
Gd3+ (5 µM) significantly increased the
frequency of AMPA mEPSCs (control, 0.6 ± 0.1 Hz;
Gd3+, 4.0 ± 0.4 Hz, n = 8, P < 0.01, Fig. 9),
suggesting that Gd3+ also had presynaptic
actions. Peak mEPSC and the 10-90% rise times were not significantly
altered by Gd3+ (Fig. 9C). However,
the time constants of decay were consistently enhanced by
Gd3+ (Fig. 9), suggesting that
Gd3+ also modulates postsynaptic AMPA receptors.
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DISCUSSION |
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The time course and amplitude of the macroscopic AMPA
receptor-mediated current reflects multiple concurrent processes
including receptor activation, deactivation, and desensitization
(Partin et al. 1996). The behavior of AMPA receptors is
complex and has been modeled using various multi-state kinetic schemes.
However, for simplicity, we characterized the effects of
Gd3+ on AMPA receptor function by describing
changes in the peak and stead-state current. Nevertheless it is
recognized that this simplistic process understates the complexity of
AMPA receptor function. Our results demonstrate that
Gd3+ reduces AMPA receptor desensitization in
hippocampal neurons since Gd3+ increased the
ratio of steady state to peak currents in whole cell recordings, slowed
the onset of desensitization in outside-out patches, and enhanced the
recovery from desensitization. The observation that the enhancement of
kainate-evoked currents by Gd3+ was blocked in
the presence of cyclothiazide, which blocks or eliminates AMPA receptor
desensitization, also suggests that Gd3+ reduces
AMPA receptor desensitization.
A small region (about 38 amino acids) of the extracellular M3-M4 loop
of AMPA receptors is formed from an alternatively spliced exon termed
the flip/flop domain (Bennett and Dingledine 1995; Hollmann et al. 1994
; Sommer et al. 1990
;
Stern-Bach et al. 1994
). This region regulates the
kinetics of deactivation (Partin et al. 1996
), the onset
of and recovery from desensitization (Lomeli et al.
1994
; Mosbacher et al. 1994
; Sommer et
al. 1990
) as well as the sensitivity to drugs such as
cyclothiazide, aniracetam, and thiocyanate (Johansen et al.
1995
; Partin et al. 1994
, 1996
). Gd3+ may also bind to this region to reduce AMPA
receptor deactivation and desensitization based on our observations
that Gd3+ slowed both deactivation and
desensitization kinetics, Gd3+ increased recovery
from desensitization, and the Gd3+-mediated
enhancement of kainate-evoked current was occluded by cyclothiazide.
In addition to reducing AMPA receptor deactivation and desensitization,
Gd3+ also had inhibitory effects on AMPA
currents. When the Gd3+ concentration was low,
the inhibition was masked by the enhanced currents caused by reduction
of AMPA receptor desensitization. The inhibitory effect was revealed by
cyclothiazide or by the use of the ultra-fast perfusion to more
accurately resolve peak currents. We failed to detect any change in the
peak of whole cell currents evoked by glutamate in response to
Gd3+. There are several possible explanations for
this discrepancy. For example, the properties (i.e.,
Gd3+ sensitivity) of the channels may have been
altered in the outside-out patch configuration as reported by
Tong and Jahr (1994). Alternatively and more likely, the
rate of solution exchange was so much slower in whole cell recordings
that the apparent peak current may simply have reflected a balance
between enhanced desensitization and inhibition.
The time course of deactivation and desensitization that we observed
for glutamate-evoked currents were in the same range as reported from
hippocampal neurons by other laboratories (Donevan and Rogawski
1998; Fleck et al. 1996
; Patneau et al.
1993
). However, they were slower than those reported for chick
nucleus magnocellularis (nMAG) neurons (Raman and
Trussel 1992
), rat MNTB relay neurons, and Bergmann glia
(Geiger et al. 1995
) or homo-oligomeric AMPA receptors
(Mosbacher et al. 1994
; Partin et al.
1996
). In contrast, the recovery from desensitization of AMPA
receptors in hippocampal neurons (
= 48 ms) was faster than
that reported for homo-oligomeric GluR1 receptors (
= 147 ms)
(Partin et al. 1996
) but slower than that for reported
for nMAG neurons (
= 16 ms) (Raman and Trussell 1992
). These differences between recombinant and native
receptors or among the native neurons of different origins may be
caused by heterogeneity in subunit composition and/or by differential RNA splicing/editing of individual subunits (Koike et al.
2000
; Lomeli et al. 1994
; Partin et al.
1994
; Sommer et al. 1990
) or perhaps due to
methodological differences such as the use of different enzymes for
isolating cells.
Application of Gd3+ to the cultured hippocampal
neurons significantly enhanced the frequency of mEPSCs, suggesting that
Gd3+ enhances release of transmitter. This result
is consistent with a previous observation (Capogna et al.
1996), although the mechanism by which
Gd3+ increases transmitter release is unclear.
Nevertheless we also observed an increase in the time constant of the
decay of mEPSC; this is consistent with actions on postsynaptic AMPA
receptors. It is unlikely that desensitization contributes to the decay
of mEPSCs, and it is more likely that the
Gd3+-induced slowing of AMPA receptors
deactivation (Colquhoun et al. 1992
; Jonas and
Sakmann 1992
) is responsible for the prolongation of decay of
mEPSCs we observed.
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FOOTNOTES |
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Address for reprint requests: S. Lei, Dept. of Physiology, University of Toronto, Medical Sciences Building, Toronto, Ontario M5S 1A8, Canada (E-mail: j.macdonald{at}utoronto.ca).
Received 4 December 2000; accepted in final form 27 March 2001.
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REFERENCES |
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